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• W1991330040 abstract "Itch is a ubiquitin ligase that has been implicated in the regulation of a number of cellular processes. We previously have identified Itch as a binding partner for the endocytic protein Endophilin and found it to be localized to endosomes. Using affinity purification coupled to mass spectrometry, we have now identified the ubiquitin-protease FAM/USP9X as a binding partner of Itch. The association between Itch and FAM/USP9X was confirmed in vitro by glutathione S-transferase pulldown and in vivo through coimmunoprecipation. Itch and FAM partially colocalize in COS-7 cells at the trans-Golgi network and in peripheral vesicles. We mapped the FAM-binding domain on Itch to the WW domains, a region known to be involved in substrate recognition. However, transient overexpression of FAM/USP9X resulted in the deubiquitylation of Itch. Moreover, we show that Itch auto-ubiquitylation leads to its degradation in the proteasome. By examining the amounts of Itch and FAM in various cell lines and rat tissues, a positive correlation was found in the expression of both proteins. This observation suggests that the levels of FAM expression could have an influence on Itch in cells. Experimental decrease in FAM levels by RNA interference leads to a significant reduction in intracellular levels of endogenous Itch, which can be prevented by treatment with the proteasome inhibitor lactacystin. Accordingly, overexpression of FAM/USP9X resulted in a marked increase in endogenous Itch levels. These results demonstrate an intriguing interplay between a ubiquitin ligase and a ubiquitin protease, based on direct interaction between the two proteins. Itch is a ubiquitin ligase that has been implicated in the regulation of a number of cellular processes. We previously have identified Itch as a binding partner for the endocytic protein Endophilin and found it to be localized to endosomes. Using affinity purification coupled to mass spectrometry, we have now identified the ubiquitin-protease FAM/USP9X as a binding partner of Itch. The association between Itch and FAM/USP9X was confirmed in vitro by glutathione S-transferase pulldown and in vivo through coimmunoprecipation. Itch and FAM partially colocalize in COS-7 cells at the trans-Golgi network and in peripheral vesicles. We mapped the FAM-binding domain on Itch to the WW domains, a region known to be involved in substrate recognition. However, transient overexpression of FAM/USP9X resulted in the deubiquitylation of Itch. Moreover, we show that Itch auto-ubiquitylation leads to its degradation in the proteasome. By examining the amounts of Itch and FAM in various cell lines and rat tissues, a positive correlation was found in the expression of both proteins. This observation suggests that the levels of FAM expression could have an influence on Itch in cells. Experimental decrease in FAM levels by RNA interference leads to a significant reduction in intracellular levels of endogenous Itch, which can be prevented by treatment with the proteasome inhibitor lactacystin. Accordingly, overexpression of FAM/USP9X resulted in a marked increase in endogenous Itch levels. These results demonstrate an intriguing interplay between a ubiquitin ligase and a ubiquitin protease, based on direct interaction between the two proteins. The ubiquitin-mediated proteolytic pathway has been implicated in multiple cellular processes, including endocytosis, cell cycle regulation, transcriptional activation, and antigen presentation. Polyubiquitylated proteins are recognized and degraded by the proteasome, a multisubunit protein degradation complex (1Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2857) Google Scholar). Protein ubiquitylation also serves regulatory functions in the cell that do not involve proteasome-mediated degradation. For example ligand-induced ubiquitylation of the Ste2 receptor in yeast triggers receptor endocytosis and targeting to vacuoles (2Hicke L. Riezman H. Cell. 1996; 84: 277-287Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar). Similarly, ubiquitylation of the epidermal growth factor receptor (EGFR) 5The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; PRD, proline-rich domain; USP, ubiquitin-specific protease(s); siRNA, small interfering RNA; GFP, green fluorescent protein; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; MS/MS, mass spectrometry; CL, cell lysate(s); JNK, c-Jun N-terminal kinase; WT, wild type. 5The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; PRD, proline-rich domain; USP, ubiquitin-specific protease(s); siRNA, small interfering RNA; GFP, green fluorescent protein; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; MS/MS, mass spectrometry; CL, cell lysate(s); JNK, c-Jun N-terminal kinase; WT, wild type. triggers its degradation in lysosomes after trafficking in the endosomal compartments (3Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1004) Google Scholar). The ubiquitin ligase Itch belongs to the Nedd4/Rsp5p HECT domain-containing family of E3 ligases characterized by a number of regions that are involved in intracellular localization and protein-protein interactions, which include an N-terminal C2 domain, four WW domains, and a C-terminal HECT domain (Fig. 1A). The N-terminal Itch C2 domain is responsible for its intracellular localization to endosomes (4Angers A. Ramjaun A.R. McPherson P.S. J. Biol. Chem. 2004; 279: 11471-11479Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar); the WW domains are responsible for the recognition of several substrate proteins (e.g. Refs. 5Chang L. Kamata H. Solinas G. Luo J.L. Maeda S. Venuprasad K. Liu Y.C. Karin M. Cell. 2006; 124: 601-613Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar, 12Qiu L. Joazeiro C. Fang N. Wang H.Y. Elly C. Altman Y. Fang D. Hunter T. Liu Y.C. J. Biol. Chem. 2000; 275: 35734-35737Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar); the C-terminal HECT domain harbors the ligase activity of the enzyme (13Liu H. Chen B. Xiong H. Huang Q.H. Zhang Q.H. Wang Z.G. Li B.L. Chen Z. Chen S.J. Oncogene. 