DEDD TFIIIC102 True Positive 11965497 Zhan Y, Hegde R, Srinivasula SM, Fernandes-Alnemri T, Alnemri ES: Death effector domain-containing proteins DEDD and FLAME-3 form nuclear complexes with the TFIIIC102 subunit of human transcription factor IIIC. Cell Death Differ. 2002 Apr;9(4):439-47. Death effector domain-containing proteins are involved in important cellular processes such as death-receptor induced apoptosis, NF-kappaB activation and ERK activation. Here we report the identification of a novel nuclear DED-containing protein, FLAME-3. FLAME-3 shares significant sequence (46.6% identical) and structural homology to another DED-containing protein, DEDD. FLAME-3 interacts with DEDD and c-FLIP (FLAME-1) but not with the other DED-containing proteins FADD, caspase-8 or caspase-10. FLAME-3 translocates to, and sequesters c-FLIP in the nucleus upon overexpression in human cell lines. Using the yeast two-hybrid system to identify DEDD-interacting proteins, the TFIIIC102 subunit of human transcription factor TFIIIC was identified as a DEDD- and FLAME-3-specific interacting protein. Co-expression of either DEDD or FLAME-3 with hTFIIIC102 in MCF-7 cells induces the translocation from the cytoplasm and sequestration of hTFIIIC102 in the nucleus, indicating that DEDD and FLAME-3 form strong heterocomplexes with hTFIIIC102 and might be important regulators of the activity of the hTFIIIC transcriptional complex. Consistent with this, overexpression of DEDD or FLAME-3 in 293 cells inhibited the expression of a luciferase-reporter gene under the control of the NF-kappaB promoter. Our data provide the first direct evidence for the involvement of DED-containing proteins in the regulation of components of the general transcription machinery in the nucleus.
DEDD RNA polymerase I True Positive 11753564 Schickling O, Stegh AH, Byrd J, Peter ME: Nuclear localization of DEDD leads to caspase-6 activation through its death effector domain and inhibition of RNA polymerase I dependent transcription. Cell Death Differ. 2001 Dec;8(12):1157-68. The death effector domain (DED) is a protein/protein interaction domain only found in proteins that are involved in apoptosis signaling. DEDD is a novel apoptosis signaling molecule that carries an N-terminal DED with complete sequence identity between the murine, rat, bovine and human domains. We previously identified two nuclear localization signals (NLS) responsible for DEDDs nuclear localization when transiently expressed. Using a new anti-DEDD antibody that allows us to stain endogenous DEDD in immunofluorescence microscopy we now detect a significant amount of DEDD in nucleoli of all cells tested. When overexpressed, DEDD localizes to nucleoli-like structures, activates caspase-6 and specifically inhibits RNA polymerase I (Pol I) dependent transcription in vivo as shown by blockage of BrUTP incorporation. The DED in DEDD is sufficient for its DNA binding, caspase-6 activating and Pol I specific transcriptional repressor activity. We have identified a third NLS in DEDD and only mutation of all three NLS generated a protein, DEDD Delta NLS1-3, that mainly localized to the cytoplasm. This protein no longer induced apoptosis, indicating that in contrast to other DED proteins, such as FADD, caspase-8 or c-FLIP, DEDD induces apoptosis from within the nucleus. This effect is abolished when specific point mutations are made within the DED. The DED in DEDD therefore represents a novel domain that is structurally similar to other DEDs but functionally different from classical DEDs found in FADD or caspase-8.
DEDD RSK2 True Positive 12796492 Vaidyanathan H, Ramos JW: RSK2 activity is regulated by its interaction with PEA-15. . J Biol Chem. 2003 Aug 22;278(34):32367-72. Epub 2003 Jun 9. The ERK MAP (mitogen-activated protein) kinase cascade modulates many cellular processes including transcription, adhesion, growth, survival, and proliferation. One target substrate of ERK involved in regulating transcription is the p90 ribosomal S6 kinase (RSK) isozyme, RSK2. Here we demonstrate that a small death effector domain-containing protein called PEA-15 binds RSK2. RSK2 and PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) co-precipitated from cells and were colocalized in the cytoplasm. Furthermore, purified PEA-15 bound in vitro translated RSK2, suggesting that these proteins interact directly. PEA-15 does not bind to RSK1 and therefore exhibits some binding specificity. RSK2 binds the COOH terminus of PEA-15 and does not interact with its NH2-terminal death effector domain. We show that this interaction has functional consequences including the inhibition of RSK2-dependent CREB transcription. PEA-15 expression also blocks histone H3 phosphorylation, an RSK2-dependent event that may contribute to effects on gene expression. These results can be attributed to two effects of PEA-15 on RSK2. First, PEA-15 blocks nuclear accumulation of RSK2 after epidermal growth factor stimulation. Second, PEA-15 inhibits RSK2 kinase activity by 50%. A mutant of PEA-15 that binds RSK2 but is localized to the nucleus had no effect on RSK2-dependent transcription. Interestingly, this mutant also did not affect RSK2 kinase activity. This may indicate that cytoplasmic retention of RSK2 is also required for PEA-15 to impair kinase activity. PEA-15 does not alter ERK phosphorylation of RSK2 and is not itself a substrate of RSK2. Hence the effects of PEA-15 on RSK2 represent a novel mechanism for the regulation of RSK2-mediated signaling.
DEDD DED True Positive 17193921 Huang X, Zhang M, Tang H, Ruo C, Cao X: Identification and characterization of DEDDL, a human-specific isoform of DEDD. Gene Expr. 2006;13(3):141-53. Death effector domain (DED) containing molecules are usually involved in the intracellular apoptosis cascade as executioners or regulators. One of these molecules, DEDD, was identified as a final target of the CD95 signaling pathway by which it would be transferred into the nucleolus to inhibit RNA polymerase I-dependent transcription. Here we describe a longer isoform of DEDD, DEDDL, produced by alternatively splicing, as an immune cell-specific DED-containing molecule. It is only expressed in human T lymphocytes and dendritic cells (DCs), and the mRNA expression in DCs was elevated upon inductive maturation. In cell lines MCF-7 and Jurkat, the overexpression of DEDDL could induce apoptosis more potently than that of DEDD. That DEDDL could bind FADD and cFLIP more potently than DEDD in vivo was revealed by cotransfection and immunoprecipitation. This may explain why DEDDL is a more potent apoptosis inducer, because DED-containing proteins usually induce apoptosis through DED binding. Finally, why DEDD and DEDDL are unstable in the overexpression and other studies may be explained by the finding that they are potential substrates of active caspases.
DEDD DED True Positive 12527898 Alcivar A, Hu S, Tang J, Yang X: DEDD and DEDD2 associate with caspase-8/10 and signal cell death. Oncogene. 2003 Jan 16;22(2):291-7. An apoptotic signal triggered by cell surface death receptors is disseminated to intracellular compartments through protein-protein interactions mediated by conserved domains such as the death effector domain (DED). A unique family of single DED-containing proteins, including DEDD and DEDD2, is targeted to the nucleolus. However, the role of DEDD/DEDD2 in apoptosis remains less understood. Here we show that DEDD and DEDD2 are highly conserved in diverse species, and that they are potent inducers of apoptosis in various cell types. Deletion analysis indicates that both the N-terminal DED domain and the C-terminal region of DEDD2 can induce apoptosis. The cell death activity of this family appears to be related to their nuclear localization. DEDD and DEDD2 bind to two tandem DED-containing caspases, caspase -8 and -10, that are engaged by death receptors. Consistent with the nuclear localization of this family, caspase-8 translocates to the nucleus during CD95-induced apoptosis. DEDD and DEDD2 also readily associate with themselves and with each other. These results suggest that DEDD and DEDD2 may be important mediators for death receptors and that they may target caspases to the nucleus.
DEDD DED True Positive 11753564 Schickling O, Stegh AH, Byrd J, Peter ME: Nuclear localization of DEDD leads to caspase-6 activation through its death effector domain and inhibition of RNA polymerase I dependent transcription. Cell Death Differ. 2001 Dec;8(12):1157-68. The death effector domain (DED) is a protein/protein interaction domain only found in proteins that are involved in apoptosis signaling. DEDD is a novel apoptosis signaling molecule that carries an N-terminal DED with complete sequence identity between the murine, rat, bovine and human domains. We previously identified two nuclear localization signals (NLS) responsible for DEDDs nuclear localization when transiently expressed. Using a new anti-DEDD antibody that allows us to stain endogenous DEDD in immunofluorescence microscopy we now detect a significant amount of DEDD in nucleoli of all cells tested. When overexpressed, DEDD localizes to nucleoli-like structures, activates caspase-6 and specifically inhibits RNA polymerase I (Pol I) dependent transcription in vivo as shown by blockage of BrUTP incorporation. The DED in DEDD is sufficient for its DNA binding, caspase-6 activating and Pol I specific transcriptional repressor activity. We have identified a third NLS in DEDD and only mutation of all three NLS generated a protein, DEDD Delta NLS1-3, that mainly localized to the cytoplasm. This protein no longer induced apoptosis, indicating that in contrast to other DED proteins, such as FADD, caspase-8 or c-FLIP, DEDD induces apoptosis from within the nucleus. This effect is abolished when specific point mutations are made within the DED. The DED in DEDD therefore represents a novel domain that is structurally similar to other DEDs but functionally different from classical DEDs found in FADD or caspase-8.
DEDD DED True Positive 11395500 Zheng L, Schickling O, Peter ME, Lenardo MJ: The death effector domain-associated factor plays distinct regulatory roles in the nucleus and cytoplasm. J Biol Chem. 2001 Aug 24;276(34):31945-52. Epub 2001 Jun 6. Homophilic interactions of death effector domains (DEDs) are crucial for the signaling pathways of death receptor-mediated apoptosis. The machinery that regulates proper oligomerization and autoactivation of procaspase-8 and/or procaspase-10 during T lymphocyte activation determines whether the cells will undergo caspase-mediated apoptosis or proliferation. We screened a yeast two-hybrid library by using the DEDs contained in the prodomains of procaspase-8 and procaspase-10 and isolated a DED-associated factor (DEDAF) that interacts with several DED-containing proteins but does not itself contain a DED. DEDAF is highly conserved between human and mouse (98% amino acid identity) and is homologous to a nuclear regulatory protein YAF-2. DEDAF is expressed at the highest levels in lymphoid tissues and placenta. DEDAF interacts with FADD, procaspase-8, and procaspase-10 in the cytosol as well as with the DED-containing DNA-binding protein (DEDD) in the nucleus. At the cell membrane, DEDAF augmented the formation of CD95-FADD-caspase-8 complexes and enhanced death receptor- as well as DED-mediated apoptosis. In the nucleus, DEDAF caused the DEDD protein to relocalize from subnuclear structures to a diffuse distribution in the nucleoplasm. Our data therefore suggest that DEDAF may be involved in the regulation of both cytoplasmic and nuclear events of apoptosis.
DEDD DED True Positive 9832420 Leo CP, Hsu SY, McGee EA, Salanova M, Hsueh AJ: DEFT, a novel death effector domain-containing molecule predominantly expressed in testicular germ cells. Endocrinology. 1998 Dec;139(12):4839-48. Apoptosis is a physiological process by which multicellular organisms eliminate unwanted cells. Death factors such as Fas ligand induce apoptosis by triggering a series of intracellular protein-protein interactions mediated by defined motifs found in the signaling molecules. One of these motifs is the death effector domain (DED), a stretch of about 80 amino acids that is shared by adaptors, regulators, and executors of the death factor pathway. We have identified the human and rat complementary DNAs encoding a novel protein termed DEFT (Death EFfector domain-containing Testicular molecule). The N-terminus of DEFT shows a high degree of homology to the DEDs found in FADD (an adaptor molecule) as well as procaspase-8/FLICE and procaspase-10/Mch4 (executors of the death program). Northern blot hybridization experiments have shown that the DEFT messenger RNA (mRNA) is expressed in a variety of human and rat tissues, with particularly abundant expression in the testis. In situ hybridization analysis further indicated the expression of DEFT mRNA in meiotic male germ cells. In a model of germ cell apoptosis induction, an increase in testis DEFT mRNA was found in immature rats after 2 days of treatment with a GnRH antagonist. Unlike FADD and procaspase-8/FLICE, overexpression of DEFT did not induce apoptosis in Chinese hamster ovary cells. Although cotransfection studies indicated that DEFT is incapable of modulating apoptosis effected by FADD and procaspase-8/FLICE, interactions between DEFT and uncharacterized DED-containing molecules in the testis remain to be studied in the future. In conclusion, we have identified a novel DED-containing protein with high expression in testis germ cells. This protein may be important in the regulation of death factor-induced apoptosis in the testis and other tissues.
DEDD DED True Positive 9774341 Stegh AH, Schickling O, Ehret A, Scaffidi C, Peterhansel C, Hofmann TG, Grummt I, Krammer PH, Peter ME: DEDD, a novel death effector domain-containing protein, targeted to the nucleolus. EMBO J. 1998 Oct 15;17(20):5974-86. The CD95 signaling pathway comprises proteins that contain one or two death effector domains (DED), such as FADD/Mort1 or caspase-8. Here we describe a novel 37 kDa protein, DEDD, that contains an N-terminal DED. DEDD is highly conserved between human and mouse (98. 7% identity) and is ubiquitously expressed. Overexpression of DEDD in 293T cells induced weak apoptosis, mainly through its DED by which it interacts with FADD and caspase-8. Endogenous DEDD was found in the cytoplasm and translocated into the nucleus upon stimulation of CD95. Immunocytological studies revealed that overexpressed DEDD directly translocated into the nucleus, where it co-localizes in the nucleolus with UBF, a basal factor required for RNA polymerase I transcription. Consistent with its nuclear localization, DEDD contains two nuclear localization signals and the C-terminal part shares sequence homology with histones. Recombinant DEDD binds to both DNA and reconstituted mononucleosomes and inhibits transcription in a reconstituted in vitro system. The results suggest that DEDD is a final target of a chain of events by which the CD95-induced apoptotic signal is transferred into the nucleolus to shut off cellular biosynthetic activities.
DEDD CLARP True Positive 10187771 Thome M, Martinon F, Hofmann K, Rubio V, Steiner V, Schneider P, Mattmann C, Tschopp J: Equine herpesvirus-2 E10 gene product, but not its cellular homologue, activates NF-kappaB transcription factor and c-Jun N-terminal kinase. J Biol Chem. 1999 Apr 9;274(15):9962-8. We have previously reported on the death effector domain containing E8 gene product from equine herpesvirus-2, designated FLICE inhibitory protein (v-FLIP), and on its cellular homologue, c-FLIP, which inhibit the activation of caspase-8 by death receptors. Here we report on the structure and function of the E10 gene product of equine herpesvirus-2, designated v-CARMEN, and on its cellular homologue, c-CARMEN, which contain a caspase-recruiting domain (CARD) motif. c-CARMEN is highly homologous to the viral protein in its N-terminal CARD motif but differs in its C-terminal extension. v-CARMEN and c-CARMEN interact directly in a CARD-dependent manner yet reveal different binding specificities toward members of the tumor necrosis factor receptor-associated factor (TRAF) family. v-CARMEN binds to TRAF6 and weakly to TRAF3 and, upon overexpression, potently induces the c-Jun N-terminal kinase (JNK), p38, and nuclear factor (NF)-kappaB transcriptional pathways. c-CARMEN or truncated versions thereof do not appear to induce JNK and NF-kappaB activation by themselves, nor do they affect the JNK and NF-kappaB activating potential of v-CARMEN. Thus, in contrast to the cellular homologue, v-CARMEN may have additional properties in its unique C terminus that allow for an autonomous activator effect on NF-kappaB and JNK. Through activation of NF-kappaB, v-CARMEN may regulate the expression of the cellular and viral genes important for viral replication.
DEDD tumor necrosis factor receptor 1 True Positive 9092488 Hu S, Vincenz C, Buller M, Dixit VM: A novel family of viral death effector domain-containing molecules that inhibit both CD-95- and tumor necrosis factor receptor-1-induced apoptosis. J Biol Chem. 1997 Apr 11;272(15):9621-4. Molluscum contagiosum virus proteins MC159 and MC160 and the equine herpesvirus 2 protein E8 share substantial homology to the death effector domain present in the adaptor molecule Fas-associated death domain protein (FADD) and the initiating death protease FADD-like interleukin-1beta-converting enzyme (FLICE) (caspase-8). FADD and FLICE participate in generating the death signal from both tumor necrosis factor receptor-1 (TNFR-1) and the CD-95 receptor. The flow of death signals from TNFR-1 occurs through the adaptor molecule tumor necrosis factor receptor-associated death domain protein (TRADD) to FADD to FLICE, whereas for CD-95 the receptor directly communicates with FADD and then FLICE. MC159 and E8 inhibited both TNFR-1- and CD-95-induced apoptosis as well as killing mediated by overexpression of the downstream adaptors TRADD and FADD. Neither viral molecule, however, inhibited FLICE-induced killing, consistent with an inhibitory action upstream of the active death protease. These data suggest the existence of a novel strategy employed by viruses to attenuate host immune killing mechanisms. Given that bovine herpesvirus 4 protein E1.1 and Kaposi's sarcoma associated-herpesvirus protein K13 also possess significant homology to the viral inhibitory molecules MC159, MC160, and E8, it may be that this class of proteins is used ubiquitously by viruses to evade host defense.
DEDD tumor necrosis factor receptor 1 True Positive 9037025 Bertin J, Armstrong RC, Ottilie S, Martin DA, Wang Y, Banks S, Wang GH, Senkevich TG, Alnemri ES, Moss B, Lenardo MJ, Tomaselli KJ, Cohen JI: Death effector domain-containing herpesvirus and poxvirus proteins inhibit both Fas- and TNFR1-induced apoptosis. Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1172-6. To identify novel antiapoptotic proteins encoded by DNA viruses, we searched viral genomes for proteins that might interfere with Fas and TNFR1 apoptotic signaling pathways. We report here that equine herpesvirus type 2 E8 protein and molluscum contagiosum virus MC159 protein both show sequence similarity to the death effector domains (DEDs) of the Fas/TNFR1 signaling components FADD and caspase-8. Yeast two-hybrid analysis revealed that E8 protein interacted with the caspase-8 prodomain whereas MC159 protein interacted with FADD. Furthermore, expression of either E8 protein or MC159 protein protected cells from Fas- and TNFR1-induced apoptosis indicating that certain herpesviruses and poxviruses use DED-mediated interactions to interfere with apoptotic signaling pathways. These findings identify a novel control point exploited by viruses to regulate Fas- and TNFR1-mediated apoptosis.
DEDD caspase 3 True Positive 16235027 Lee JC, Wang GX, Schickling O, Peter ME: Fusing DEDD with ubiquitin changes its intracellular localization and apoptotic potential. Apoptosis. 2005 Dec;10(6):1483-95. DEDD, a highly conserved and ubiquitous death effector domain containing protein, exists in non, mono, and diubiquitinated forms. We previously reported that endogenous unmodified DEDD is only found in nucleoli and that mono- and diubiquitinated DEDD associate with caspase-3 in the cytosol suggesting that ubiquitination may be important to the apoptosis regulating functions of DEDD in the cytosol. We now demonstrate that many of its 16 lysine residues can serve as alternative acceptors for ubiquitination to maintain the monoubiquitination status of DEDD. A central region in DEDD (amino acids 109-305) outside the death effector domain was found to be essential for ubiquitination and/or the docking of the ubiquitination machinery. Fusion of ubiquitin to the C-terminus of DEDD to mimic monoubiquitinated DEDD relocated DEDD from nucleoli to the cytosol. This fusion protein also demonstrated a greater apoptosis potential than unmodified DEDD. Finally, we show that both mono- and polyubiquitination of DEDD can be achieved by the cellular inhibitor of apoptosis proteins 1 and 2 (cIAP-1/2). In addition, the cotransfection of DEDD with cIAP-1 or cIAP-2 results in the relocalization of the IAPs to the nucleoli. Our data suggest that monoubiquitination of DEDD regulates both its cytoplasmic localization and its proapoptotic potential and that IAP proteins can regulate DEDD's ubiquitination status.
DEDD caspase 3 True Positive 12235123 Lee JC, Schickling O, Stegh AH, Oshima RG, Dinsdale D, Cohen GM, Peter ME: DEDD regulates degradation of intermediate filaments during apoptosis. J Cell Biol. 2002 Sep 16;158(6):1051-66. Epub 2002 Sep 16. Apoptosis depends critically on regulated cytoskeletal reorganization events in a cell. We demonstrate that death effector domain containing DNA binding protein (DEDD), a highly conserved and ubiquitous death effector domain containing protein, exists predominantly as mono- or diubiquitinated, and that diubiquitinated DEDD interacts with both the K8/18 intermediate filament network and pro-caspase-3. Early in apoptosis, both cytosolic DEDD and its close homologue DEDD2 formed filaments that colocalized with and depended on K8/18 and active caspase-3. Subsequently, these filamentous structures collapsed into intracellular inclusions that migrated into cytoplasmic blebs and contained DEDD, DEDD2, active caspase-3, and caspase-3-cleaved K18 late in apoptosis. Biochemical studies further confirmed that DEDD coimmunoprecipitated with both K18 and pro-caspase-3, and kinetic analyses placed apoptotic DEDD staining prior to caspase-3 activation and K18 cleavage. In addition, both caspase-3 activation and K18 cleavage was inhibited by expression of DEDDDeltaNLS1-3, a cytosolic form of DEDD that cannot be ubiquitinated. Finally, siRNA mediated DEDD knockdown cells exhibited inhibition of staurosporine-induced DNA degradation. Our data suggest that DEDD represents a novel scaffold protein that directs the effector caspase-3 to certain substrates facilitating their ordered degradation during apoptosis.
DEDD IAP True Positive 16235027 Lee JC, Wang GX, Schickling O, Peter ME: Fusing DEDD with ubiquitin changes its intracellular localization and apoptotic potential. Apoptosis. 2005 Dec;10(6):1483-95. DEDD, a highly conserved and ubiquitous death effector domain containing protein, exists in non, mono, and diubiquitinated forms. We previously reported that endogenous unmodified DEDD is only found in nucleoli and that mono- and diubiquitinated DEDD associate with caspase-3 in the cytosol suggesting that ubiquitination may be important to the apoptosis regulating functions of DEDD in the cytosol. We now demonstrate that many of its 16 lysine residues can serve as alternative acceptors for ubiquitination to maintain the monoubiquitination status of DEDD. A central region in DEDD (amino acids 109-305) outside the death effector domain was found to be essential for ubiquitination and/or the docking of the ubiquitination machinery. Fusion of ubiquitin to the C-terminus of DEDD to mimic monoubiquitinated DEDD relocated DEDD from nucleoli to the cytosol. This fusion protein also demonstrated a greater apoptosis potential than unmodified DEDD. Finally, we show that both mono- and polyubiquitination of DEDD can be achieved by the cellular inhibitor of apoptosis proteins 1 and 2 (cIAP-1/2). In addition, the cotransfection of DEDD with cIAP-1 or cIAP-2 results in the relocalization of the IAPs to the nucleoli. Our data suggest that monoubiquitination of DEDD regulates both its cytoplasmic localization and its proapoptotic potential and that IAP proteins can regulate DEDD's ubiquitination status.
DEDD BRAL 1 False Positive 15582580 Yeom M, Shim I, Lee HJ, Hahm DH: Proteomic analysis of nicotine-associated protein expression in the striatum of repeated nicotine-treated rats. Biochem Biophys Res Commun. 2005 Jan 14;326(2):321-8. Through the proteomic analysis using 2-dimensional electrophoresis, the nicotine addiction-associated proteins were extensively screened in the striatum of rat brains. The nicotine addiction was developed by repeated nicotine injection (0.4mg/kg s.c.), twice daily for 7 days, followed by one challenge injection after a 3 day withdrawal period, and then confirmed by observing a 2.3-fold increase in locomoter activity. The 3 up- and 4 down-regulated proteins were selected and identified to be zinc-finger binding protein-89 (ZBP-89), 2'3'-cyclic nucleotide 3'-phosphodiesterase 1, deoxyribonuclease 1-like 3 (DNase1l3), tandem pore domain halothane inhibited K (+) channel (THIK-2), brain-specific hyaluronan-binding protein (BRAL-1), death effector domain-containing DNA binding protein (DEDD), and brain-derived neurotrophic factor (BDNF) by mass spectrophotometric fingerprinting. Among them, the expression patterns of ZEB-89, DNase1l3, THIK-2, DEDD, and BDNF mRNAs were found to be coincident with those of cognate proteins, by using RT-PCR analysis. These proteins could be suggested as drug targets to develop a new therapy for nicotine-associated diseases, as well as the clues to understand the mechanism of nicotine.
DEDD IFN True Positive 16514659 Espert L, Eldin P, Gongora C, Bayard B, Harper F, Chelbi-Alix MK, Bertrand E, Degols G, Mechti N: The exonuclease ISG20 mainly localizes in the nucleolus and the Cajal (Coiled) bodies and is associated with nuclear SMN protein-containing complexes. J Cell Biochem. 2006 Aug 1;98(5):1320-33. We have previously shown that ISG20, an interferon (IFN)-induced gene, encodes a 3' to 5' exoribonuclease member of the DEDD superfamily of exonucleases. ISG20 specifically degrades single-stranded RNA. In this report, using immunofluorescence analysis, we demonstrate that in addition to a diffuse cytoplasmic and nucleoplasmic localization, the endogenous ISG20 protein was present in the nucleus both in the nucleolus and in the Cajal bodies (CBs). In addition, we show that the ectopic expression of the CBs signature protein, coilin, fused to the red fluorescent protein (coilin-dsRed) increased the number of nuclear dots containing both ISG20 and coilin-dsRed. Using electron microcopy analysis, ISG20 appeared principally concentrated in the dense fibrillar component of the nucleolus, the major site for rRNA processing. We also present evidences that ISG20 was associated with survival of motor neuron (SMN)-containing macromolecular nuclear complexes required for the biogenesis of various small nuclear ribonucleoproteins. Finally, we demonstrate that ISG20 was associated with U1 and U2 snRNAs, and U3 snoRNA. The accumulation of ISG20 in the CBs after IFN treatment strongly suggests its involvement in a new route for IFN-mediated inhibition of protein synthesis by modulating snRNA and rRNA maturation.
DEDD inv True Positive 15866929 Buchner JM, Robertson AE, Poynter DJ, Denniston SS, Karls AC: Piv site-specific invertase requires a DEDD motif analogous to the catalytic center of the RuvC Holliday junction resolvases. J Bacteriol. 2005 May;187(10):3431-7. Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.
DEDD caspase 6 True Positive 11753564 Schickling O, Stegh AH, Byrd J, Peter ME: Nuclear localization of DEDD leads to caspase-6 activation through its death effector domain and inhibition of RNA polymerase I dependent transcription. Cell Death Differ. 2001 Dec;8(12):1157-68. The death effector domain (DED) is a protein/protein interaction domain only found in proteins that are involved in apoptosis signaling. DEDD is a novel apoptosis signaling molecule that carries an N-terminal DED with complete sequence identity between the murine, rat, bovine and human domains. We previously identified two nuclear localization signals (NLS) responsible for DEDDs nuclear localization when transiently expressed. Using a new anti-DEDD antibody that allows us to stain endogenous DEDD in immunofluorescence microscopy we now detect a significant amount of DEDD in nucleoli of all cells tested. When overexpressed, DEDD localizes to nucleoli-like structures, activates caspase-6 and specifically inhibits RNA polymerase I (Pol I) dependent transcription in vivo as shown by blockage of BrUTP incorporation. The DED in DEDD is sufficient for its DNA binding, caspase-6 activating and Pol I specific transcriptional repressor activity. We have identified a third NLS in DEDD and only mutation of all three NLS generated a protein, DEDD Delta NLS1-3, that mainly localized to the cytoplasm. This protein no longer induced apoptosis, indicating that in contrast to other DED proteins, such as FADD, caspase-8 or c-FLIP, DEDD induces apoptosis from within the nucleus. This effect is abolished when specific point mutations are made within the DED. The DED in DEDD therefore represents a novel domain that is structurally similar to other DEDs but functionally different from classical DEDs found in FADD or caspase-8.
DEDD ubiquitin True Positive 14742246 Dinsdale D, Lee JC, Dewson G, Cohen GM, Peter ME: Intermediate filaments control the intracellular distribution of caspases during apoptosis. Am J Pathol. 2004 Feb;164(2):395-407. Caspases are responsible for a cascade of events controlling the disassembly of apoptotic cells. We now demonstrate that caspase-9 is activated at an early stage of apoptosis in epithelial cells and all its detectable, catalytically active large subunits (both the p35 and p37) are concentrated on cytokeratin fibrils. Immunolabeling of distinctive neoepitopes, exposed by cleavage of procaspase-9 at either Asp315 or Asp330, was co-localized on these fibrils with active caspase-3, caspase-cleaved cytokeratin-18, death-effector-domain containing DNA-binding protein and ubiquitin. Cytokeratin filaments may thus provide a scaffold whereby active subunits of caspase-9 can activate caspase-3 which, in turn, can activate more caspase-9 so forming an amplification loop to facilitate cleavage of cytokeratin-18, disruption of the cytoskeleton and the ensuing formation of cytoplasmic inclusions. These inclusions, formed from the collapse of fibrils, together with their associated components, also contain ubiquitinated proteins, vimentin, heat-shock protein 72, and tumor necrosis factor receptor type-1-associated death domain protein. Many of their constituents, including active caspases, remain sequestered within these inclusions, even after detergent treatment and isolation. Thus, such inclusions do not merely accumulate disrupted cytokeratins but also sequestrate potentially noxious proteins that could injure healthy neighboring cells.
DEDD cyclin B1 True Positive 17592253 Miyazaki T, Arai S: Two distinct controls of mitotic cdk1/cyclin B1 activity requisite for cell growth prior to cell division. Cell Cycle. 2007 Jun 15;6(12):1419-25. Epub 2007 May 7. Cell growth prior to cell division is restricted by the activity of cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complexes. Recently, we identified that the death-effector domain (DED) containing protein, DEDD, acts as a novel inhibitor of mitotic Cdk1/cyclin B1, influencing cell size. Like cyclin B1, DEDD protein levels specifically peak during the G (2)/M phase. In the nucleus, DEDD associates with Cdk1/cyclin B1 complexes, via direct binding to cyclin B1, and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD (-/-)) embryonic fibroblasts is increased compared to that in DEDD (+/+) cells. This accelerates mitotic progression in DEDD (-/-) cells, with a shortened G (2)/M phase, reduced rRNA, and diminished cell volume. Likewise, DEDD (-/-) mice show decreased body and organ weights relative to DEDD (+/+) mice. Interestingly, the DED domain is not involved in the association of DEDD with Cdk1/cyclin B1, but is indispensable for the cell sizing function of DEDD. Together, in addition to the well-established machinery for activation of Cdk1 through dephosphorylation of its inhibitory-residues, we propose a novel mechanism for impeditive regulation of mitotic Cdk1/cyclin B1 mediated by DEDD within the nucleus, which allows sufficient cell growth prior to cell division.
DEDD cyclin B1 True Positive 17283331 Arai S, Miyake K, Voit R, Nemoto S, Wakeland EK, Grummt I, Miyazaki T: Death-effector domain-containing protein DEDD is an inhibitor of mitotic Cdk1/cyclin B1. Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2289-94. Epub 2007 Feb 5. Accumulating evidence has shown that many molecules, including some cyclin-dependent kinases (Cdks) and cyclins, as well as the death-effector domain (DED)-containing FADD, function for both apoptosis and cell cycle. Here we identified that DEDD, which also possesses the DED domain, acts as a novel inhibitor of the mitotic Cdk1/cyclin B1 complex. DEDD associates with mitotic Cdk1/cyclin B1 complexes via direct binding to cyclin B1 and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD-/-) embryonic fibroblasts is increased compared with that in DEDD+/+ cells, which results in accelerated mitotic progression, thus exhibiting a shortened G2/M stage. Interestingly, DEDD-/- cells also demonstrated decreased G1 duration, which perhaps enhanced the overall reduction in rRNA amounts and cell volume, primarily caused by the rapid termination of rRNA synthesis before cell division. Likewise, DEDD-/- mice show decreased body and organ weights relative to DEDD+/+ mice. Thus, DEDD is an impeder of cell mitosis, and its absence critically influences cell and body size via modulation of rRNA synthesis.
DEDD SMA False Positive 9829180 Tortorello ML, Reineke KF, Stewart DS, Raybourne RB: Comparison of methods for determining the presence of Escherichia coli O157:H7 in apple juice. J Food Prot. 1998 Nov;61(11):1425-30. Six methods were compared for detection of three strains of Escherichia coli O157:H7 in enrichments of inoculated apple juice. Juice was inoculated at levels varying from 0.1 to 100 CFU/ml and centrifuged after overnight storage at 4 degrees C, and pellets were incubated at 37 degrees C in nonselective enrichment broth. At hourly intervals between 5 and 10 h and at 24 h, the enrichments were tested for E. coli O157:H7 by direct fluorescent antibody (DFA), antibody-direct epifluorescent filter technique (Ab-DEFT), direct selective plating on sorbitol MacConkey agar (SMA), immunomagnetic separation coupled to either selective plating (IMS-SMA) or the polymerase chain reaction (IMS-PCR), and flow cytometry (FC). The most consistent detection of 0.1 CFU/ml of the slowest growing strain of the pathogen was provided by the IMS-SMA and IMS-PCR after 8 h of enrichment. The time required for detection at the level of 0.1 CFU/ml for each assay was Ab-DEFT, 11 h; IMS-PCR, 16 h; FC, 24 h; IMS-SMA, 32 h; and SMA, 48 h. Absolute detection limits (without enrichment) were: IMS-PCR, 10 (3) CFU/ml; Ab-DEFT and IMS-SMA, 10 (4) CFU/ml; SMA, 10 (5) CFU/ml; and DFA, 10 (6) CFU/ml. Recovery of the pathogen (10 CFU/ml) in apple juice after 28 days of 4 degrees C storage was possible by means of an 8-h enrichment and Ab-DEFT, IMS-PCR, or IMS-SMA.
DEDD FLICE True Positive 12527898 Alcivar A, Hu S, Tang J, Yang X: DEDD and DEDD2 associate with caspase-8/10 and signal cell death. Oncogene. 2003 Jan 16;22(2):291-7. An apoptotic signal triggered by cell surface death receptors is disseminated to intracellular compartments through protein-protein interactions mediated by conserved domains such as the death effector domain (DED). A unique family of single DED-containing proteins, including DEDD and DEDD2, is targeted to the nucleolus. However, the role of DEDD/DEDD2 in apoptosis remains less understood. Here we show that DEDD and DEDD2 are highly conserved in diverse species, and that they are potent inducers of apoptosis in various cell types. Deletion analysis indicates that both the N-terminal DED domain and the C-terminal region of DEDD2 can induce apoptosis. The cell death activity of this family appears to be related to their nuclear localization. DEDD and DEDD2 bind to two tandem DED-containing caspases, caspase -8 and -10, that are engaged by death receptors. Consistent with the nuclear localization of this family, caspase-8 translocates to the nucleus during CD95-induced apoptosis. DEDD and DEDD2 also readily associate with themselves and with each other. These results suggest that DEDD and DEDD2 may be important mediators for death receptors and that they may target caspases to the nucleus.
DEDD FLICE True Positive 9832420 Leo CP, Hsu SY, McGee EA, Salanova M, Hsueh AJ: DEFT, a novel death effector domain-containing molecule predominantly expressed in testicular germ cells. Endocrinology. 1998 Dec;139(12):4839-48. Apoptosis is a physiological process by which multicellular organisms eliminate unwanted cells. Death factors such as Fas ligand induce apoptosis by triggering a series of intracellular protein-protein interactions mediated by defined motifs found in the signaling molecules. One of these motifs is the death effector domain (DED), a stretch of about 80 amino acids that is shared by adaptors, regulators, and executors of the death factor pathway. We have identified the human and rat complementary DNAs encoding a novel protein termed DEFT (Death EFfector domain-containing Testicular molecule). The N-terminus of DEFT shows a high degree of homology to the DEDs found in FADD (an adaptor molecule) as well as procaspase-8/FLICE and procaspase-10/Mch4 (executors of the death program). Northern blot hybridization experiments have shown that the DEFT messenger RNA (mRNA) is expressed in a variety of human and rat tissues, with particularly abundant expression in the testis. In situ hybridization analysis further indicated the expression of DEFT mRNA in meiotic male germ cells. In a model of germ cell apoptosis induction, an increase in testis DEFT mRNA was found in immature rats after 2 days of treatment with a GnRH antagonist. Unlike FADD and procaspase-8/FLICE, overexpression of DEFT did not induce apoptosis in Chinese hamster ovary cells. Although cotransfection studies indicated that DEFT is incapable of modulating apoptosis effected by FADD and procaspase-8/FLICE, interactions between DEFT and uncharacterized DED-containing molecules in the testis remain to be studied in the future. In conclusion, we have identified a novel DED-containing protein with high expression in testis germ cells. This protein may be important in the regulation of death factor-induced apoptosis in the testis and other tissues.
DEDD FLICE True Positive 9380701 Inohara N, Koseki T, Hu Y, Chen S, Nunez G: CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis. Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10717-22. We have identified and characterized CLARP, a caspase-like apoptosis-regulatory protein. Sequence analysis revealed that human CLARP contains two amino-terminal death effector domains fused to a carboxyl-terminal caspase-like domain. The structure and amino acid sequence of CLARP resemble those of caspase-8, caspase-10, and DCP2, a Drosophila melanogaster protein identified in this study. Unlike caspase-8, caspase-10, and DCP2, however, two important residues predicted to be involved in catalysis were lost in the caspase-like domain of CLARP. Analysis with fluorogenic substrates for caspase activity confirmed that CLARP is catalytically inactive. CLARP was found to interact with caspase-8 but not with FADD/MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathway. Expression of CLARP induced apoptosis, which was blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk). Moreover, CLARP augmented the killing ability of caspase-8 and FADD/MORT-1 in mammalian cells. The human clarp gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis.
DEDD cytokeratin True Positive 16820959 Schutte B, Henfling M, Ramaekers FC: DEDD association with cytokeratin filaments correlates with sensitivity to apoptosis. Apoptosis. 2006 Sep;11(9):1561-72. The cytokeratin 8/18 (CK8/18) cytoskeleton network is an early target for caspase cleavage during apoptosis. Recent reports suggest that the highly conserved and ubiquitous death effector domain containing DNA binding protein (DEDD) plays a role in the recruitment of procaspase-9 and -3 at this CK8/18 scaffold. DEDD interacts with both the CK8/18 intermediate filament network and procaspase-3 and -9. It is suggested that the CK8/18 fibrils may provide a scaffold for the proximity-induced autocleavage and activation of procaspase-9 in close association with caspase-3.We addressed this issue by investigating DEDD staining patterns in various cell lines and by correlating these expression patterns with the sensitivity of these cell lines for roscovitine-induced apoptosis. We showed that in some cell lines DEDD revealed a bright filamentous staining pattern in others DEDD staining was weak and diffusely distributed in the cytoplasm of the cells. The difference in staining patterns was irrespective of the phosphorylation status of the cytokeratin filaments. In cells showing a filamentous staining pattern, DEDD was strongly associated with the CK8/18 cytokeratin filaments as evidenced by double immunofluorescence and its resistance to extraction with Triton X-100. Subcellular fractionation indicates that DEDD co-purifies with CK18, which corroborates a strong association of DEDD and the cytokeratin network. DEDD was either mono- or diubiquinated. Cells showing a filamentous DEDD distribution are more apoptosis-prone as evidenced by the rapid appearance of M30 CytoDeath-positive cells after induction of apoptosis. The sensitivity towards apoptosis is irrespective of the procaspase-3 content of the cells. Our data support the notion that DEDD-mediated accumulation of procaspases at the cytokeratin scaffold leads to an increase in the local concentration, which renders cells more apoptosis-prone.
DEDD CD95 True Positive 17193921 Huang X, Zhang M, Tang H, Ruo C, Cao X: Identification and characterization of DEDDL, a human-specific isoform of DEDD. Gene Expr. 2006;13(3):141-53. Death effector domain (DED) containing molecules are usually involved in the intracellular apoptosis cascade as executioners or regulators. One of these molecules, DEDD, was identified as a final target of the CD95 signaling pathway by which it would be transferred into the nucleolus to inhibit RNA polymerase I-dependent transcription. Here we describe a longer isoform of DEDD, DEDDL, produced by alternatively splicing, as an immune cell-specific DED-containing molecule. It is only expressed in human T lymphocytes and dendritic cells (DCs), and the mRNA expression in DCs was elevated upon inductive maturation. In cell lines MCF-7 and Jurkat, the overexpression of DEDDL could induce apoptosis more potently than that of DEDD. That DEDDL could bind FADD and cFLIP more potently than DEDD in vivo was revealed by cotransfection and immunoprecipitation. This may explain why DEDDL is a more potent apoptosis inducer, because DED-containing proteins usually induce apoptosis through DED binding. Finally, why DEDD and DEDDL are unstable in the overexpression and other studies may be explained by the finding that they are potential substrates of active caspases.
DEDD CD95 True Positive 9774341 Stegh AH, Schickling O, Ehret A, Scaffidi C, Peterhansel C, Hofmann TG, Grummt I, Krammer PH, Peter ME: DEDD, a novel death effector domain-containing protein, targeted to the nucleolus. EMBO J. 1998 Oct 15;17(20):5974-86. The CD95 signaling pathway comprises proteins that contain one or two death effector domains (DED), such as FADD/Mort1 or caspase-8. Here we describe a novel 37 kDa protein, DEDD, that contains an N-terminal DED. DEDD is highly conserved between human and mouse (98. 7% identity) and is ubiquitously expressed. Overexpression of DEDD in 293T cells induced weak apoptosis, mainly through its DED by which it interacts with FADD and caspase-8. Endogenous DEDD was found in the cytoplasm and translocated into the nucleus upon stimulation of CD95. Immunocytological studies revealed that overexpressed DEDD directly translocated into the nucleus, where it co-localizes in the nucleolus with UBF, a basal factor required for RNA polymerase I transcription. Consistent with its nuclear localization, DEDD contains two nuclear localization signals and the C-terminal part shares sequence homology with histones. Recombinant DEDD binds to both DNA and reconstituted mononucleosomes and inhibits transcription in a reconstituted in vitro system. The results suggest that DEDD is a final target of a chain of events by which the CD95-induced apoptotic signal is transferred into the nucleolus to shut off cellular biosynthetic activities.
DEDD brain derived neurotrophic factor False Positive 15582580 Yeom M, Shim I, Lee HJ, Hahm DH: Proteomic analysis of nicotine-associated protein expression in the striatum of repeated nicotine-treated rats. Biochem Biophys Res Commun. 2005 Jan 14;326(2):321-8. Through the proteomic analysis using 2-dimensional electrophoresis, the nicotine addiction-associated proteins were extensively screened in the striatum of rat brains. The nicotine addiction was developed by repeated nicotine injection (0.4mg/kg s.c.), twice daily for 7 days, followed by one challenge injection after a 3 day withdrawal period, and then confirmed by observing a 2.3-fold increase in locomoter activity. The 3 up- and 4 down-regulated proteins were selected and identified to be zinc-finger binding protein-89 (ZBP-89), 2'3'-cyclic nucleotide 3'-phosphodiesterase 1, deoxyribonuclease 1-like 3 (DNase1l3), tandem pore domain halothane inhibited K (+) channel (THIK-2), brain-specific hyaluronan-binding protein (BRAL-1), death effector domain-containing DNA binding protein (DEDD), and brain-derived neurotrophic factor (BDNF) by mass spectrophotometric fingerprinting. Among them, the expression patterns of ZEB-89, DNase1l3, THIK-2, DEDD, and BDNF mRNAs were found to be coincident with those of cognate proteins, by using RT-PCR analysis. These proteins could be suggested as drug targets to develop a new therapy for nicotine-associated diseases, as well as the clues to understand the mechanism of nicotine.
DEDD TNF True Positive 11346652 You Z, Ouyang H, Lopatin D, Polver PJ, Wang CY: Nuclear factor-kappa B-inducible death effector domain-containing protein suppresses tumor necrosis factor-mediated apoptosis by inhibiting caspase-8 activity. J Biol Chem. 2001 Jul 13;276(28):26398-404. Epub 2001 May 9. Activation of the transcription factor nuclear factor-kappa B (NF-kappa B) has been found to play an essential role in the inhibition of tumor necrosis factor (TNF)-mediated apoptosis. NF-kappa B regulates several antiapoptotic molecules including inhibitors of apoptosis, Bcl-2 family proteins (A1 and Bcl-X (L))(,) and IEX-IL. Here we report that the expression of a small death effector domain (DED)-containing protein, NDED (NF-kappa B-inducible DED-containing protein), depends on the activation of NF-kappa B. The inhibition of NF-kappa B by I kappa B alpha, a natural inhibitor of NF-kappa B, suppressed NDED mRNA expression induced by TNF. The restoration of NDED in NF-kappa B null cells inhibited TNF-induced apoptosis. Intriguingly, unlike the caspase-8 inhibitor cellular FADD-like interleukin-1 beta converting enzyme-inhibitory protein (c-FLIP), NDED suppressed TNF-mediated apoptosis by inhibiting TNF-induced caspase-8 enzymatic activity but not the processing of caspase-8. Furthermore, NDED could not inhibit etoposide-mediated apoptosis that is independent of caspase-8 activation. Our results provide the first demonstration that NF-kappa B transcriptionally induces the DED-containing protein to suppress TNF-mediated apoptosis by inhibiting caspase-8 activity, which offers new insight into the antiapoptotic mechanism of NF-kappa B.
DEDD K18 True Positive 12235123 Lee JC, Schickling O, Stegh AH, Oshima RG, Dinsdale D, Cohen GM, Peter ME: DEDD regulates degradation of intermediate filaments during apoptosis. J Cell Biol. 2002 Sep 16;158(6):1051-66. Epub 2002 Sep 16. Apoptosis depends critically on regulated cytoskeletal reorganization events in a cell. We demonstrate that death effector domain containing DNA binding protein (DEDD), a highly conserved and ubiquitous death effector domain containing protein, exists predominantly as mono- or diubiquitinated, and that diubiquitinated DEDD interacts with both the K8/18 intermediate filament network and pro-caspase-3. Early in apoptosis, both cytosolic DEDD and its close homologue DEDD2 formed filaments that colocalized with and depended on K8/18 and active caspase-3. Subsequently, these filamentous structures collapsed into intracellular inclusions that migrated into cytoplasmic blebs and contained DEDD, DEDD2, active caspase-3, and caspase-3-cleaved K18 late in apoptosis. Biochemical studies further confirmed that DEDD coimmunoprecipitated with both K18 and pro-caspase-3, and kinetic analyses placed apoptotic DEDD staining prior to caspase-3 activation and K18 cleavage. In addition, both caspase-3 activation and K18 cleavage was inhibited by expression of DEDDDeltaNLS1-3, a cytosolic form of DEDD that cannot be ubiquitinated. Finally, siRNA mediated DEDD knockdown cells exhibited inhibition of staurosporine-induced DNA degradation. Our data suggest that DEDD represents a novel scaffold protein that directs the effector caspase-3 to certain substrates facilitating their ordered degradation during apoptosis.
DEDD PAB1 True Positive 15044470 Viswanathan P, Ohn T, Chiang YC, Chen J, Denis CL: Mouse CAF1 can function as a processive deadenylase/3'-5'-exonuclease in vitro but in yeast the deadenylase function of CAF1 is not required for mRNA poly (A) removal. J Biol Chem. 2004 Jun 4;279(23):23988-95. Epub 2004 Mar 23. The mouse CAF1 (mCAF1) is an ortholog of the yeast (y) CAF1 protein, which is a component of the CCR4-NOT complex, the major cytoplasmic deadenylase of Saccharomyces cerevisiae. Although CAF1 protein belongs to the DEDDh family of RNases, CCR4 appears to be the principle deadenylase of the CCR4-NOT complex. Here, we present evidence that mCAF1 is a processive, 3'-5'-RNase with a preference for poly (A) substrates. Like CCR4, increased length of RNA substrates converted mCAF1 into a processive enzyme. In contrast to two other DEDD family members, PAN2 and PARN, mCAF1 was not activated either by PAB1 or capped RNA substrates. The rate of deadenylation in vitro by yCCR4 and mCAF1 were both strongly influenced by secondary structures present in sequences adjacent to the poly (A) tail, suggesting that the ability of both enzymes to deadenylate might be affected by the context of the mRNA 3'-untranslated region sequences. The ability of mCAF1 to complement a ycaf1 deletion in yeast, however, did not require the RNase function of mCAF1. Importantly, yCAF1 mutations, which have been shown to block its RNase activity in vitro, did not inactivate yCAF1 in vivo, and mRNAs were deadenylated in vivo at nearly the same rate as found for wild type yCAF1. These results indicate that at least in yeast the CAF1 RNase activity is not required for its in vivo function.
DEDD cytokeratin 8 True Positive 16820959 Schutte B, Henfling M, Ramaekers FC: DEDD association with cytokeratin filaments correlates with sensitivity to apoptosis. Apoptosis. 2006 Sep;11(9):1561-72. The cytokeratin 8/18 (CK8/18) cytoskeleton network is an early target for caspase cleavage during apoptosis. Recent reports suggest that the highly conserved and ubiquitous death effector domain containing DNA binding protein (DEDD) plays a role in the recruitment of procaspase-9 and -3 at this CK8/18 scaffold. DEDD interacts with both the CK8/18 intermediate filament network and procaspase-3 and -9. It is suggested that the CK8/18 fibrils may provide a scaffold for the proximity-induced autocleavage and activation of procaspase-9 in close association with caspase-3.We addressed this issue by investigating DEDD staining patterns in various cell lines and by correlating these expression patterns with the sensitivity of these cell lines for roscovitine-induced apoptosis. We showed that in some cell lines DEDD revealed a bright filamentous staining pattern in others DEDD staining was weak and diffusely distributed in the cytoplasm of the cells. The difference in staining patterns was irrespective of the phosphorylation status of the cytokeratin filaments. In cells showing a filamentous staining pattern, DEDD was strongly associated with the CK8/18 cytokeratin filaments as evidenced by double immunofluorescence and its resistance to extraction with Triton X-100. Subcellular fractionation indicates that DEDD co-purifies with CK18, which corroborates a strong association of DEDD and the cytokeratin network. DEDD was either mono- or diubiquinated. Cells showing a filamentous DEDD distribution are more apoptosis-prone as evidenced by the rapid appearance of M30 CytoDeath-positive cells after induction of apoptosis. The sensitivity towards apoptosis is irrespective of the procaspase-3 content of the cells. Our data support the notion that DEDD-mediated accumulation of procaspases at the cytokeratin scaffold leads to an increase in the local concentration, which renders cells more apoptosis-prone.
DEDD DEDD2 True Positive 11965497 Zhan Y, Hegde R, Srinivasula SM, Fernandes-Alnemri T, Alnemri ES: Death effector domain-containing proteins DEDD and FLAME-3 form nuclear complexes with the TFIIIC102 subunit of human transcription factor IIIC. Cell Death Differ. 2002 Apr;9(4):439-47. Death effector domain-containing proteins are involved in important cellular processes such as death-receptor induced apoptosis, NF-kappaB activation and ERK activation. Here we report the identification of a novel nuclear DED-containing protein, FLAME-3. FLAME-3 shares significant sequence (46.6% identical) and structural homology to another DED-containing protein, DEDD. FLAME-3 interacts with DEDD and c-FLIP (FLAME-1) but not with the other DED-containing proteins FADD, caspase-8 or caspase-10. FLAME-3 translocates to, and sequesters c-FLIP in the nucleus upon overexpression in human cell lines. Using the yeast two-hybrid system to identify DEDD-interacting proteins, the TFIIIC102 subunit of human transcription factor TFIIIC was identified as a DEDD- and FLAME-3-specific interacting protein. Co-expression of either DEDD or FLAME-3 with hTFIIIC102 in MCF-7 cells induces the translocation from the cytoplasm and sequestration of hTFIIIC102 in the nucleus, indicating that DEDD and FLAME-3 form strong heterocomplexes with hTFIIIC102 and might be important regulators of the activity of the hTFIIIC transcriptional complex. Consistent with this, overexpression of DEDD or FLAME-3 in 293 cells inhibited the expression of a luciferase-reporter gene under the control of the NF-kappaB promoter. Our data provide the first direct evidence for the involvement of DED-containing proteins in the regulation of components of the general transcription machinery in the nucleus.
DEDD FADD True Positive 9832420 Leo CP, Hsu SY, McGee EA, Salanova M, Hsueh AJ: DEFT, a novel death effector domain-containing molecule predominantly expressed in testicular germ cells. Endocrinology. 1998 Dec;139(12):4839-48. Apoptosis is a physiological process by which multicellular organisms eliminate unwanted cells. Death factors such as Fas ligand induce apoptosis by triggering a series of intracellular protein-protein interactions mediated by defined motifs found in the signaling molecules. One of these motifs is the death effector domain (DED), a stretch of about 80 amino acids that is shared by adaptors, regulators, and executors of the death factor pathway. We have identified the human and rat complementary DNAs encoding a novel protein termed DEFT (Death EFfector domain-containing Testicular molecule). The N-terminus of DEFT shows a high degree of homology to the DEDs found in FADD (an adaptor molecule) as well as procaspase-8/FLICE and procaspase-10/Mch4 (executors of the death program). Northern blot hybridization experiments have shown that the DEFT messenger RNA (mRNA) is expressed in a variety of human and rat tissues, with particularly abundant expression in the testis. In situ hybridization analysis further indicated the expression of DEFT mRNA in meiotic male germ cells. In a model of germ cell apoptosis induction, an increase in testis DEFT mRNA was found in immature rats after 2 days of treatment with a GnRH antagonist. Unlike FADD and procaspase-8/FLICE, overexpression of DEFT did not induce apoptosis in Chinese hamster ovary cells. Although cotransfection studies indicated that DEFT is incapable of modulating apoptosis effected by FADD and procaspase-8/FLICE, interactions between DEFT and uncharacterized DED-containing molecules in the testis remain to be studied in the future. In conclusion, we have identified a novel DED-containing protein with high expression in testis germ cells. This protein may be important in the regulation of death factor-induced apoptosis in the testis and other tissues.
DEDD ISG20 True Positive 16514659 Espert L, Eldin P, Gongora C, Bayard B, Harper F, Chelbi-Alix MK, Bertrand E, Degols G, Mechti N: The exonuclease ISG20 mainly localizes in the nucleolus and the Cajal (Coiled) bodies and is associated with nuclear SMN protein-containing complexes. J Cell Biochem. 2006 Aug 1;98(5):1320-33. We have previously shown that ISG20, an interferon (IFN)-induced gene, encodes a 3' to 5' exoribonuclease member of the DEDD superfamily of exonucleases. ISG20 specifically degrades single-stranded RNA. In this report, using immunofluorescence analysis, we demonstrate that in addition to a diffuse cytoplasmic and nucleoplasmic localization, the endogenous ISG20 protein was present in the nucleus both in the nucleolus and in the Cajal bodies (CBs). In addition, we show that the ectopic expression of the CBs signature protein, coilin, fused to the red fluorescent protein (coilin-dsRed) increased the number of nuclear dots containing both ISG20 and coilin-dsRed. Using electron microcopy analysis, ISG20 appeared principally concentrated in the dense fibrillar component of the nucleolus, the major site for rRNA processing. We also present evidences that ISG20 was associated with survival of motor neuron (SMN)-containing macromolecular nuclear complexes required for the biogenesis of various small nuclear ribonucleoproteins. Finally, we demonstrate that ISG20 was associated with U1 and U2 snRNAs, and U3 snoRNA. The accumulation of ISG20 in the CBs after IFN treatment strongly suggests its involvement in a new route for IFN-mediated inhibition of protein synthesis by modulating snRNA and rRNA maturation.
DEDD THIK 2 False Positive 15582580 Yeom M, Shim I, Lee HJ, Hahm DH: Proteomic analysis of nicotine-associated protein expression in the striatum of repeated nicotine-treated rats. Biochem Biophys Res Commun. 2005 Jan 14;326(2):321-8. Through the proteomic analysis using 2-dimensional electrophoresis, the nicotine addiction-associated proteins were extensively screened in the striatum of rat brains. The nicotine addiction was developed by repeated nicotine injection (0.4mg/kg s.c.), twice daily for 7 days, followed by one challenge injection after a 3 day withdrawal period, and then confirmed by observing a 2.3-fold increase in locomoter activity. The 3 up- and 4 down-regulated proteins were selected and identified to be zinc-finger binding protein-89 (ZBP-89), 2'3'-cyclic nucleotide 3'-phosphodiesterase 1, deoxyribonuclease 1-like 3 (DNase1l3), tandem pore domain halothane inhibited K (+) channel (THIK-2), brain-specific hyaluronan-binding protein (BRAL-1), death effector domain-containing DNA binding protein (DEDD), and brain-derived neurotrophic factor (BDNF) by mass spectrophotometric fingerprinting. Among them, the expression patterns of ZEB-89, DNase1l3, THIK-2, DEDD, and BDNF mRNAs were found to be coincident with those of cognate proteins, by using RT-PCR analysis. These proteins could be suggested as drug targets to develop a new therapy for nicotine-associated diseases, as well as the clues to understand the mechanism of nicotine.
DEDD ERK True Positive 11965497 Zhan Y, Hegde R, Srinivasula SM, Fernandes-Alnemri T, Alnemri ES: Death effector domain-containing proteins DEDD and FLAME-3 form nuclear complexes with the TFIIIC102 subunit of human transcription factor IIIC. Cell Death Differ. 2002 Apr;9(4):439-47. Death effector domain-containing proteins are involved in important cellular processes such as death-receptor induced apoptosis, NF-kappaB activation and ERK activation. Here we report the identification of a novel nuclear DED-containing protein, FLAME-3. FLAME-3 shares significant sequence (46.6% identical) and structural homology to another DED-containing protein, DEDD. FLAME-3 interacts with DEDD and c-FLIP (FLAME-1) but not with the other DED-containing proteins FADD, caspase-8 or caspase-10. FLAME-3 translocates to, and sequesters c-FLIP in the nucleus upon overexpression in human cell lines. Using the yeast two-hybrid system to identify DEDD-interacting proteins, the TFIIIC102 subunit of human transcription factor TFIIIC was identified as a DEDD- and FLAME-3-specific interacting protein. Co-expression of either DEDD or FLAME-3 with hTFIIIC102 in MCF-7 cells induces the translocation from the cytoplasm and sequestration of hTFIIIC102 in the nucleus, indicating that DEDD and FLAME-3 form strong heterocomplexes with hTFIIIC102 and might be important regulators of the activity of the hTFIIIC transcriptional complex. Consistent with this, overexpression of DEDD or FLAME-3 in 293 cells inhibited the expression of a luciferase-reporter gene under the control of the NF-kappaB promoter. Our data provide the first direct evidence for the involvement of DED-containing proteins in the regulation of components of the general transcription machinery in the nucleus.
DEDD ERK True Positive 10926929 Zhang Y, Redina O, Altshuller YM, Yamazaki M, Ramos J, Chneiweiss H, Kanaho Y, Frohman MA: Regulation of expression of phospholipase D1 and D2 by PEA-15, a novel protein that interacts with them. J Biol Chem. 2000 Nov 10;275(45):35224-32. Phospholipase D (PLD), a signal-transducing membrane-associated enzyme, is implicated in diverse processes including apoptosis, ERK activation, and glucose transport. Prior studies have identified specific PLD activators and repressors that directly regulate its enzymatic activity. Using two-hybrid screens, we have identified PEA-15 as a PLD interactor that unexpectedly functions to alter its level of expression. PEA-15 is a widely expressed death effector domain-containing phosphoprotein involved in signal transduction, apoptosis, ERK activation, and glucose transport. The PLD1-interacting site on PEA-15 consists of part of the death effector domain domain plus additional C-terminal flanking sequences, whereas the PEA-15-interacting site on PLD1 overlaps the previously identified RhoA-interacting site. PEA-15 did not affect basal or stimulated in vitro PLD1 enzymatic activation. However, co-expression of PEA-15 increased levels of PLD1 activity. This increased activation correlated with higher PLD1 protein expression levels, as marked by faster accumulation and longer persistence of PLD1 when PEA-15 was present. PEA-15 similarly increased protein expressions level of PLD2 and co-immunoprecipitated with it. These results suggest that PEA-15 may stabilize PLD or act as a PLD chaperone. The common involvement of PEA-15 and PLD in apoptosis, ERK activation, and glucose transport additionally suggests functional significance.
DEDD death effector domain associated factor True Positive 11395500 Zheng L, Schickling O, Peter ME, Lenardo MJ: The death effector domain-associated factor plays distinct regulatory roles in the nucleus and cytoplasm. J Biol Chem. 2001 Aug 24;276(34):31945-52. Epub 2001 Jun 6. Homophilic interactions of death effector domains (DEDs) are crucial for the signaling pathways of death receptor-mediated apoptosis. The machinery that regulates proper oligomerization and autoactivation of procaspase-8 and/or procaspase-10 during T lymphocyte activation determines whether the cells will undergo caspase-mediated apoptosis or proliferation. We screened a yeast two-hybrid library by using the DEDs contained in the prodomains of procaspase-8 and procaspase-10 and isolated a DED-associated factor (DEDAF) that interacts with several DED-containing proteins but does not itself contain a DED. DEDAF is highly conserved between human and mouse (98% amino acid identity) and is homologous to a nuclear regulatory protein YAF-2. DEDAF is expressed at the highest levels in lymphoid tissues and placenta. DEDAF interacts with FADD, procaspase-8, and procaspase-10 in the cytosol as well as with the DED-containing DNA-binding protein (DEDD) in the nucleus. At the cell membrane, DEDAF augmented the formation of CD95-FADD-caspase-8 complexes and enhanced death receptor- as well as DED-mediated apoptosis. In the nucleus, DEDAF caused the DEDD protein to relocalize from subnuclear structures to a diffuse distribution in the nucleoplasm. Our data therefore suggest that DEDAF may be involved in the regulation of both cytoplasmic and nuclear events of apoptosis.
DEDD retrocyclin False Positive 1820740 Canese R, Podo F: [Localized nuclear magnetic resonance spectroscopy in vivo. Ann Ist Super Sanita. 1991;27(4):675-708. Physical principles and quantitative problems]. Recent technological developments extended the use of nuclear magnetic resonance to clinical imaging (MRI), as well as to in vivo metabolic studies on tissues and organs by means of localized spectroscopy (MRS). The quantitative determination of absolute metabolic concentrations by in vivo MRS still represents a challenge for technological efforts and biochemical investigations. In fact, the different methodologies today available for spectral acquisition from localized regions within a body organ or pathological lesion should ideally lead to a complete suppression of signals from surrounding tissues, without any appreciable signal loss from the volume of interest. Accurate assessment of deviations of the real from the ideal case represents an essential requirement for either relative or absolute quantification of in vivo localized MR spectra. A review will be presented of the most utilized MRS methodologies, together with a discussion on their potentialities and limitations. Techniques have been classified according to the use of either r.f. (B1) and/or static magnetic field (B0) gradients. The last session will be devoted to the presentation of some results obtained in our laboratory on the use of a particular test-object (constructed at the Deft Institute of Technology) for assessing signal localization efficiency provided by a surface coil in a small scale MRS/MRI equipment (for in vivo biochemical studies on small animals). The surface coil was used in conjunction with a number of sequence (t1-90 degrees-acq; 1-D "chemical shift imaging"; "depth") of either square or adiabatic r.f. pulses. The results allow the selection, on a quantitative basis, of the most appropriate pulse sequence (s) to be used for metabolic studies on superficial experimental tumours (implanted s.c. in small animals), according to the mean area and thickness of the neoplastic lesion. The latter parameters can be non-invasively assessed by previous MRI analysis of the tumour. This study was carried out in the frame of the EC COMAC-BME Concerted Research Project on Tissue Characterization by MRS and MRI.
DEDD TFIIIC True Positive 11965497 Zhan Y, Hegde R, Srinivasula SM, Fernandes-Alnemri T, Alnemri ES: Death effector domain-containing proteins DEDD and FLAME-3 form nuclear complexes with the TFIIIC102 subunit of human transcription factor IIIC. Cell Death Differ. 2002 Apr;9(4):439-47. Death effector domain-containing proteins are involved in important cellular processes such as death-receptor induced apoptosis, NF-kappaB activation and ERK activation. Here we report the identification of a novel nuclear DED-containing protein, FLAME-3. FLAME-3 shares significant sequence (46.6% identical) and structural homology to another DED-containing protein, DEDD. FLAME-3 interacts with DEDD and c-FLIP (FLAME-1) but not with the other DED-containing proteins FADD, caspase-8 or caspase-10. FLAME-3 translocates to, and sequesters c-FLIP in the nucleus upon overexpression in human cell lines. Using the yeast two-hybrid system to identify DEDD-interacting proteins, the TFIIIC102 subunit of human transcription factor TFIIIC was identified as a DEDD- and FLAME-3-specific interacting protein. Co-expression of either DEDD or FLAME-3 with hTFIIIC102 in MCF-7 cells induces the translocation from the cytoplasm and sequestration of hTFIIIC102 in the nucleus, indicating that DEDD and FLAME-3 form strong heterocomplexes with hTFIIIC102 and might be important regulators of the activity of the hTFIIIC transcriptional complex. Consistent with this, overexpression of DEDD or FLAME-3 in 293 cells inhibited the expression of a luciferase-reporter gene under the control of the NF-kappaB promoter. Our data provide the first direct evidence for the involvement of DED-containing proteins in the regulation of components of the general transcription machinery in the nucleus.
DEDD caspase True Positive 16820959 Schutte B, Henfling M, Ramaekers FC: DEDD association with cytokeratin filaments correlates with sensitivity to apoptosis. Apoptosis. 2006 Sep;11(9):1561-72. The cytokeratin 8/18 (CK8/18) cytoskeleton network is an early target for caspase cleavage during apoptosis. Recent reports suggest that the highly conserved and ubiquitous death effector domain containing DNA binding protein (DEDD) plays a role in the recruitment of procaspase-9 and -3 at this CK8/18 scaffold. DEDD interacts with both the CK8/18 intermediate filament network and procaspase-3 and -9. It is suggested that the CK8/18 fibrils may provide a scaffold for the proximity-induced autocleavage and activation of procaspase-9 in close association with caspase-3.We addressed this issue by investigating DEDD staining patterns in various cell lines and by correlating these expression patterns with the sensitivity of these cell lines for roscovitine-induced apoptosis. We showed that in some cell lines DEDD revealed a bright filamentous staining pattern in others DEDD staining was weak and diffusely distributed in the cytoplasm of the cells. The difference in staining patterns was irrespective of the phosphorylation status of the cytokeratin filaments. In cells showing a filamentous staining pattern, DEDD was strongly associated with the CK8/18 cytokeratin filaments as evidenced by double immunofluorescence and its resistance to extraction with Triton X-100. Subcellular fractionation indicates that DEDD co-purifies with CK18, which corroborates a strong association of DEDD and the cytokeratin network. DEDD was either mono- or diubiquinated. Cells showing a filamentous DEDD distribution are more apoptosis-prone as evidenced by the rapid appearance of M30 CytoDeath-positive cells after induction of apoptosis. The sensitivity towards apoptosis is irrespective of the procaspase-3 content of the cells. Our data support the notion that DEDD-mediated accumulation of procaspases at the cytokeratin scaffold leads to an increase in the local concentration, which renders cells more apoptosis-prone.
DEDD caspase True Positive 14742246 Dinsdale D, Lee JC, Dewson G, Cohen GM, Peter ME: Intermediate filaments control the intracellular distribution of caspases during apoptosis. Am J Pathol. 2004 Feb;164(2):395-407. Caspases are responsible for a cascade of events controlling the disassembly of apoptotic cells. We now demonstrate that caspase-9 is activated at an early stage of apoptosis in epithelial cells and all its detectable, catalytically active large subunits (both the p35 and p37) are concentrated on cytokeratin fibrils. Immunolabeling of distinctive neoepitopes, exposed by cleavage of procaspase-9 at either Asp315 or Asp330, was co-localized on these fibrils with active caspase-3, caspase-cleaved cytokeratin-18, death-effector-domain containing DNA-binding protein and ubiquitin. Cytokeratin filaments may thus provide a scaffold whereby active subunits of caspase-9 can activate caspase-3 which, in turn, can activate more caspase-9 so forming an amplification loop to facilitate cleavage of cytokeratin-18, disruption of the cytoskeleton and the ensuing formation of cytoplasmic inclusions. These inclusions, formed from the collapse of fibrils, together with their associated components, also contain ubiquitinated proteins, vimentin, heat-shock protein 72, and tumor necrosis factor receptor type-1-associated death domain protein. Many of their constituents, including active caspases, remain sequestered within these inclusions, even after detergent treatment and isolation. Thus, such inclusions do not merely accumulate disrupted cytokeratins but also sequestrate potentially noxious proteins that could injure healthy neighboring cells.
DEDD caspase True Positive 12527898 Alcivar A, Hu S, Tang J, Yang X: DEDD and DEDD2 associate with caspase-8/10 and signal cell death. Oncogene. 2003 Jan 16;22(2):291-7. An apoptotic signal triggered by cell surface death receptors is disseminated to intracellular compartments through protein-protein interactions mediated by conserved domains such as the death effector domain (DED). A unique family of single DED-containing proteins, including DEDD and DEDD2, is targeted to the nucleolus. However, the role of DEDD/DEDD2 in apoptosis remains less understood. Here we show that DEDD and DEDD2 are highly conserved in diverse species, and that they are potent inducers of apoptosis in various cell types. Deletion analysis indicates that both the N-terminal DED domain and the C-terminal region of DEDD2 can induce apoptosis. The cell death activity of this family appears to be related to their nuclear localization. DEDD and DEDD2 bind to two tandem DED-containing caspases, caspase -8 and -10, that are engaged by death receptors. Consistent with the nuclear localization of this family, caspase-8 translocates to the nucleus during CD95-induced apoptosis. DEDD and DEDD2 also readily associate with themselves and with each other. These results suggest that DEDD and DEDD2 may be important mediators for death receptors and that they may target caspases to the nucleus.
DEDD caspase True Positive 9326610 Han DK, Chaudhary PM, Wright ME, Friedman C, Trask BJ, Riedel RT, Baskin DG, Schwartz SM, Hood L: MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclXL and initiates cell death. Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11333-8. Activation of the cascade of proteolytic caspases has been identified as the final common pathway of apoptosis in diverse biological systems. We have isolated a gene, termed MRIT, that possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases. However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC (R/Q) G. Nonetheless MRIT activates caspase-dependent death. Using yeast two-hybrid assays, we demonstrate that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition, MRIT simultaneously and independently interacts with BclXL and FLICE in mammalian cells. Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member.
DEDD Cdk1 True Positive 17592253 Miyazaki T, Arai S: Two distinct controls of mitotic cdk1/cyclin B1 activity requisite for cell growth prior to cell division. Cell Cycle. 2007 Jun 15;6(12):1419-25. Epub 2007 May 7. Cell growth prior to cell division is restricted by the activity of cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complexes. Recently, we identified that the death-effector domain (DED) containing protein, DEDD, acts as a novel inhibitor of mitotic Cdk1/cyclin B1, influencing cell size. Like cyclin B1, DEDD protein levels specifically peak during the G (2)/M phase. In the nucleus, DEDD associates with Cdk1/cyclin B1 complexes, via direct binding to cyclin B1, and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD (-/-)) embryonic fibroblasts is increased compared to that in DEDD (+/+) cells. This accelerates mitotic progression in DEDD (-/-) cells, with a shortened G (2)/M phase, reduced rRNA, and diminished cell volume. Likewise, DEDD (-/-) mice show decreased body and organ weights relative to DEDD (+/+) mice. Interestingly, the DED domain is not involved in the association of DEDD with Cdk1/cyclin B1, but is indispensable for the cell sizing function of DEDD. Together, in addition to the well-established machinery for activation of Cdk1 through dephosphorylation of its inhibitory-residues, we propose a novel mechanism for impeditive regulation of mitotic Cdk1/cyclin B1 mediated by DEDD within the nucleus, which allows sufficient cell growth prior to cell division.
DEDD Cdk1 True Positive 17283331 Arai S, Miyake K, Voit R, Nemoto S, Wakeland EK, Grummt I, Miyazaki T: Death-effector domain-containing protein DEDD is an inhibitor of mitotic Cdk1/cyclin B1. Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2289-94. Epub 2007 Feb 5. Accumulating evidence has shown that many molecules, including some cyclin-dependent kinases (Cdks) and cyclins, as well as the death-effector domain (DED)-containing FADD, function for both apoptosis and cell cycle. Here we identified that DEDD, which also possesses the DED domain, acts as a novel inhibitor of the mitotic Cdk1/cyclin B1 complex. DEDD associates with mitotic Cdk1/cyclin B1 complexes via direct binding to cyclin B1 and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD-/-) embryonic fibroblasts is increased compared with that in DEDD+/+ cells, which results in accelerated mitotic progression, thus exhibiting a shortened G2/M stage. Interestingly, DEDD-/- cells also demonstrated decreased G1 duration, which perhaps enhanced the overall reduction in rRNA amounts and cell volume, primarily caused by the rapid termination of rRNA synthesis before cell division. Likewise, DEDD-/- mice show decreased body and organ weights relative to DEDD+/+ mice. Thus, DEDD is an impeder of cell mitosis, and its absence critically influences cell and body size via modulation of rRNA synthesis.
KIF5C importin True Positive 12191015 Mavlyutov TA, Cai Y, Ferreira PA: Identification of RanBP2- and kinesin-mediated transport pathways with restricted neuronal and subcellular localization. Traffic. 2002 Sep;3(9):630-40. Ran-binding proteins, karyopherins, and RanGTPase mediate and impart directionality to nucleocytoplasmic transport processes. This biological process remains elusive in neurons. RanBP2 has been localized at the nuclear pore complexes and is very abundant in the neuroretina. RanBP2 mediates the assembly of a large complex comprising RanGTPase, CRM1/exportin-1, importin-beta, KIF5-motor proteins, components of the 19S cap of the 26S proteasome, ubc9 and opsin. Here, we show RanBP2 is abundant in the ellipsoid compartment of photoreceptors and RanGTPase-positive particles in cytoplasmic tracks extending away from the nuclear envelope of subpopulations of ganglion cells, suggesting RanBP2's release from nuclear pore complexes. KIF5C and KIF5B are specifically expressed in a subset of neuroretinal cells and differentially localize with RanBP2 and importin-beta in distinct compartments. The C-terminal domains of KIF5B and KIF5C, but not KIF5A, associate directly with importin-beta in a RanGTPase-dependent fashion in vivo and in vitro, indicating importin-beta is an endogenous cargo for a subset of KIF5s in retinal neurons. The KIF5 transport pathway is absent from the myoid region of a topographically distinct subclass of blue cones and the distribution of kinesin-light chains is largely distinct from its KIF5 partners. Altogether, the results identify the existence of neuronal- and subtype-specific kinesin-mediated transport pathways of importin-beta-bound cargoes to and/or from RanBP2 and indicate RanBP2 itself may also constitute a scaffold carrier for some of its associated partners. The implications of these findings in protein kinesis and pathogenesis of degenerative neuropathies are discussed.
KIF5C Ran binding protein 2 True Positive 11553612 Cai Y, Singh BB, Aslanukov A, Zhao H, Ferreira PA: The docking of kinesins, KIF5B and KIF5C, to Ran-binding protein 2 (RanBP2) is mediated via a novel RanBP2 domain. J Biol Chem. 2001 Nov 9;276(45):41594-602. Epub 2001 Sep 11. The Ran-binding protein 2 (RanBP2) is a vertebrate mosaic protein composed of four interspersed RanGTPase binding domains (RBDs), a variable and species-specific zinc finger cluster domain, leucine-rich, cyclophilin, and cyclophilin-like (CLD) domains. Functional mapping of RanBP2 showed that the domains, zinc finger and CLD, between RBD1 and RBD2, and RBD3 and RBD4, respectively, associate specifically with the nuclear export receptor, CRM1/exportin-1, and components of the 19 S regulatory particle of the 26 S proteasome. Now, we report the mapping of a novel RanBP2 domain located between RBD2 and RBD3, which is also conserved in the partially duplicated isoform RanBP2L1. Yet, this domain leads to the neuronal association of only RanBP2 with two kinesin microtubule-based motor proteins, KIF5B and KIF5C. These kinesins associate directly in vitro and in vivo with RanBP2. Moreover, the kinesin light chain and RanGTPase are part of this RanBP2 macroassembly complex. These data provide evidence of a specific docking site in RanBP2 for KIF5B and KIF5C. A model emerges whereby RanBP2 acts as a selective signal integrator of nuclear and cytoplasmic trafficking pathways in neurons.
KIF5C KIF5A False Positive 10964943 Kanai Y, Okada Y, Tanaka Y, Harada A, Terada S, Hirokawa N: KIF5C, a novel neuronal kinesin enriched in motor neurons. J Neurosci. 2000 Sep 1;20(17):6374-84. Kinesin superfamily proteins (KIFs) are the molecular motors conveying cargos along microtubules. KIF5s, the heavy chains of conventional kinesin (KHC), are originally identified members of KIFs, and neuronal KIF5A and ubiquitous KIF5B have been identified so far. In the present work, we cloned a novel member of KIF5, KIF5C, and generated specific antibodies against three KIF5s to investigate their distribution and functions. KIF5A showed pan-neuronal distribution in the nervous system. KIF5B showed a glial cell distribution pattern in general; however, interestingly, its expression was strongly upregulated in axon-elongating neurons, such as olfactory primary neurons and mossy fibers. KIF5C was also a neuronal KIF5 like KIF5A but was highly expressed in lower motor neurons in 2-week-old or older mice, suggesting its important roles in the maintenance of motor neurons rather than in their formation, such as axonal elongation. Because a large part of KIF5s in adult motor neurons were expected to be KIF5C, we generated mice lacking the kif5C gene to investigate the functions of KIF5C in neurons in living animals. The mutant mice showed smaller brain size but were viable and did not show gross changes in the nervous system. Closer examinations revealed the relative loss of motor neurons to sensory neurons. Because three KIF5s showed high similarity in the amino acid sequence, could rescue the KIF5B mutant cells, and could form heterodimers, we think that there are functional redundancy among the three KIF5s and that KIF5A and KIF5B prevented the KIF5C null mice from the severe phenotype.
KIF5C OIP106 True Positive 15644324 Brickley K, Smith MJ, Beck M, Stephenson FA: GRIF-1 and OIP106, members of a novel gene family of coiled-coil domain proteins: association in vivo and in vitro with kinesin. J Biol Chem. 2005 Apr 15;280(15):14723-32. Epub 2005 Jan 11. Gamma-aminobutyric acid (A) receptor-interacting factor (GRIF-1) is a 913-amino acid protein proposed to function as a GABA (A) receptor beta (2) subunit-interacting, trafficking protein. GRIF-1 shares approximately 44% amino acid sequence identity with O-linked N-acetylglucosamine transferase interacting protein 106, OIP106. Both proteins contain predicted coiled-coil domains and probably constitute a novel gene family. The Drosophila orthologue of this family of proteins may be Milton. Milton shares approximately 44% amino acid homology with GRIF-1. Milton is proposed to function in kinesin-mediated transport of mitochondria to nerve terminals. We report here that GRIF-1 and OIP106 also associate with kinesin and mitochondria. Following expression in human embryonic kidney 293 cells, both GRIF-1 and OIP106 were shown by co-immunoprecipitation to be specifically associated with an endogenous kinesin heavy chain species of 115 kDa and exogenous KIF5C. Association of GRIF-1 with kinesin was also evident in native brain and heart tissue. In the brain, anti-GRIF-1-(8-633) antibodies specifically co-immunoprecipitated two kinesin-immunoreactive species with molecular masses of 118 and 115 kDa, and in the heart, one kinesin-immunoreactive species, 115 kDa, was immunoprecipitated. Further studies revealed that GRIF-1 was predominantly associated with KIF5A in the brain and with KIF5B in both the heart and in HEK 293 cells. Yeast two-hybrid interaction assays and immunoprecipitations showed that GRIF-1 associated directly with KIF5C with the GRIF-1/KIF5C interaction domain localized to GRIF-1-(124-283). These results further support a role for GRIF-1 and OIP106 in protein and/or organelle transport in excitable cells in a manner analogous to glutamate receptor-interacting-protein 1, in the motor-dependent transport of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate excitatory neurotransmitter receptors to dendrites.
KIF5C GRIF 1 True Positive 16835241 Smith MJ, Pozo K, Brickley K, Stephenson FA: Mapping the GRIF-1 binding domain of the kinesin, KIF5C, substantiates a role for GRIF-1 as an adaptor protein in the anterograde trafficking of cargoes. J Biol Chem. 2006 Sep 15;281(37):27216-28. Epub 2006 Jul 11. Gamma-aminobutyric acid, type A (GABAA) receptor interacting factor-1 (GRIF-1) and N-acetylglucosamine transferase interacting protein (OIP) 106 are both members of a newly identified coiled-coil family of proteins. They are kinesin-associated proteins proposed to function as adaptors in the anterograde trafficking of organelles to synapses. Here we have studied in more detail the interaction between the prototypic kinesin heavy chain, KIF5C, kinesin light chain, and GRIF-1. The GRIF-1 binding site of KIF5C was mapped using truncation constructs in yeast two-hybrid interaction assays, co-immunoprecipitations, and co-localization studies following expression in mammalian cells. Using these approaches, it was shown that GRIF-1 and the KIF5C binding domain of GRIF-1, GRIF-1-(124-283), associated with the KIF5C non-motor domain. Refined studies using yeast two-hybrid interactions and co-immunoprecipitations showed that GRIF-1 and GRIF-1-(124-283) associated with the cargo binding region within the KIF5C non-motor domain. Substantiation that the GRIF-1-KIF5C interaction was direct was shown by fluorescence resonance energy transfer analyses using fluorescently tagged GRIF-1 and KIF5C constructs. A significant fluorescence resonance energy transfer value was found between the C-terminal EYFP-tagged KIF5C and ECFP-GRIF-1, the C-terminal EYFP-tagged KIF5C non-motor domain and ECFP-GRIF-1, but not between the N-terminal EYFP-tagged KIF5C nor the EYFP-KIF5C motor domain and ECFP-GRIF-1, thus confirming direct association between the two proteins at the KIF5C C-terminal and GRIF-1 N-terminal regions. Co-immunoprecipitation and confocal imaging strategies further showed that GRIF-1 can bind to the tetrameric kinesin light-chain/kinesin heavy-chain complex. These findings support a role for GRIF-1 as a kinesin adaptor molecule requisite for the anterograde delivery of defined cargoes such as mitochondria and/or vesicles incorporating beta2 subunit-containing GABAA receptors, in the brain.
KIF5C GRIF 1 True Positive 15644324 Brickley K, Smith MJ, Beck M, Stephenson FA: GRIF-1 and OIP106, members of a novel gene family of coiled-coil domain proteins: association in vivo and in vitro with kinesin. J Biol Chem. 2005 Apr 15;280(15):14723-32. Epub 2005 Jan 11. Gamma-aminobutyric acid (A) receptor-interacting factor (GRIF-1) is a 913-amino acid protein proposed to function as a GABA (A) receptor beta (2) subunit-interacting, trafficking protein. GRIF-1 shares approximately 44% amino acid sequence identity with O-linked N-acetylglucosamine transferase interacting protein 106, OIP106. Both proteins contain predicted coiled-coil domains and probably constitute a novel gene family. The Drosophila orthologue of this family of proteins may be Milton. Milton shares approximately 44% amino acid homology with GRIF-1. Milton is proposed to function in kinesin-mediated transport of mitochondria to nerve terminals. We report here that GRIF-1 and OIP106 also associate with kinesin and mitochondria. Following expression in human embryonic kidney 293 cells, both GRIF-1 and OIP106 were shown by co-immunoprecipitation to be specifically associated with an endogenous kinesin heavy chain species of 115 kDa and exogenous KIF5C. Association of GRIF-1 with kinesin was also evident in native brain and heart tissue. In the brain, anti-GRIF-1-(8-633) antibodies specifically co-immunoprecipitated two kinesin-immunoreactive species with molecular masses of 118 and 115 kDa, and in the heart, one kinesin-immunoreactive species, 115 kDa, was immunoprecipitated. Further studies revealed that GRIF-1 was predominantly associated with KIF5A in the brain and with KIF5B in both the heart and in HEK 293 cells. Yeast two-hybrid interaction assays and immunoprecipitations showed that GRIF-1 associated directly with KIF5C with the GRIF-1/KIF5C interaction domain localized to GRIF-1-(124-283). These results further support a role for GRIF-1 and OIP106 in protein and/or organelle transport in excitable cells in a manner analogous to glutamate receptor-interacting-protein 1, in the motor-dependent transport of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate excitatory neurotransmitter receptors to dendrites.
KIF5C kinesin heavy chain True Positive 16835241 Smith MJ, Pozo K, Brickley K, Stephenson FA: Mapping the GRIF-1 binding domain of the kinesin, KIF5C, substantiates a role for GRIF-1 as an adaptor protein in the anterograde trafficking of cargoes. J Biol Chem. 2006 Sep 15;281(37):27216-28. Epub 2006 Jul 11. Gamma-aminobutyric acid, type A (GABAA) receptor interacting factor-1 (GRIF-1) and N-acetylglucosamine transferase interacting protein (OIP) 106 are both members of a newly identified coiled-coil family of proteins. They are kinesin-associated proteins proposed to function as adaptors in the anterograde trafficking of organelles to synapses. Here we have studied in more detail the interaction between the prototypic kinesin heavy chain, KIF5C, kinesin light chain, and GRIF-1. The GRIF-1 binding site of KIF5C was mapped using truncation constructs in yeast two-hybrid interaction assays, co-immunoprecipitations, and co-localization studies following expression in mammalian cells. Using these approaches, it was shown that GRIF-1 and the KIF5C binding domain of GRIF-1, GRIF-1-(124-283), associated with the KIF5C non-motor domain. Refined studies using yeast two-hybrid interactions and co-immunoprecipitations showed that GRIF-1 and GRIF-1-(124-283) associated with the cargo binding region within the KIF5C non-motor domain. Substantiation that the GRIF-1-KIF5C interaction was direct was shown by fluorescence resonance energy transfer analyses using fluorescently tagged GRIF-1 and KIF5C constructs. A significant fluorescence resonance energy transfer value was found between the C-terminal EYFP-tagged KIF5C and ECFP-GRIF-1, the C-terminal EYFP-tagged KIF5C non-motor domain and ECFP-GRIF-1, but not between the N-terminal EYFP-tagged KIF5C nor the EYFP-KIF5C motor domain and ECFP-GRIF-1, thus confirming direct association between the two proteins at the KIF5C C-terminal and GRIF-1 N-terminal regions. Co-immunoprecipitation and confocal imaging strategies further showed that GRIF-1 can bind to the tetrameric kinesin light-chain/kinesin heavy-chain complex. These findings support a role for GRIF-1 as a kinesin adaptor molecule requisite for the anterograde delivery of defined cargoes such as mitochondria and/or vesicles incorporating beta2 subunit-containing GABAA receptors, in the brain.
KIF5C kinesin heavy chain True Positive 10964943 Kanai Y, Okada Y, Tanaka Y, Harada A, Terada S, Hirokawa N: KIF5C, a novel neuronal kinesin enriched in motor neurons. J Neurosci. 2000 Sep 1;20(17):6374-84. Kinesin superfamily proteins (KIFs) are the molecular motors conveying cargos along microtubules. KIF5s, the heavy chains of conventional kinesin (KHC), are originally identified members of KIFs, and neuronal KIF5A and ubiquitous KIF5B have been identified so far. In the present work, we cloned a novel member of KIF5, KIF5C, and generated specific antibodies against three KIF5s to investigate their distribution and functions. KIF5A showed pan-neuronal distribution in the nervous system. KIF5B showed a glial cell distribution pattern in general; however, interestingly, its expression was strongly upregulated in axon-elongating neurons, such as olfactory primary neurons and mossy fibers. KIF5C was also a neuronal KIF5 like KIF5A but was highly expressed in lower motor neurons in 2-week-old or older mice, suggesting its important roles in the maintenance of motor neurons rather than in their formation, such as axonal elongation. Because a large part of KIF5s in adult motor neurons were expected to be KIF5C, we generated mice lacking the kif5C gene to investigate the functions of KIF5C in neurons in living animals. The mutant mice showed smaller brain size but were viable and did not show gross changes in the nervous system. Closer examinations revealed the relative loss of motor neurons to sensory neurons. Because three KIF5s showed high similarity in the amino acid sequence, could rescue the KIF5B mutant cells, and could form heterodimers, we think that there are functional redundancy among the three KIF5s and that KIF5A and KIF5B prevented the KIF5C null mice from the severe phenotype.
PITPNM2 sterol carrier protein 2 False Positive 16828756 Wirtz KW: Phospholipid transfer proteins in perspective. FEBS Lett. 2006 Oct 9;580(23):5436-41. Epub 2006 Jun 30. Since their discovery and subsequent purification from mammalian tissues more than 30 years ago an impressive number of studies have been carried out to characterize and elucidate the biological functions of phosphatidylcholine transfer protein (PC-TP), phosphatidylinositol transfer protein (PI-TP) and non-specific lipid transfer protein, more commonly known as sterol carrier protein 2 (SCP-2). Here I will present information to show that these soluble, low-molecular weight proteins constitute domain structures in StArR-related lipid transfer (START) proteins (i.e. PC-TP), in retinal degeneration protein, type B (RdgB)-related PI-TPs (e.g. Dm RdgB, Nir2, Nir3) and in peroxisomal beta-oxidation enzyme-related SCP-2 (i.e. 3-oxoacyl-CoA thiolase, also denoted as SCP-X and the 80-kDa D-bifunctional protein). Further I will summarize the most recent studies pertaining to the physiological function of these soluble phospholipid transfer proteins in metazoa.
PITPNM2 VAP B True Positive 15545272 Amarilio R, Ramachandran S, Sabanay H, Lev S: Differential regulation of endoplasmic reticulum structure through VAP-Nir protein interaction. J Biol Chem. 2005 Feb 18;280(7):5934-44. Epub 2004 Nov 15. The endoplasmic reticulum (ER) exhibits a characteristic tubular structure that is dynamically rearranged in response to specific physiological demands. However, the mechanisms by which the ER maintains its characteristic structure are largely unknown. Here we show that the integral ER-membrane protein VAP-B causes a striking rearrangement of the ER through interaction with the Nir2 and Nir3 proteins. We provide evidence that Nir (Nir1, Nir2, and Nir3)-VAP-B interactions are mediated through the conserved FFAT (two phenylalanines (FF) in acidic tract) motif present in Nir proteins. However, each interaction affects the structural integrity of the ER differently. Whereas the Nir2-VAP-B interaction induces the formation of stacked ER membrane arrays, the Nir3-VAP-B interaction leads to a gross remodeling of the ER and the bundling of thick microtubules along the altered ER membranes. In contrast, the Nir1-VAP-B interaction has no apparent effect on ER structure. We also show that the Nir2-VAP-B interaction attenuates protein export from the ER. These results demonstrate new mechanisms for the regulation of ER structure, all of which are mediated through interaction with an identical integral ER-membrane protein.
PITPNM2 Nir2 True Positive 15545272 Amarilio R, Ramachandran S, Sabanay H, Lev S: Differential regulation of endoplasmic reticulum structure through VAP-Nir protein interaction. J Biol Chem. 2005 Feb 18;280(7):5934-44. Epub 2004 Nov 15. The endoplasmic reticulum (ER) exhibits a characteristic tubular structure that is dynamically rearranged in response to specific physiological demands. However, the mechanisms by which the ER maintains its characteristic structure are largely unknown. Here we show that the integral ER-membrane protein VAP-B causes a striking rearrangement of the ER through interaction with the Nir2 and Nir3 proteins. We provide evidence that Nir (Nir1, Nir2, and Nir3)-VAP-B interactions are mediated through the conserved FFAT (two phenylalanines (FF) in acidic tract) motif present in Nir proteins. However, each interaction affects the structural integrity of the ER differently. Whereas the Nir2-VAP-B interaction induces the formation of stacked ER membrane arrays, the Nir3-VAP-B interaction leads to a gross remodeling of the ER and the bundling of thick microtubules along the altered ER membranes. In contrast, the Nir1-VAP-B interaction has no apparent effect on ER structure. We also show that the Nir2-VAP-B interaction attenuates protein export from the ER. These results demonstrate new mechanisms for the regulation of ER structure, all of which are mediated through interaction with an identical integral ER-membrane protein.
PITPNM2 PYK2 True Positive 10022914 Lev S, Hernandez J, Martinez R, Chen A, Plowman G, Schlessinger J: Identification of a novel family of targets of PYK2 related to Drosophila retinal degeneration B (rdgB) protein. Mol Cell Biol. 1999 Mar;19(3):2278-88. The protein tyrosine kinase PYK2 has been implicated in signaling pathways activated by G-protein-coupled receptors, intracellular calcium, and stress signals. Here we describe the molecular cloning and characterization of a novel family of PYK2-binding proteins designated Nirs (PYK2 N-terminal domain-interacting receptors). The three Nir proteins (Nir1, Nir2, and Nir3) bind to the amino-terminal domain of PYK2 via a conserved sequence motif located in the carboxy terminus. The primary structures of Nirs reveal six putative transmembrane domains, a region homologous to phosphatidylinositol (PI) transfer protein, and an acidic domain. The Nir proteins are the human homologues of the Drosophila retinal degeneration B protein (rdgB), a protein implicated in the visual transduction pathway in flies. We demonstrate that Nirs are calcium-binding proteins that exhibit PI transfer activity in vivo. Activation of PYK2 by agents that elevate intracellular calcium or by phorbol ester induce tyrosine phosphorylation of Nirs. Moreover, PYK2 and Nirs exhibit similar expression patterns in several regions of the brain and retina. In addition, PYK2-Nir complexes are detected in lysates prepared from cultured cells or from brain tissues. Finally, the Nir1-encoding gene is located at human chromosome 17p13.1, in proximity to a locus responsible for several human retinal diseases. We propose that the Nir and rdgB proteins represent a new family of evolutionarily conserved PYK2-binding proteins that play a role in the control of calcium and phosphoinositide metabolism downstream of G-protein-coupled receptors.
PITPNM2 3 oxoacyl CoA thiolase False Positive 16828756 Wirtz KW: Phospholipid transfer proteins in perspective. FEBS Lett. 2006 Oct 9;580(23):5436-41. Epub 2006 Jun 30. Since their discovery and subsequent purification from mammalian tissues more than 30 years ago an impressive number of studies have been carried out to characterize and elucidate the biological functions of phosphatidylcholine transfer protein (PC-TP), phosphatidylinositol transfer protein (PI-TP) and non-specific lipid transfer protein, more commonly known as sterol carrier protein 2 (SCP-2). Here I will present information to show that these soluble, low-molecular weight proteins constitute domain structures in StArR-related lipid transfer (START) proteins (i.e. PC-TP), in retinal degeneration protein, type B (RdgB)-related PI-TPs (e.g. Dm RdgB, Nir2, Nir3) and in peroxisomal beta-oxidation enzyme-related SCP-2 (i.e. 3-oxoacyl-CoA thiolase, also denoted as SCP-X and the 80-kDa D-bifunctional protein). Further I will summarize the most recent studies pertaining to the physiological function of these soluble phospholipid transfer proteins in metazoa.
PITPNM2 SCP 2 False Positive 16828756 Wirtz KW: Phospholipid transfer proteins in perspective. FEBS Lett. 2006 Oct 9;580(23):5436-41. Epub 2006 Jun 30. Since their discovery and subsequent purification from mammalian tissues more than 30 years ago an impressive number of studies have been carried out to characterize and elucidate the biological functions of phosphatidylcholine transfer protein (PC-TP), phosphatidylinositol transfer protein (PI-TP) and non-specific lipid transfer protein, more commonly known as sterol carrier protein 2 (SCP-2). Here I will present information to show that these soluble, low-molecular weight proteins constitute domain structures in StArR-related lipid transfer (START) proteins (i.e. PC-TP), in retinal degeneration protein, type B (RdgB)-related PI-TPs (e.g. Dm RdgB, Nir2, Nir3) and in peroxisomal beta-oxidation enzyme-related SCP-2 (i.e. 3-oxoacyl-CoA thiolase, also denoted as SCP-X and the 80-kDa D-bifunctional protein). Further I will summarize the most recent studies pertaining to the physiological function of these soluble phospholipid transfer proteins in metazoa.
PITPNM2 SNAP 25 True Positive 12037004 Tian D, Lev S: Cellular and developmental distribution of human homologues of the Drosophilia rdgB protein in the rat retina. Invest Ophthalmol Vis Sci. 2002 Jun;43(6):1946-53. PURPOSE: The Nirs (Nir1, Nir2, and Nir3), human homologues of Drosophila retinal degeneration B (rdgB), have been considered candidate genes for human inherited retinal degeneration diseases. To gain a better understanding of their functions in the retina and their putative roles in retinal degeneration diseases, this study was undertaken to determine their distribution profile in developing and mature rat retinas. METHODS: Specific antibodies against each of the Nir proteins were raised in rabbits and used in indirect immunofluorescence analysis to determine the distribution profile of the three proteins. Eyes from Wistar rats at various developmental stages (embryonic day [E] 18 to postnatal day [P] 16) were sectioned vertically and immunostained with anti-Nir antibodies. Coimmunostaining for Nirs and several specific cellular and subcellular markers was used to determine precisely the cellular and subcellular distribution of the Nirs. Sections were observed under a confocal laser microscope, and image analysis was performed with the standard operating software provided with the microscope. RESULTS: Confocal microscopic analysis of Nir1 immunoreactivity revealed that it was predominantly expressed in premature Muller cells at birth and that it was upregulated during Muller cell maturation. In contrast, Nir2 and Nir3 were homogeneously distributed in undifferentiated neuroblasts and ganglion cells at birth and later became distinctly distributed in newly differentiated neuronal cells. From P4, Nir2 and Nir3 were highly expressed in neuronal cells and their processes, coinciding with the formation of synaptic layers and ongoing synaptogenesis. From P12, Nir2 was uniformly expressed in all classes of retinal neuronal cells, including ganglion cells, horizontal cells, amacrine cells, bipolar cells, and photoreceptor cells. In the adult rat retina, Nir2 was preferentially localized to the somata of all classes of retinal neurons, whereas Nir3 was highly expressed in the synaptic terminals. This specific localization of Nir3 was confirmed by double immunostaining with the presynaptic protein synaptosomal-associated protein (SNAP)-25. In photoreceptor cells, both Nir2 and Nir3 were found to be highly expressed in the inner segments but were not detectably expressed in the outer segments. CONCLUSIONS: These findings suggest that the three Nir proteins are highly expressed in the developing retina, each exhibiting a distinct distribution profile. The different distribution patterns of these closely related proteins during development and at maturity may reflect their different cellular functions in vivo and their different roles in retinal cell survival or degeneration.
SNX2 TGN True Positive 17498660 Utskarpen A, Slagsvold HH, Dyve AB, Skanland SS, Sandvig K: SNX1 and SNX2 mediate retrograde transport of Shiga toxin. Biochem Biophys Res Commun. 2007 Jun 29;358(2):566-70. Epub 2007 May 4. The bacterial toxin Shiga toxin (Stx) is transported retrogradely from early endosomes to the Golgi apparatus on its way to the endoplasmic reticulum (ER) and the cytosol. In this study we explored the functions of the two phosphoinositide binding proteins Sorting nexin 1 (SNX1) and Sorting nexin 2 (SNX2) in endosomal sorting of the toxin. When Vero cells were depleted of either SNX1 or SNX2 by small interfering RNA (siRNA), Stx transport to the trans-Golgi network (TGN) was impaired by > or = 40%, whereas combined depletion of SNX1 and SNX2 gave a total inhibition of approximately 80%. Inhibition of PI (3) P formation by wortmannin resulted in a similar reduction. Thus, although being partly redundant, both SNX1 and SNX2 are required for efficient Stx trafficking to the Golgi apparatus.
SNX2 TGN True Positive 16179610 Carlton JG, Bujny MV, Peter BJ, Oorschot VM, Rutherford A, Arkell RS, Klumperman J, McMahon HT, Cullen PJ: Sorting nexin-2 is associated with tubular elements of the early endosome, but is not essential for retromer-mediated endosome-to-TGN transport. J Cell Sci. 2005 Oct 1;118(Pt 19):4527-39. Sorting nexins are a large family of phox-homology-domain-containing proteins that have been implicated in the control of endosomal sorting. Sorting nexin-1 is a component of the mammalian retromer complex that regulates retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network. In yeast, retromer is composed of Vps5p (the orthologue of sorting nexin-1), Vps17p (a related sorting nexin) and a cargo selective subcomplex composed of Vps26p, Vps29p and Vps35p. With the exception of Vps17p, mammalian orthologues of all yeast retromer components have been identified. For Vps17p, one potential mammalian orthologue is sorting nexin-2. Here we show that, like sorting nexin-1, sorting nexin-2 binds phosphatidylinositol 3-monophosphate and phosphatidylinositol 3,5-bisphosphate, and possesses a Bin/Amphiphysin/Rvs domain that can sense membrane curvature. However, in contrast to sorting nexin-1, sorting nexin-2 could not induce membrane tubulation in vitro or in vivo. Functionally, we show that endogenous sorting nexin-1 and sorting nexin-2 co-localise on high curvature tubular elements of the 3-phosphoinositide-enriched early endosome, and that suppression of sorting nexin-2 does not perturb the degradative sorting of receptors for epidermal growth factor or transferrin, nor the steady-state distribution of the cation-independent mannose 6-phosphate receptor. However, suppression of sorting nexin-2 results in a subtle alteration in the kinetics of cation-independent mannose 6-phosphate receptor retrieval. These data suggest that although sorting nexin-2 may be a component of the retromer complex, its presence is not essential for the regulation of endosome-to-trans Golgi network retrieval of the cation-independent mannose 6-phosphate receptor.
SNX2 Amphiphysin True Positive 16179610 Carlton JG, Bujny MV, Peter BJ, Oorschot VM, Rutherford A, Arkell RS, Klumperman J, McMahon HT, Cullen PJ: Sorting nexin-2 is associated with tubular elements of the early endosome, but is not essential for retromer-mediated endosome-to-TGN transport. J Cell Sci. 2005 Oct 1;118(Pt 19):4527-39. Sorting nexins are a large family of phox-homology-domain-containing proteins that have been implicated in the control of endosomal sorting. Sorting nexin-1 is a component of the mammalian retromer complex that regulates retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network. In yeast, retromer is composed of Vps5p (the orthologue of sorting nexin-1), Vps17p (a related sorting nexin) and a cargo selective subcomplex composed of Vps26p, Vps29p and Vps35p. With the exception of Vps17p, mammalian orthologues of all yeast retromer components have been identified. For Vps17p, one potential mammalian orthologue is sorting nexin-2. Here we show that, like sorting nexin-1, sorting nexin-2 binds phosphatidylinositol 3-monophosphate and phosphatidylinositol 3,5-bisphosphate, and possesses a Bin/Amphiphysin/Rvs domain that can sense membrane curvature. However, in contrast to sorting nexin-1, sorting nexin-2 could not induce membrane tubulation in vitro or in vivo. Functionally, we show that endogenous sorting nexin-1 and sorting nexin-2 co-localise on high curvature tubular elements of the 3-phosphoinositide-enriched early endosome, and that suppression of sorting nexin-2 does not perturb the degradative sorting of receptors for epidermal growth factor or transferrin, nor the steady-state distribution of the cation-independent mannose 6-phosphate receptor. However, suppression of sorting nexin-2 results in a subtle alteration in the kinetics of cation-independent mannose 6-phosphate receptor retrieval. These data suggest that although sorting nexin-2 may be a component of the retromer complex, its presence is not essential for the regulation of endosome-to-trans Golgi network retrieval of the cation-independent mannose 6-phosphate receptor.
SNX2 epidermal growth factor receptor True Positive 14978220 Gullapalli A, Garrett TA, Paing MM, Griffin CT, Yang Y, Trejo J: A role for sorting nexin 2 in epidermal growth factor receptor down-regulation: evidence for distinct functions of sorting nexin 1 and 2 in protein trafficking. Mol Biol Cell. 2004 May;15(5):2143-55. Epub 2004 Feb 20. Sorting nexin 1 (SNX1) and SNX2, homologues of the yeast vacuolar protein-sorting (Vps) 5p, contain a phospholipid-binding motif termed the phox homology (PX) domain and a carboxyl terminal coiled-coil region. A role for SNX1 in trafficking of cell surface receptors from endosomes to lysosomes has been proposed; however, the function of SNX2 remains unknown. Toward understanding the function of SNX2, we first examined the distribution of endogenous protein in HeLa cells. We show that SNX2 resides primarily in early endosomes, whereas SNX1 is found partially in early endosomes and in tubulovesicular-like structures distributed throughout the cytoplasm. We also demonstrate that SNX1 interacts with the mammalian retromer complex through its amino terminal domain, whereas SNX2 does not. Moreover, activated endogenous epidermal growth factor receptor (EGFR) colocalizes markedly with SNX2-positive endosomes, but minimally with SNX1-containing vesicles. To assess SNX2 function, we examined the effect of a PX domain-mutated SNX2 that is defective in vesicle localization on EGFR trafficking. Mutant SNX2 markedly inhibited agonist-induced EGFR degradation, whereas internalization remained intact. In contrast, SNX1 PX domain mutants failed to effect EGFR degradation, whereas a SNX1 deletion mutant significantly inhibited receptor down-regulation. Interestingly, knockdown of SNX1 and SNX2 expression by RNA interference failed to alter agonist-induced EGFR down-regulation. Together, these findings suggest that both SNX1 and SNX2 are involved in regulating lysosomal sorting of internalized EGFR, but neither protein is essential for this process. These studies are the first to demonstrate a function for SNX2 in protein trafficking.
SNX2 Vps26A True Positive 16214895 Griffin CT, Trejo J, Magnuson T: Genetic evidence for a mammalian retromer complex containing sorting nexins 1 and 2. Proc Natl Acad Sci U S A. 2005 Oct 18;102(42):15173-7. Epub 2005 Oct 7. We have previously shown that the putative mammalian retromer components sorting nexins 1 and 2 (Snx1 and Snx2) result in embryonic lethality when simultaneously targeted for deletion in mice, whereas others have shown that Hbeta58 (also known as mVps26), another retromer component, results in similar lethality when targeted for deletion. In the current study, we address the genetic interaction of these mammalian retromer components in mice. Our findings reveal a functional interaction between Hbeta58, SNX1, and SNX2 and strongly suggest that SNX2 plays a more critical role than SNX1 in retromer activity during embryonic development. This genetic evidence supports the existence of mammalian retromer complexes containing SNX1 and SNX2 and identifies SNX2 as an important mediator of retromer biology. Moreover, we find that mammalian retromer complexes containing SNX1 and SNX2 have an essential role in embryonic development that is independent of cation-independent mannose 6-phosphate receptor trafficking.
SNX2 SNX1 True Positive 17101778 Rojas R, Kametaka S, Haft CR, Bonifacino JS: Interchangeable but essential functions of SNX1 and SNX2 in the association of retromer with endosomes and the trafficking of mannose 6-phosphate receptors. Mol Cell Biol. 2007 Feb;27(3):1112-24. Epub 2006 Nov 13. The retromer is a cytosolic/peripheral membrane protein complex that mediates the retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network (TGN) in mammalian cells. Previous studies showed that the mammalian retromer comprises three proteins, named Vps26, Vps29, and Vps35, plus the sorting nexin, SNX1. There is conflicting evidence, however, as to whether a homologous sorting nexin, SNX2, is truly a component of the retromer. In addition, the nature of the subunit interactions and assembly of the mammalian retromer complex are poorly understood. We have addressed these issues by performing biochemical and functional analyses of endogenous retromers in the human cell line HeLa. We found that the mammalian retromer complex consists of two autonomously assembling subcomplexes, namely, a Vps26-Vps29-Vps35 obligate heterotrimer and a SNX1/2 alternative heterodimer or homodimer. The association of Vps26-Vps29-Vps35 with endosomes requires the presence of either SNX1 or SNX2, whereas SNX1/2 can be recruited to endosomes independently of Vps26-Vps29-Vps35. We also found that the presence of either SNX1 or SNX2 is essential for the retrieval of the cation-independent mannose 6-phosphate receptor to the TGN. These observations indicate that the mammalian retromer complex assembles by sequential association of SNX1/2 and Vps26-Vps29-Vps35 subcomplexes on endosomal membranes and that SNX1 and SNX2 play interchangeable but essential roles in retromer structure and function.
SNX2 SNX1 True Positive 16732284 Shi H, Rojas R, Bonifacino JS, Hurley JH: The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain. Nat Struct Mol Biol. 2006 Jun;13(6):540-8. Epub 2006 May 28. The mammalian retromer complex consists of SNX1, SNX2, Vps26, Vps29 and Vps35, and retrieves lysosomal enzyme receptors from endosomes to the trans-Golgi network. The structure of human Vps26A at 2.1-A resolution reveals two curved beta-sandwich domains connected by a polar core and a flexible linker. Vps26 has an unpredicted structural relationship to arrestins. The Vps35-binding site on Vps26 maps to a mobile loop spanning residues 235-246, near the tip of the C-terminal domain. The loop is phylogenetically conserved and provides a mechanism for Vps26 integration into the complex that leaves the rest of the structure free for engagements with membranes and for conformational changes. Hydrophobic residues and a glycine in this loop are required for integration into the retromer complex and endosomal localization of human Vps26, and for the function of yeast Vps26 in carboxypeptidase Y sorting.
SNX2 SNX1 True Positive 16214895 Griffin CT, Trejo J, Magnuson T: Genetic evidence for a mammalian retromer complex containing sorting nexins 1 and 2. Proc Natl Acad Sci U S A. 2005 Oct 18;102(42):15173-7. Epub 2005 Oct 7. We have previously shown that the putative mammalian retromer components sorting nexins 1 and 2 (Snx1 and Snx2) result in embryonic lethality when simultaneously targeted for deletion in mice, whereas others have shown that Hbeta58 (also known as mVps26), another retromer component, results in similar lethality when targeted for deletion. In the current study, we address the genetic interaction of these mammalian retromer components in mice. Our findings reveal a functional interaction between Hbeta58, SNX1, and SNX2 and strongly suggest that SNX2 plays a more critical role than SNX1 in retromer activity during embryonic development. This genetic evidence supports the existence of mammalian retromer complexes containing SNX1 and SNX2 and identifies SNX2 as an important mediator of retromer biology. Moreover, we find that mammalian retromer complexes containing SNX1 and SNX2 have an essential role in embryonic development that is independent of cation-independent mannose 6-phosphate receptor trafficking.
SNX2 SNX1 True Positive 14978220 Gullapalli A, Garrett TA, Paing MM, Griffin CT, Yang Y, Trejo J: A role for sorting nexin 2 in epidermal growth factor receptor down-regulation: evidence for distinct functions of sorting nexin 1 and 2 in protein trafficking. Mol Biol Cell. 2004 May;15(5):2143-55. Epub 2004 Feb 20. Sorting nexin 1 (SNX1) and SNX2, homologues of the yeast vacuolar protein-sorting (Vps) 5p, contain a phospholipid-binding motif termed the phox homology (PX) domain and a carboxyl terminal coiled-coil region. A role for SNX1 in trafficking of cell surface receptors from endosomes to lysosomes has been proposed; however, the function of SNX2 remains unknown. Toward understanding the function of SNX2, we first examined the distribution of endogenous protein in HeLa cells. We show that SNX2 resides primarily in early endosomes, whereas SNX1 is found partially in early endosomes and in tubulovesicular-like structures distributed throughout the cytoplasm. We also demonstrate that SNX1 interacts with the mammalian retromer complex through its amino terminal domain, whereas SNX2 does not. Moreover, activated endogenous epidermal growth factor receptor (EGFR) colocalizes markedly with SNX2-positive endosomes, but minimally with SNX1-containing vesicles. To assess SNX2 function, we examined the effect of a PX domain-mutated SNX2 that is defective in vesicle localization on EGFR trafficking. Mutant SNX2 markedly inhibited agonist-induced EGFR degradation, whereas internalization remained intact. In contrast, SNX1 PX domain mutants failed to effect EGFR degradation, whereas a SNX1 deletion mutant significantly inhibited receptor down-regulation. Interestingly, knockdown of SNX1 and SNX2 expression by RNA interference failed to alter agonist-induced EGFR down-regulation. Together, these findings suggest that both SNX1 and SNX2 are involved in regulating lysosomal sorting of internalized EGFR, but neither protein is essential for this process. These studies are the first to demonstrate a function for SNX2 in protein trafficking.
SNX2 PAR1 True Positive 16407403 Gullapalli A, Wolfe BL, Griffin CT, Magnuson T, Trejo J: An essential role for SNX1 in lysosomal sorting of protease-activated receptor-1: evidence for retromer-, Hrs-, and Tsg101-independent functions of sorting nexins. Mol Biol Cell. 2006 Mar;17(3):1228-38. Epub 2006 Jan 11. Sorting nexin 1 (SNX1) and SNX2 are the mammalian homologues of the yeast Vps5p retromer component that functions in endosome-to-Golgi trafficking. SNX1 is also implicated in endosome-to-lysosome sorting of cell surface receptors, although its requirement in this process remains to be determined. To assess SNX1 function in endocytic sorting of protease-activated receptor-1 (PAR1), we used siRNA to deplete HeLa cells of endogenous SNX1 protein. PAR1, a G-protein-coupled receptor, is proteolytically activated by thrombin, internalized, sorted predominantly to lysosomes, and efficiently degraded. Strikingly, depletion of endogenous SNX1 by siRNA markedly inhibited agonist-induced PAR1 degradation, whereas expression of a SNX1 siRNA-resistant mutant protein restored agonist-promoted PAR1 degradation in cells lacking endogenous SNX1, indicating that SNX1 is necessary for lysosomal degradation of PAR1. SNX1 is known to interact with components of the mammalian retromer complex and Hrs, an early endosomal membrane-associated protein. However, activated PAR1 degradation was not affected in cells depleted of retromer Vps26/Vps35 subunits, Hrs or Tsg101, an Hrs-interacting protein. We further show that SNX2, which dimerizes with SNX1, is not essential for lysosomal sorting of PAR1, but rather can regulate PAR1 degradation by disrupting endosomal localization of endogenous SNX1 when ectopically expressed. Together, our findings establish an essential role for endogenous SNX1 in sorting activated PAR1 to a distinct lysosomal degradative pathway that is independent of retromer, Hrs, and Tsg101.
SNX2 formin binding protein 17 True Positive 14502124 Morin F, Vannier B, Houdart F, Regnacq M, Berges T, Voisin P: A proline-rich domain in the gamma subunit of phosphodiesterase 6 mediates interaction with SH3-containing proteins. Mol Vis. 2003 Sep 18;9:449-59. PURPOSE: Phosphodiesterase 6 (PDE6) is the primary effector of phototransduction in vertebrate photoreceptors. Previous studies described the expression of the regulatory subunit of rod PDE6 (Pgamma-rod) in non-photosensitive tissues of the adult rat and the effects of this protein on MAP kinase pathways. Upon examination of the Pgamma-rod sequence, we detected a proline-rich domain that might reveal its ability to interact with SH3-containing proteins. Therefore, the present study was initiated to identify new protein partners of Pgamma-rod. METHODS: A yeast two-hybrid screen of a rat brain cDNA library was performed using Pgamma-rod as a bait. Pgamma-rod-SH3 interaction was confirmed by GST pull-down of in vitro-translated proteins. The aminoacids involved in the interaction were mapped by site-directed mutagenesis. Rnase protection assay, RT-PCR and western blot analysis were used to detect Pgamma-rod expression in various rat tissues. RESULTS: A clone was isolated twice, that consisted essentially of the SH3 domain of the formin-binding protein 17 (FBP17). This interaction was confirmed by GST pull-down. Mutational analysis of the Pgamma-rod-FBP17 interaction confirmed it involved the proline-rich domain of Pgamma-rod and the SH3 domain of FBP17. This proline-rich domain also allowed Pgamma-rod to interact with Cdc42-interacting protein 4 (CIP4), another SH3-containing protein. RT-PCR and Rnase protection assay detected different amounts of Pgamma-rod mRNA in adult and embryonic rat tissues. Western blots confirmed the presence of low levels of Pgamma-rod protein only in embryonic tissues. CONCLUSIONS: Our data suggest that Pgamma-rod participates in SH3-mediated cellular pathways and may therefore play a wider role than previously appreciated. One possibility is that FBP17 interaction with sorting nexin 2 might connect Pgamma-rod to receptor tyrosine kinase recycling. However, further studies are still required to identify the diversity of SH3-containing proteins that interact with Pgamma-rod. This effort should provide a rationale to understand how Pgamma-rod can affect receptor internalization-dependent MAP kinase activity.
SNX2 formin binding protein 17 True Positive 11438682 Fuchs U, Rehkamp G, Haas OA, Slany R, Konig M, Bojesen S, Bohle RM, Damm-Welk C, Ludwig WD, Harbott J, Borkhardt A: The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8756-61. Epub 2001 Jul 3. We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins (11;9)(q23;q34) inv (11)(q13q23). The rearrangement resulted in a 5'-MLL/FBP17-3' fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.
SNX2 cation independent mannose 6 phosphate receptor True Positive 16179610 Carlton JG, Bujny MV, Peter BJ, Oorschot VM, Rutherford A, Arkell RS, Klumperman J, McMahon HT, Cullen PJ: Sorting nexin-2 is associated with tubular elements of the early endosome, but is not essential for retromer-mediated endosome-to-TGN transport. J Cell Sci. 2005 Oct 1;118(Pt 19):4527-39. Sorting nexins are a large family of phox-homology-domain-containing proteins that have been implicated in the control of endosomal sorting. Sorting nexin-1 is a component of the mammalian retromer complex that regulates retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network. In yeast, retromer is composed of Vps5p (the orthologue of sorting nexin-1), Vps17p (a related sorting nexin) and a cargo selective subcomplex composed of Vps26p, Vps29p and Vps35p. With the exception of Vps17p, mammalian orthologues of all yeast retromer components have been identified. For Vps17p, one potential mammalian orthologue is sorting nexin-2. Here we show that, like sorting nexin-1, sorting nexin-2 binds phosphatidylinositol 3-monophosphate and phosphatidylinositol 3,5-bisphosphate, and possesses a Bin/Amphiphysin/Rvs domain that can sense membrane curvature. However, in contrast to sorting nexin-1, sorting nexin-2 could not induce membrane tubulation in vitro or in vivo. Functionally, we show that endogenous sorting nexin-1 and sorting nexin-2 co-localise on high curvature tubular elements of the 3-phosphoinositide-enriched early endosome, and that suppression of sorting nexin-2 does not perturb the degradative sorting of receptors for epidermal growth factor or transferrin, nor the steady-state distribution of the cation-independent mannose 6-phosphate receptor. However, suppression of sorting nexin-2 results in a subtle alteration in the kinetics of cation-independent mannose 6-phosphate receptor retrieval. These data suggest that although sorting nexin-2 may be a component of the retromer complex, its presence is not essential for the regulation of endosome-to-trans Golgi network retrieval of the cation-independent mannose 6-phosphate receptor.
SNX2 nexin True Positive 11438682 Fuchs U, Rehkamp G, Haas OA, Slany R, Konig M, Bojesen S, Bohle RM, Damm-Welk C, Ludwig WD, Harbott J, Borkhardt A: The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8756-61. Epub 2001 Jul 3. We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins (11;9)(q23;q34) inv (11)(q13q23). The rearrangement resulted in a 5'-MLL/FBP17-3' fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.
WNT6 SPC False Positive 11948913 Itaranta P, Lin Y, Perasaari J, Roel G, Destree O, Vainio S: Wnt-6 is expressed in the ureter bud and induces kidney tubule development in vitro. Genesis. 2002 Apr;32(4):259-68. The embryonic kidney is a classic developmental model system for studying inductive tissue interactions that govern organogenesis. We report here that Wnt-6 is expressed in the ureter bud, and that cell lines expressing Wnt-6 induce nephrogenesis in vitro. Wnt-6 cells induce tubules with similar kinetics to spinal cord (SPC) and lead to induced expression of Pax2, Pax8, Sfrp2, and E-cadherin genes, early markers of tubulogenesis. Moreover, Wnt-6 signaling rescues tubulogenesis in mesenchyme separated from Wnt-4 mutant embryos and leads to activation of Wnt-4 transcription. Wnt-6 also induces a secondary axis in early Xenopus embryos. We conclude that Wnt-6 is a candidate for the ureter epithelium-derived signal that leads to activation of kidney tubulogenesis via Wnt-4.
WNT6 Pax3 True Positive 16506066 Otto A, Schmidt C, Patel K: Pax3 and Pax7 expression and regulation in the avian embryo. Anat Embryol. 2006 Aug;211(4):293-310. Epub 2006 Feb 28. Satellite cells are essential for postnatal growth and repair of skeletal muscle. The paired-box transcription factors Pax3 and Pax7 are expressed in emerging muscle precursors. Recent studies have traced the origin of satellite cells to the embryonic dermomyotome, however, their developmental regulation throughout embryogenesis remains unclear. We show the overlying surface ectoderm and lateral plate are essential for Pax3 expression, and that the overlying surface ectoderm and neural tube are necessary for Pax7 expression within the dorsal somite. Furthermore we show that the notochord acts to down regulate the expression of both genes. Moreover, we identify diffusible factors within these tissues that act to maintain expression of Pax3 ( + ) and Pax7 (+) muscle precursors. We show that Wnt1, 3a, 4 and 6 proteins are able to up regulate and expand the expression of Pax3 and Pax7 within the dorsal somite. Finally, we show that Wnt6 can mimic the effect of the dorsal ectoderm to maintain Pax3 and Pax7 expression.
WNT6 Pax3 True Positive 16271265 Geetha-Loganathan P, Nimmagadda S, Prols F, Patel K, Scaal M, Huang R, Christ B: Ectodermal Wnt-6 promotes Myf5-dependent avian limb myogenesis. Dev Biol. 2005 Dec 1;288(1):221-33. Epub 2005 Nov 2. Limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Although several signaling molecules have been reported to be involved in the process of limb myogenesis, none of their activities has led to a consolidate idea about the limb myogenic pathway. Particularly, the role of ectodermal signals in limb myogenesis is still obscure. Here, we investigated the role of the ectoderm and ectodermal Wnt-6 during limb muscle development. We found that ectopic expression of Wnt-6 in the limb bud specifically extends the expression domains of Pax3, Paraxis, Myf5, Myogenin, Desmin and Myosin heavy chain (MyHC) but inhibits MyoD expression. Ectoderm removal results in a loss of expression of all of these myogenic markers. We show that Wnt-6 can compensate the absence of the ectoderm by rescuing the expression of Pax3, Paraxis, Myf5, Myogenin, Desmin and MyHC but not MyoD. These results show that, in chick, at least two signals from the limb ectoderm are necessary for muscle development. One of the signals is Wnt-6, which plays a unique role in promoting limb myogenesis via Pax3/Paraxis-Myf5, whereas the other putative signaling pathway involving MyoD expression is negatively regulated by Wnt-6 signaling.
WNT6 Wnt4 True Positive 11948913 Itaranta P, Lin Y, Perasaari J, Roel G, Destree O, Vainio S: Wnt-6 is expressed in the ureter bud and induces kidney tubule development in vitro. Genesis. 2002 Apr;32(4):259-68. The embryonic kidney is a classic developmental model system for studying inductive tissue interactions that govern organogenesis. We report here that Wnt-6 is expressed in the ureter bud, and that cell lines expressing Wnt-6 induce nephrogenesis in vitro. Wnt-6 cells induce tubules with similar kinetics to spinal cord (SPC) and lead to induced expression of Pax2, Pax8, Sfrp2, and E-cadherin genes, early markers of tubulogenesis. Moreover, Wnt-6 signaling rescues tubulogenesis in mesenchyme separated from Wnt-4 mutant embryos and leads to activation of Wnt-4 transcription. Wnt-6 also induces a secondary axis in early Xenopus embryos. We conclude that Wnt-6 is a candidate for the ureter epithelium-derived signal that leads to activation of kidney tubulogenesis via Wnt-4.
WNT6 WNT3 False Positive 16083717 Gregorieff A, Pinto D, Begthel H, Destree O, Kielman M, Clevers H: Expression pattern of Wnt signaling components in the adult intestine. Gastroenterology. 2005 Aug;129(2):626-38. BACKGROUND & AIMS: In the intestine, the canonical Wnt signaling cascade plays a crucial role in driving the proliferation of epithelial cells. Furthermore, aberrant activation of Wnt signaling is strongly associated with the development of colorectal cancer. Despite this evidence, little is known about the precise identity and localization of Wnts and their downstream effectors in the adult intestine. To address this issue, we examined the expression pattern of all Wnts, Frizzleds (Fzs), low-density lipoprotein receptor-related proteins, Wnt antagonists, and T-cell factors in the murine small intestine and colon and adenomas. METHODS: Embryonic, postnatal, and adult intestinal samples were subjected to in situ hybridization by using specific RNA probes for the various genes tested. RESULTS: Our analysis showed high expression of several signaling components (including Wnt-3, Wnt-6, Wnt-9b, Frizzled 4, Frizzled 6, Frizzled 7, low-density lipoprotein receptor-related protein 5, and secreted Frizzled-related protein 5) in crypt epithelial cells. We also detected Wnt-2b, Wnt-4, Wnt-5a, Wnt-5b, Frizzled 4, and Frizzled 6 in differentiated epithelial and mesenchymal cells of the small intestine and colon. Finally, several factors (Frizzled 4, T-cell factor 1, lymphoid enhancer factor, Dickkopf 2, Dickkopf 3, and Wnt-interacting factor) displayed differential expression in normal vs neoplastic tissue. CONCLUSIONS: Our study predicts a much broader role for Wnt signaling in gut development and homeostasis than was previously anticipated from available genetic studies and identifies novel factors likely involved in promoting canonical and noncanonical Wnt signals in the intestine.
WNT6 TGFA False Positive 16953426 Beaty TH, Hetmanski JB, Fallin MD, Park JW, Sull JW, McIntosh I, Liang KY, Vanderkolk CA, Redett RJ, Boyadjiev SA, Jabs EW, Chong SS, Cheah FS, Wu-Chou YH, Chen PK, Chiu YF, Yeow V, Ng IS, Cheng J, Huang S, Ye X, Wang H, Ingersoll R, Scott AF: Analysis of candidate genes on chromosome 2 in oral cleft case-parent trios from three populations. Hum Genet. 2006 Nov;120(4):501-18. Epub 2006 Sep 5. Isolated oral clefts, including cleft lip with/without cleft palate (CL/P) and cleft palate (CP), have a complex and heterogeneous etiology. Case-parent trios from three populations were used to study genes spanning chromosome 2, where single nucleotide polymorphic (SNP) markers were analyzed individually and as haplotypes. Case-parent trios from three populations (74 from Maryland, 64 from Singapore and 95 from Taiwan) were genotyped for 962 SNPs in 104 genes on chromosome 2, including two well-recognized candidate genes: TGFA and SATB2. Individual SNPs and haplotypes (in sliding windows of 2-5 SNPs) were used to test for linkage and disequilibrium separately in CL/P and CP trios. A novel candidate gene (ZNF533) showed consistent evidence of linkage and disequilibrium in all three populations for both CL/P and CP. SNPs in key regions of ZNF533 showed considerable variability in estimated genotypic odds ratios and their significance, suggesting allelic heterogeneity. Haplotype frequencies for regions of ZNF533 were estimated and used to partition genetic variance into among-and within-population components. Wright's fixation index, a measure of genetic diversity, showed little difference between Singapore and Taiwan compared with Maryland. The tensin-1 gene (TNS1) also showed evidence of linkage and disequilibrium among both CL/P and CP trios in all three populations, albeit at a lower level of significance. Additional genes (VAX2, GLI2, ZHFX1B on 2p; WNT6-WNT10A and COL4A3-COL4A4 on 2q) showed consistent evidence of linkage and disequilibrium only among CL/P trios in all three populations, and TGFA showed significant evidence in two of three populations.
WNT6 WNT2 False Positive 12469206 Katoh M: Expression and regulation of WNT1 in human cancer: up-regulation of WNT1 by beta-estradiol in MCF-7 cells. Int J Oncol. 2003 Jan;22(1):209-12. WNT family of secreted-type glycoproteins play key roles in carcinogenesis and embryogenesis. We have cloned and characterized human WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14 and WNT14B/WNT15 using bioinformatics and cDNA-PCR, and also reported frequent up-regulation of WNT2 in primary gastric cancer. Here, expression and regulation of WNT1 in human cancer were investigated using cDNA-PCR. WNT1 mRNA was relatively highly expressed in OKAJIMA cells (gastric cancer) and BxPC-3 cells (pancreatic cancer). Expression of WNT1 mRNA was up-regulated in 5 out of 10 cases of primary gastric cancer. Effects of beta-estradiol on expression of human WNT1 in MCF-7 cells (breast cancer) was next investigated, because mouse Wnt-1 induces mammary carcinogenesis even in estrogen receptor alpha (ERalpha) knockout mice. Expression of WNT1 mRNA was significantly up-regulated by beta-estradiol in MCF-7 cells. WNT1 was found to be one of estrogen target genes in human MCF-7 cells, which in part explains Wnt1-induced mammary carcinogenesis in ERalpha knockout mice.
WNT6 Myf5 True Positive 16271265 Geetha-Loganathan P, Nimmagadda S, Prols F, Patel K, Scaal M, Huang R, Christ B: Ectodermal Wnt-6 promotes Myf5-dependent avian limb myogenesis. Dev Biol. 2005 Dec 1;288(1):221-33. Epub 2005 Nov 2. Limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Although several signaling molecules have been reported to be involved in the process of limb myogenesis, none of their activities has led to a consolidate idea about the limb myogenic pathway. Particularly, the role of ectodermal signals in limb myogenesis is still obscure. Here, we investigated the role of the ectoderm and ectodermal Wnt-6 during limb muscle development. We found that ectopic expression of Wnt-6 in the limb bud specifically extends the expression domains of Pax3, Paraxis, Myf5, Myogenin, Desmin and Myosin heavy chain (MyHC) but inhibits MyoD expression. Ectoderm removal results in a loss of expression of all of these myogenic markers. We show that Wnt-6 can compensate the absence of the ectoderm by rescuing the expression of Pax3, Paraxis, Myf5, Myogenin, Desmin and MyHC but not MyoD. These results show that, in chick, at least two signals from the limb ectoderm are necessary for muscle development. One of the signals is Wnt-6, which plays a unique role in promoting limb myogenesis via Pax3/Paraxis-Myf5, whereas the other putative signaling pathway involving MyoD expression is negatively regulated by Wnt-6 signaling.
WNT6 Myf5 True Positive 9753670 Tajbakhsh S, Borello U, Vivarelli E, Kelly R, Papkoff J, Duprez D, Buckingham M, Cossu G: Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5. Development. 1998 Nov;125(21):4155-62. Activation of myogenesis in newly formed somites is dependent upon signals derived from neighboring tissues, namely axial structures (neural tube and notochord) and dorsal ectoderm. In explants of paraxial mesoderm from mouse embryos, axial structures preferentially activate myogenesis through a Myf5-dependent pathway and dorsal ectoderm preferentially through a MyoD-dependent pathway. Here we report that cells expressing Wnt1 will preferentially activate Myf5 while cells expressing Wnt7a will preferentially activate MyoD. Wnt1 is expressed in the dorsal neural tube and Wnt7a in dorsal ectoderm in the early embryo, therefore both can potentially act in vivo to activate Myf5 and MyoD, respectively. Wnt4, Wnt5a and Wnt6 exert an intermediate effect activating both Myf5 and MyoD equivalently in paraxial mesoderm. Sonic Hedgehog synergises with both Wnt1 and Wnt7a in explants from E8.5 paraxial mesoderm but not in explants from E9.5 embryos. Signaling through different myogenic pathways may explain the rescue of muscle formation in Myf5 null embryos, which do not form an early myotome but later develop both epaxial and hypaxial musculature. Explants of unsegmented paraxial mesoderm contain myogenic precursors capable of expressing MyoD in response to signaling from a neural tube isolated from E10.5 embryos, the developmental stage when MyoD is present throughout the embryo. Myogenic cells cannot activate MyoD in response to signaling from a less mature neural tube. Together these data suggest that different Wnt molecules can activate myogenesis through different pathways such that commitment of myogenic precursors is precisely regulated in space and time to achieve the correct pattern of skeletal muscle development.
WNT6 beta catenin True Positive 11350055 Kirikoshi H, Sekihara H, Katoh M: WNT10A and WNT6, clustered in human chromosome 2q35 region with head-to-tail manner, are strongly coexpressed in SW480 cells. Biochem Biophys Res Commun. 2001 May 18;283(4):798-805. Human WNT10A and WNT6 were cloned and characterized. WNT10A encoded a 417-amino-acid polypeptide with WNT core domain, and WNT6 encoded a 365-amino-acid polypeptide with N-terminal signal peptide, WNT core domain, and RGD motif. WNT10A and WNT6 genes were clustered in the head-to-tail manner with an interval less than 7.0 kb in human chromosome 2q35 region. Among human WNT family, WNT10A was most homologous to WNT10B (59.2% amino-acid identity), and WNT6 was most homologous to WNT1 (47.4% amino-acid identity). WNT10B and WNT1 genes were also clustered in human chromosome 12q13 region. Two WNT gene clusters in human chromosome 2q35 and 12q13 regions might be generated due to duplication of ancestral gene cluster. The 3.0- and 2.4-kb WNT10A mRNAs were expressed in fetal kidney, placenta, adult spleen and kidney. The 2.0-kb WNT6 mRNA was coexpressed with WNT10A in placenta and adult spleen. WNT10A and WNT6 were strongly coexpressed in SW480 (colorectal cancer). In addition to SW480, WNT10A was strongly expressed in HL-60 (promyelocytic leukemia) and Raji (Burkitt's lymphoma), and WNT6 in HeLa S3 (cervical cancer). Overexpression WNT10A and WNT6 might play key roles in human carcinogenesis through activation of WNT-beta-catenin-TCF signaling pathway, just like Wnt10b and Wnt1.
WNT6 WNT2B False Positive 12239632 Kirikoshi H, Katoh M: Expression of WNT7A in human normal tissues and cancer, and regulation of WNT7A and WNT7B in human cancer. Int J Oncol. 2002 Oct;21(4):895-900. WNT signals are transduced through seven-transmembrane-type WNT receptors encoded by Frizzled (FZD) genes to the beta-catenin - TCF pathway, the JNK pathway or the Ca2+-releasing pathway. WNT signaling molecules are potent targets for diagnosis of cancer (susceptibility, metastasis, and prognosis), for prevention and treatment of cancer, and for regenerative medicine or tissue engineering. We have so far cloned and characterized human WNT signaling molecules WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14, WNT14B/WNT15, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD10, FRAT1, FRAT2, NKD1, NKD2, VANGL1/STB2, ARHU/WRCH1, ARHV/WRCH2, GIPC2, GIPC3, betaTRCP2/FBXW1B, SOX17, and TCF-3 using bioinformatics, cDNA-library screening, and cDNA-PCR. Here, expression of WNT7A in human normal tissues and cancer, and regulation of WNT7A and WNT7B in human cancer were investigated. WNT7A was highly expressed in fetal lung, adult testis, lymph node, and peripheral blood leukocytes. WNT7A was relatively highly expressed in temporal lobe, occipital lobe, parietal lobe, paracentral gyrus of cerebral cortex, caudate nucleus, hippocampus, medulla oblongata and putamen within adult brain. WNT7A was highly expressed in SW480 (colorectal cancer), BxPC-3 and Hs766T (pancreatic cancer), and was also expressed in MKN7 and MKN45 (gastric cancer). WNT7B rather than WNT7A was expressed in MCF-7 (breast cancer) and NT2 (embryonal tumor). beta-estradiol did not affect expression levels of WNT7A and WNT7B in MCF-7 cells. WNT7B, but not WNT7A, was slightly up-regulated by all-trans retinoic acid in NT2 cells.
WNT6 Desmin True Positive 16271265 Geetha-Loganathan P, Nimmagadda S, Prols F, Patel K, Scaal M, Huang R, Christ B: Ectodermal Wnt-6 promotes Myf5-dependent avian limb myogenesis. Dev Biol. 2005 Dec 1;288(1):221-33. Epub 2005 Nov 2. Limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Although several signaling molecules have been reported to be involved in the process of limb myogenesis, none of their activities has led to a consolidate idea about the limb myogenic pathway. Particularly, the role of ectodermal signals in limb myogenesis is still obscure. Here, we investigated the role of the ectoderm and ectodermal Wnt-6 during limb muscle development. We found that ectopic expression of Wnt-6 in the limb bud specifically extends the expression domains of Pax3, Paraxis, Myf5, Myogenin, Desmin and Myosin heavy chain (MyHC) but inhibits MyoD expression. Ectoderm removal results in a loss of expression of all of these myogenic markers. We show that Wnt-6 can compensate the absence of the ectoderm by rescuing the expression of Pax3, Paraxis, Myf5, Myogenin, Desmin and MyHC but not MyoD. These results show that, in chick, at least two signals from the limb ectoderm are necessary for muscle development. One of the signals is Wnt-6, which plays a unique role in promoting limb myogenesis via Pax3/Paraxis-Myf5, whereas the other putative signaling pathway involving MyoD expression is negatively regulated by Wnt-6 signaling.
WNT6 WNT10A True Positive 11494032 Kirikoshi H, Sekihara H, Katoh M: Up-regulation of WNT10A by tumor necrosis factor alpha and Helicobacter pylori in gastric cancer. Int J Oncol. 2001 Sep;19(3):533-6. WNT signaling pathway is implicated in carcinogenesis and embryogenesis. We have previously cloned and characterized WNT10A and WNT6, which are clustered in human chromosome 2q35 region. In this study, we investigated expression of WNT10A and WNT6 in gastric cancer. The 3.0- and 2.4-kb WNT10A mRNAs were expressed in gastric cancer cell lines MKN7, MKN45 and MKN74. The 2.0-kb WNT6 mRNA was expressed in gastric cancer cell lines MKN28 and MKN74. WNT10A was up-regulated in 3 out of 6 cases of primary gastric cancer, while WNT6 was not up-regulated in primary gastric cancer. Effects of inflammatory cytokines and Helicobacter pylori (H. pylori) on expression of WNT10A and WNT6 were next investigated. Interferon gamma (IFNgamma) failed to induce up-regulation of WNT10A and WNT6. Tumor necrosis factor alpha (TNFalpha) induced up-regulation of WNT10A in MKN45 cells. Up-regulation of WNT10A reached maximum at 6 h after TNFalpha treatment. H. pylori also induced up-regulation of WNT10A in MKN45 cells. These results strongly suggest that up-regulation of WNT10A induced by TNFalpha and H. pylori might play key roles in human gastric cancer through activation of WNT--beta-catenin--TCF signaling pathway.
WNT6 Paraxis True Positive 16271265 Geetha-Loganathan P, Nimmagadda S, Prols F, Patel K, Scaal M, Huang R, Christ B: Ectodermal Wnt-6 promotes Myf5-dependent avian limb myogenesis. Dev Biol. 2005 Dec 1;288(1):221-33. Epub 2005 Nov 2. Limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Although several signaling molecules have been reported to be involved in the process of limb myogenesis, none of their activities has led to a consolidate idea about the limb myogenic pathway. Particularly, the role of ectodermal signals in limb myogenesis is still obscure. Here, we investigated the role of the ectoderm and ectodermal Wnt-6 during limb muscle development. We found that ectopic expression of Wnt-6 in the limb bud specifically extends the expression domains of Pax3, Paraxis, Myf5, Myogenin, Desmin and Myosin heavy chain (MyHC) but inhibits MyoD expression. Ectoderm removal results in a loss of expression of all of these myogenic markers. We show that Wnt-6 can compensate the absence of the ectoderm by rescuing the expression of Pax3, Paraxis, Myf5, Myogenin, Desmin and MyHC but not MyoD. These results show that, in chick, at least two signals from the limb ectoderm are necessary for muscle development. One of the signals is Wnt-6, which plays a unique role in promoting limb myogenesis via Pax3/Paraxis-Myf5, whereas the other putative signaling pathway involving MyoD expression is negatively regulated by Wnt-6 signaling.
WNT6 Paraxis True Positive 16100089 Linker C, Lesbros C, Gros J, Burrus LW, Rawls A, Marcelle C: beta-Catenin-dependent Wnt signalling controls the epithelial organisation of somites through the activation of paraxis. Development. 2005 Sep;132(17):3895-905. The regulation of cell adhesion in epithelia is a fundamental process governing morphogenesis in embryos and a key step in the progression of invasive cancers. Here, we have analysed the molecular pathways controlling the epithelial organisation of somites. Somites are mesodermal epithelial structures of vertebrate embryos that undergo several changes in cell adhesion during early embryonic life. We show that Wnt6 in the ectoderm overlaying the somites, but not Wnt1 in the neighbouring neural tube, is the most likely candidate molecule responsible for the maintenance of the epithelial structure of the dorsal compartment of the somite: the dermomyotome. We characterised the signalling pathway that mediates Wnt6 activity. Our experiments suggest that the Wnt receptor molecule Frizzled7 probably transduces the Wnt6 signal. Intracellularly, this leads to the activation of the beta-catenin/LEF1-dependent pathway. Finally, we demonstrate that the bHLH transcription factor paraxis, which was previously shown to be a major player in the epithelial organisation of somites, is a target of the beta-catenin signal. We conclude that beta-catenin activity, initiated by Wnt6 and mediated by paraxis, is required for the maintenance of the epithelial structure of somites.
WNT6 Myogenin True Positive 16271265 Geetha-Loganathan P, Nimmagadda S, Prols F, Patel K, Scaal M, Huang R, Christ B: Ectodermal Wnt-6 promotes Myf5-dependent avian limb myogenesis. Dev Biol. 2005 Dec 1;288(1):221-33. Epub 2005 Nov 2. Limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Although several signaling molecules have been reported to be involved in the process of limb myogenesis, none of their activities has led to a consolidate idea about the limb myogenic pathway. Particularly, the role of ectodermal signals in limb myogenesis is still obscure. Here, we investigated the role of the ectoderm and ectodermal Wnt-6 during limb muscle development. We found that ectopic expression of Wnt-6 in the limb bud specifically extends the expression domains of Pax3, Paraxis, Myf5, Myogenin, Desmin and Myosin heavy chain (MyHC) but inhibits MyoD expression. Ectoderm removal results in a loss of expression of all of these myogenic markers. We show that Wnt-6 can compensate the absence of the ectoderm by rescuing the expression of Pax3, Paraxis, Myf5, Myogenin, Desmin and MyHC but not MyoD. These results show that, in chick, at least two signals from the limb ectoderm are necessary for muscle development. One of the signals is Wnt-6, which plays a unique role in promoting limb myogenesis via Pax3/Paraxis-Myf5, whereas the other putative signaling pathway involving MyoD expression is negatively regulated by Wnt-6 signaling.
WNT6 ectodysplasin True Positive 11203701 Laurikkala J, Mikkola M, Mustonen T, Aberg T, Koppinen P, Pispa J, Nieminen P, Galceran J, Grosschedl R, Thesleff I: TNF signaling via the ligand-receptor pair ectodysplasin and edar controls the function of epithelial signaling centers and is regulated by Wnt and activin during tooth organogenesis. Dev Biol. 2001 Jan 15;229(2):443-55. Ectodermal dysplasia syndromes affect the development of several organs, including hair, teeth, and glands. The recent cloning of two genes responsible for these syndromes has led to the identification of a novel TNF family ligand, ectodysplasin, and TNF receptor, edar. This has indicated a developmental regulatory role for TNFs for the first time. Our in situ hybridization analysis of the expression of ectodysplasin (encoded by the Tabby gene) and edar (encoded by the downless gene) during mouse tooth morphogenesis showed that they are expressed in complementary patterns exclusively in ectodermal tissue layer. Edar was expressed reiteratively in signaling centers regulating key steps in morphogenesis. The analysis of the effects of eight signaling molecules in the TGFbeta, FGF, Hh, Wnt, and EGF families in tooth explant cultures revealed that the expression of edar was induced by activinbetaA, whereas Wnt6 induced ectodysplasin expression. Moreover, ectodysplasin expression was downregulated in branchial arch epithelium and in tooth germs of Lef1 mutant mice, suggesting that signaling by ectodysplasin is regulated by LEF-1-mediated Wnt signals. The analysis of the signaling centers in tooth germs of Tabby mice (ectodysplasin null mutants) indicated that in the absence of ectodysplasin the signaling centers were small. However, no downstream targets of ectodysplasin signaling were identified among several genes expressed in the signaling centers. We conclude that ectodysplasin functions as a planar signal between ectodermal compartments and regulates the function, but not the induction, of epithelial signaling centers. This TNF signaling is tightly associated with epithelial-mesenchymal interactions and with other signaling pathways regulating organogenesis. We suggest that activin signaling from mesenchyme induces the expression of the TNF receptor edar in the epithelial signaling centers, thus making them responsive to Wnt-induced ectodysplasin from the nearby ectoderm. This is the first demonstration of integration of the Wnt, activin, and TNF signaling pathways.
WNT6 Wnt1 True Positive 16818447 Geetha-Loganathan P, Nimmagadda S, Huang R, Christ B, Scaal M: Regulation of ectodermal Wnt6 expression by the neural tube is transduced by dermomyotomal Wnt11: a mechanism of dermomyotomal lip sustainment. Development. 2006 Aug;133(15):2897-904. Epub 2006 Jul 3. Ectodermal Wnt6 plays an important role during development of the somites and the lateral plate mesoderm. In the course of development, Wnt6 expression shows a dynamic pattern. At the level of the segmental plate and the epithelial somites, Wnt6 is expressed in the entire ectoderm overlying the neural tube, the paraxial mesoderm and the lateral plate mesoderm. With somite maturation, expression becomes restricted to the lateral ectoderm covering the ventrolateral lip of the dermomyotome and the lateral plate mesoderm. To study the regulation of Wnt6 expression, we have interfered with neighboring signaling pathways. We show that Wnt1 and Wnt3a signaling from the neural tube inhibit Wnt6 expression in the medial surface ectoderm via dermomyotomal Wnt11. We demonstrate that Wnt11 is an epithelialization factor acting on the medial dermomyotome, and present a model suggesting Wnt11 and Wnt6 as factors maintaining the epithelial nature of the dorsomedial and ventrolateral lips of the dermomyotome, respectively, during dermomyotomal growth.
WNT6 Wnt1 True Positive 9505170 Erdreich-Epstein A, Shackleford GM: Differential expression of Wnt genes in normal and flat variants of PC12 cells, a cell line responsive to ectopic Wnt1 expression. Growth Factors. 1998;15(2):149-58. Wnt genes encode secreted growth factor-like proteins that participate in growth regulation, differentiation and tumorigenesis. Ectopic expression of Wnt1 converts the PC12 neural crest-derived rat pheochromocytoma cell line from a round phenotype that express chromaffin markers to flat adherent cells (termed PC12/Wnt1) that do not express them. A pool of spontaneously flat variants of PC12 cells (PC12/flat) is phenotypically similar to the PC12/Wnt1 cells, but does not express Wnt1. Here we describe the expression of 13 Wnt genes in wild type PC12, PC12/flat and PC12/Wnt1 cells. Wild type PC12 expressed Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt6, Wnt10a and Wnt11. Compared with expression in wild type cells, both PC12/flat and PC12/Wnt1 cells lost most or all expression of Wnt3a and Wnt4 and gained expression of Wnt7b. Wnt5a and Wnt6 expression was higher in PC12/Wnt1 cells than in PC12 or PC12/flat. Wnt3 was expressed at low levels in both PC12 and PC12/flat, but was absent in PC12/Wnt1 cells. Wnt10a and Wnt11 were approximately equally expressed in the three groups, and Wnt2, Wnt5b, Wnt7a, Wnt10b and endogenous Wnt1 mRNAs were not detected. These results demonstrate that the expression of some Wnt genes changes in PC12 cells upon conversion to the flat phenotype, and suggest that Wnt1 may modulate expression of several other Wnt genes in these cells.
WNT6 FGF 10 False Positive 17196193 Nyeng P, Norgaard GA, Kobberup S, Jensen J: FGF10 signaling controls stomach morphogenesis. Dev Biol. 2007 Mar 1;303(1):295-310. Epub 2006 Nov 17. Maintenance of progenitor cell properties in development is required for proper organogenesis of most organs, including those derived from the endoderm. FGF10 has been shown to play a role in both lung and pancreatic development. Here we find that FGF10 signaling controls stomach progenitor maintenance, morphogenesis and cellular differentiation. Through a characterization of the initiation of terminal differentiation of the three major gastric regions in the mouse, forestomach, corpus and antrum, we first describe the existence of a "secondary transition" event occurring in mouse stomach between E15.5 and E16.5. This includes the formation of terminally differentiated squamous cells, parietal, chief and gastric endocrine cells from a pre-patterned gastric progenitor epithelium. Expression analysis of both FGF and Notch signaling components suggested a role of these networks in such progenitors, which was tested through ectopically expressing FGF10 in the developing posterior stomach. These data provide evidence that gastric gland specification and progenitor cell maintenance is controlled by FGF10. The glandular proliferative niche was disrupted in pPDX-FGF10 (FLAG) mice leading to aberrant gland formation, and endocrine and parietal cell differentiation was attenuated. These effects were paralleled by changes in Hes1, Shh and Wnt6 expression, suggesting that FGF10 acts in concert with multiple morphogenetic signaling systems during gastric development.
WNT6 FGF 10 False Positive 11066089 Kettunen P, Laurikkala J, Itaranta P, Vainio S, Itoh N, Thesleff I: Associations of FGF-3 and FGF-10 with signaling networks regulating tooth morphogenesis. Dev Dyn. 2000 Nov;219(3):322-32. The morphogenesis and cell differentiation in developing teeth is governed by interactions between the oral epithelium and neural crest-derived ectomesenchyme. The fibroblast growth factors FGF-4, -8, and -9 have been implicated as epithelial signals regulating mesenchymal gene expression and cell proliferation during tooth initiation and later during epithelial folding morphogenesis and the establishment of tooth shape. To further evaluate the roles of FGFs in tooth development, we analyzed the roles of FGF-3, FGF-7, and FGF-10 in developing mouse teeth. In situ hybridization analysis showed developmentally regulated expression during tooth formation for Fgf-3 and Fgf-10 that was mainly restricted to the dental papilla mesenchymal cells. Fgf-7 transcripts were restricted to the developing bone surrounding the developing tooth germ. Fgf-10 expression was observed in the presumptive dental epithelium and mesenchyme during tooth initiation, whereas Fgf-3 expression appeared in the dental mesenchyme at the late bud stage. During the cap and bell stage, both Fgf-3 and Fgf-10 were intensely expressed in the dental papilla mesenchymal cells both in incisors and molars. It is of interest that Fgf-3 expression was also observed in the primary enamel knot, a putative signaling center of the tooth, whereas no transcripts were seen in the secondary enamel knots that appear in the tips of future cusps of the bell stage tooth germs. Down-regulation of Fgf-3 and Fgf-10 expression in postmitotic odontoblasts correlated with the terminal differentiation of the odontoblasts and the neighboring ameloblasts. In the incisors, mesenchymal cells of the cervical loop area showed partially overlapping expression patterns for all studied Fgfs. In vitro analyses showed that expression of Fgf-3 and Fgf-10 in the dental mesenchyme was dependent on dental epithelium and that epithelially expressed FGFs, FGF-4 and -8 induced Fgf-3 but not Fgf-10 expression in the isolated dental mesenchyme. Beads soaked in Shh, BMP-2, and TGF-beta 1 protein did not induce either Fgf-3 or Fgf-10 expression. Cells expressing Wnt-6 did not induce Fgf-10 expression. Furthermore, FGF-10 protein stimulated cell proliferation in the dental epithelium but not in the mesenchyme. These results suggest that FGF-3 and FGF-10 have redundant functions as mesenchymal signals regulating epithelial morphogenesis of the tooth and that their expressions appear to be differentially regulated. In addition, FGF-3 may participate in signaling functions of the primary enamel knot. The dynamic expression patterns of different Fgfs in dental epithelium and mesenchyme and their interactions suggest existence of regulatory signaling cascades between epithelial and mesenchymal FGFs during tooth development.
WNT6 MyoD True Positive 16271265 Geetha-Loganathan P, Nimmagadda S, Prols F, Patel K, Scaal M, Huang R, Christ B: Ectodermal Wnt-6 promotes Myf5-dependent avian limb myogenesis. Dev Biol. 2005 Dec 1;288(1):221-33. Epub 2005 Nov 2. Limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Although several signaling molecules have been reported to be involved in the process of limb myogenesis, none of their activities has led to a consolidate idea about the limb myogenic pathway. Particularly, the role of ectodermal signals in limb myogenesis is still obscure. Here, we investigated the role of the ectoderm and ectodermal Wnt-6 during limb muscle development. We found that ectopic expression of Wnt-6 in the limb bud specifically extends the expression domains of Pax3, Paraxis, Myf5, Myogenin, Desmin and Myosin heavy chain (MyHC) but inhibits MyoD expression. Ectoderm removal results in a loss of expression of all of these myogenic markers. We show that Wnt-6 can compensate the absence of the ectoderm by rescuing the expression of Pax3, Paraxis, Myf5, Myogenin, Desmin and MyHC but not MyoD. These results show that, in chick, at least two signals from the limb ectoderm are necessary for muscle development. One of the signals is Wnt-6, which plays a unique role in promoting limb myogenesis via Pax3/Paraxis-Myf5, whereas the other putative signaling pathway involving MyoD expression is negatively regulated by Wnt-6 signaling.
WNT6 MyoD True Positive 9753670 Tajbakhsh S, Borello U, Vivarelli E, Kelly R, Papkoff J, Duprez D, Buckingham M, Cossu G: Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5. Development. 1998 Nov;125(21):4155-62. Activation of myogenesis in newly formed somites is dependent upon signals derived from neighboring tissues, namely axial structures (neural tube and notochord) and dorsal ectoderm. In explants of paraxial mesoderm from mouse embryos, axial structures preferentially activate myogenesis through a Myf5-dependent pathway and dorsal ectoderm preferentially through a MyoD-dependent pathway. Here we report that cells expressing Wnt1 will preferentially activate Myf5 while cells expressing Wnt7a will preferentially activate MyoD. Wnt1 is expressed in the dorsal neural tube and Wnt7a in dorsal ectoderm in the early embryo, therefore both can potentially act in vivo to activate Myf5 and MyoD, respectively. Wnt4, Wnt5a and Wnt6 exert an intermediate effect activating both Myf5 and MyoD equivalently in paraxial mesoderm. Sonic Hedgehog synergises with both Wnt1 and Wnt7a in explants from E8.5 paraxial mesoderm but not in explants from E9.5 embryos. Signaling through different myogenic pathways may explain the rescue of muscle formation in Myf5 null embryos, which do not form an early myotome but later develop both epaxial and hypaxial musculature. Explants of unsegmented paraxial mesoderm contain myogenic precursors capable of expressing MyoD in response to signaling from a neural tube isolated from E10.5 embryos, the developmental stage when MyoD is present throughout the embryo. Myogenic cells cannot activate MyoD in response to signaling from a less mature neural tube. Together these data suggest that different Wnt molecules can activate myogenesis through different pathways such that commitment of myogenic precursors is precisely regulated in space and time to achieve the correct pattern of skeletal muscle development.
WNT6 WNT10B False Positive 14745960 Veltmaat JM, Van Veelen W, Thiery JP, Bellusci S: Identification of the mammary line in mouse by Wnt10b expression. Dev Dyn. 2004 Feb;229(2):349-56. Mammogenesis in rabbit, rat, and human begins with the formation of an elevated ectodermal ridge in the embryo. Attempts to demonstrate a morphologically or histologically equivalent mammary line in the mouse have yielded controversial results. We show here that a mammary line exists in the mouse embryo at embryonic day (E) 11.25 as a concise line of Wnt10b expression and a broader band of Wnt6 expression in the surface ectoderm, between the subaxillary and suprainguinal region of each flank. Additional streaks of Wnt10b expression in the axillary and inguinal region join the mammary line on the flank slightly later. Expression of Wnt10b and Wnt6 becomes restricted to the placodes within 1.5 days. The ectoderm of the mammary line is organized as a pseudostratified epithelium connecting the developing mammary placodes at around E11.5, whereas all other surface ectoderm is single-layered. Thus, the mammary line expressing Wnt10b defines a distinct ectodermal region that we consider the equivalent of the ectodermal ridge in, for example, rabbit. To date, the formation of the mammary line expressing Wnt10b is the earliest discernible ectodermal event in murine embryonic mammary gland development.
WNT6 WNT True Positive 12239632 Kirikoshi H, Katoh M: Expression of WNT7A in human normal tissues and cancer, and regulation of WNT7A and WNT7B in human cancer. Int J Oncol. 2002 Oct;21(4):895-900. WNT signals are transduced through seven-transmembrane-type WNT receptors encoded by Frizzled (FZD) genes to the beta-catenin - TCF pathway, the JNK pathway or the Ca2+-releasing pathway. WNT signaling molecules are potent targets for diagnosis of cancer (susceptibility, metastasis, and prognosis), for prevention and treatment of cancer, and for regenerative medicine or tissue engineering. We have so far cloned and characterized human WNT signaling molecules WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14, WNT14B/WNT15, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD10, FRAT1, FRAT2, NKD1, NKD2, VANGL1/STB2, ARHU/WRCH1, ARHV/WRCH2, GIPC2, GIPC3, betaTRCP2/FBXW1B, SOX17, and TCF-3 using bioinformatics, cDNA-library screening, and cDNA-PCR. Here, expression of WNT7A in human normal tissues and cancer, and regulation of WNT7A and WNT7B in human cancer were investigated. WNT7A was highly expressed in fetal lung, adult testis, lymph node, and peripheral blood leukocytes. WNT7A was relatively highly expressed in temporal lobe, occipital lobe, parietal lobe, paracentral gyrus of cerebral cortex, caudate nucleus, hippocampus, medulla oblongata and putamen within adult brain. WNT7A was highly expressed in SW480 (colorectal cancer), BxPC-3 and Hs766T (pancreatic cancer), and was also expressed in MKN7 and MKN45 (gastric cancer). WNT7B rather than WNT7A was expressed in MCF-7 (breast cancer) and NT2 (embryonal tumor). beta-estradiol did not affect expression levels of WNT7A and WNT7B in MCF-7 cells. WNT7B, but not WNT7A, was slightly up-regulated by all-trans retinoic acid in NT2 cells.
WNT6 WNT True Positive 11350055 Kirikoshi H, Sekihara H, Katoh M: WNT10A and WNT6, clustered in human chromosome 2q35 region with head-to-tail manner, are strongly coexpressed in SW480 cells. Biochem Biophys Res Commun. 2001 May 18;283(4):798-805. Human WNT10A and WNT6 were cloned and characterized. WNT10A encoded a 417-amino-acid polypeptide with WNT core domain, and WNT6 encoded a 365-amino-acid polypeptide with N-terminal signal peptide, WNT core domain, and RGD motif. WNT10A and WNT6 genes were clustered in the head-to-tail manner with an interval less than 7.0 kb in human chromosome 2q35 region. Among human WNT family, WNT10A was most homologous to WNT10B (59.2% amino-acid identity), and WNT6 was most homologous to WNT1 (47.4% amino-acid identity). WNT10B and WNT1 genes were also clustered in human chromosome 12q13 region. Two WNT gene clusters in human chromosome 2q35 and 12q13 regions might be generated due to duplication of ancestral gene cluster. The 3.0- and 2.4-kb WNT10A mRNAs were expressed in fetal kidney, placenta, adult spleen and kidney. The 2.0-kb WNT6 mRNA was coexpressed with WNT10A in placenta and adult spleen. WNT10A and WNT6 were strongly coexpressed in SW480 (colorectal cancer). In addition to SW480, WNT10A was strongly expressed in HL-60 (promyelocytic leukemia) and Raji (Burkitt's lymphoma), and WNT6 in HeLa S3 (cervical cancer). Overexpression WNT10A and WNT6 might play key roles in human carcinogenesis through activation of WNT-beta-catenin-TCF signaling pathway, just like Wnt10b and Wnt1.
WNT6 TCF True Positive 11350055 Kirikoshi H, Sekihara H, Katoh M: WNT10A and WNT6, clustered in human chromosome 2q35 region with head-to-tail manner, are strongly coexpressed in SW480 cells. Biochem Biophys Res Commun. 2001 May 18;283(4):798-805. Human WNT10A and WNT6 were cloned and characterized. WNT10A encoded a 417-amino-acid polypeptide with WNT core domain, and WNT6 encoded a 365-amino-acid polypeptide with N-terminal signal peptide, WNT core domain, and RGD motif. WNT10A and WNT6 genes were clustered in the head-to-tail manner with an interval less than 7.0 kb in human chromosome 2q35 region. Among human WNT family, WNT10A was most homologous to WNT10B (59.2% amino-acid identity), and WNT6 was most homologous to WNT1 (47.4% amino-acid identity). WNT10B and WNT1 genes were also clustered in human chromosome 12q13 region. Two WNT gene clusters in human chromosome 2q35 and 12q13 regions might be generated due to duplication of ancestral gene cluster. The 3.0- and 2.4-kb WNT10A mRNAs were expressed in fetal kidney, placenta, adult spleen and kidney. The 2.0-kb WNT6 mRNA was coexpressed with WNT10A in placenta and adult spleen. WNT10A and WNT6 were strongly coexpressed in SW480 (colorectal cancer). In addition to SW480, WNT10A was strongly expressed in HL-60 (promyelocytic leukemia) and Raji (Burkitt's lymphoma), and WNT6 in HeLa S3 (cervical cancer). Overexpression WNT10A and WNT6 might play key roles in human carcinogenesis through activation of WNT-beta-catenin-TCF signaling pathway, just like Wnt10b and Wnt1.
WNT6 FOXC1 True Positive 15505035 Tamimi Y, Lines M, Coca-Prados M, Walter MA: Identification of target genes regulated by FOXC1 using nickel agarose-based chromatin enrichment. Invest Ophthalmol Vis Sci. 2004 Nov;45(11):3904-13. PURPOSE: To overcome the problem of antibody availability, often encountered during chromatin immunoprecipitation (ChIP) assays, nickel agarose-based chromatin enrichment (NACE) was developed. Based on the affinity of (His)-6-tagged proteins for the nickel ion, this modified form of ChIP allows the isolation of chromatin in the absence of specific antibodies. METHODS: Nonpigmented ciliary epithelium cells were transfected with (His)-6-tagged FOXC1. FOXC1-enriched chromatin complexes were isolated by using the tight electrostatic interaction between histidine residues of the recombinant FOXC1 protein and nickel. One hundred fifty NACE-enriched clones were sequenced and subjected to in silico and biochemical analyses. RESULTS: Twenty-six clones were detected near known genes: Eight were near predicted but uncharacterized genes, eight were within areas where neither known nor predicted genes have yet been mapped, four were chimeric, and the rest were either repetitive (n=81) or poor-quality (n=23) sequences. Twenty of the 26 known genes were expressed in the eye. Five of the NACE-enriched clones (BMP2K, DACH, FVT-1, SIX-1, and PGE-2 receptor), as well as nine clones selected from the literature, were validated by PCR amplification in two independent lots of NACE-enriched chromatin. All five NACE-selected genes were detected in two independent assays, as well as four (BMP7, SMAD2, TGF-B1, and WNT6) of the nine genes selected from the literature, consistent with these genes' being regulated by FOXC1. CONCLUSIONS: NACE is a useful technique allowing specific chromatin enrichment in cases where antibodies are unavailable. Specific recovery of PTGER, DACH1, WNT6, and FVT-1 implicates FOXC1 in a variety of cellular events including modulation of intraocular pressure, cell cycle, ocular development, and oncogenesis.