2004; 23: 3385-3394Crossref PubMed Scopus (29) Google Scholar) (Fig. 1A). Previously we have demonstrated that Endophilin A1, an Src homology 3 (SH3) domain-containing protein that functions in clathrin-mediated endocytosis is a substrate for Itch. Interestingly, Itch does not interact with Endophilin via its WW domains; rather through a short 18-amino acid proline-rich domain (PRD) (4Angers A. Ramjaun A.R. McPherson P.S. J. Biol. Chem. 2004; 279: 11471-11479Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Because of the prominent role of ubiquitylation in endocytosis and our observation that both Itch and Endophilin are highly expressed in the brain, we performed pulldown experiments from rat brain extracts to identify novel substrates of Itch. Interestingly, one of the protein bands isolated was identified as the ubiquitin specific protease FAM, also known as USP9X. Ubiquitin proteases, or deubiquitylating enzymes, form a large class of proteins whose activity is to mediate the disassembly of ubiquitin-protein conjugates. Based on sequence motif similarity, more than 90 deubiquitylating enzymes have been identified in the human genome and were originally classified into two main families: the ubiquitin C-terminal hydrolases and ubiquitin-specific proteases (USP) (14Baek K.H. Exp. Mol. Med. 2003; 35: 1-7Crossref PubMed Scopus (51) Google Scholar). Proteases belonging to the C-terminal hydrolase family are generally small (20-30 kDa) and are associated with the proteasomes. They are involved in the generation/recycling of ubiquitin monomers from polyubiquitin chains by removing it from proteins degraded in proteasomes (15Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-223Crossref PubMed Scopus (775) Google Scholar). Members of the USP family, which includes FAM/USP9X, are large (60-300 kDa) and are able to hydrolyze ubiquitin from ubiquitylated substrates prior to proteasomal entry, thus extending the half-life of the protein and therefore modulate their activities in the cell. This type demonstrates tissue specific pattern of expression and has been shown to exhibit substrate specificity. USP9X/FAM is the mammalian counterpart of the product of the Drosophila fat-facets gene (faf) that is essential for normal eye development and viability of the early embryo (16Fischer-Vize J.A. Rubin G.M. Lehmann R. Development (Camb.). 1992; 116: 985-1000Crossref PubMed Google Scholar). Genetic experiments have identified the Liquid facets protein (Lqf) as one critical substrate of Faf in the eye (17Cadavid A.L. Ginzel A. Fischer J.A. Development (Camb.). 2000; 127: 1727-1736Crossref PubMed Google Scholar). In mammalian cells, a few substrates have been identified for USP9X/FAM. It binds to and stabilizes β-catenin and AF-6, important components of cell-cell adhesions and members of a transcription factor complex. Another substrate for FAM/USP9X is the vertebrate homologue of Lqf, Epsin1 (17Cadavid A.L. Ginzel A. Fischer J.A. Development (Camb.). 2000; 127: 1727-1736Crossref PubMed Google Scholar, 19Chen H. Polo S. Di Fiore P.P. De Camilli P.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14908-14913Crossref PubMed Scopus (108) Google Scholar). Epsin 1, a major binding partner for Eps15 (EGFR-phosphorylated protein 15), functions as an important adaptor in clathrin coat assembly and may have additional roles in growth factor receptor signaling and transcription (20De Camilli P. Chen H. Hyman J. Panepucci E. Bateman A. Brunger A.T. FEBS Lett. 2002; 513: 11-18Crossref PubMed Scopus (120) Google Scholar). In this report, we characterize the interaction between Itch and FAM/Usp9X. Similar to other HECT domain ubiquitin ligases, Itch activity leads to its auto-catalytic ubiquitylation (21Gao M. Labuda T. Xia Y. Gallagher E. Fang D. Liu Y.C. Karin M. Science. 2004; 306: 271-275Crossref PubMed Scopus (332) Google Scholar, 23Verdecia M.A. Joazeiro C.A. Wells N.J. Ferrer J.L. Bowman M.E. Hunter T. Noel J.P. Mol. Cell. 2003; 11: 249-259Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). We show that this auto-ubiquitylation activity leads to proteasomal degradation of Itch. Interaction between Itch and FAM reverses Itch auto-ubiquitylation and protects the ligase from proteasomal degradation. Consistent with this observation, experimental manipulation of FAM levels by overexpression or siRNA-induced down-regulation directly impact on endogenous Itch levels. Accordingly, the expression levels of both proteins are correlated in a variety of tissues and cell lines. Together, these results suggest that FAM directly regulates the level of expression of Itch, which suggests that Itch-dependent cellular processes would also be dependent on FAM expression. cDNA Constructs and Mutagenesis—Full-length Itch WT, Itch ΔC2, Itch ΔC2/ΔPRD, Itch C2, Itch PRD, and ligasedead Itch CA constructs were described in Angers et al. (4Angers A. Ramjaun A.R. McPherson P.S. J. Biol. Chem. 2004; 279: 11471-11479Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Other Itch cDNA constructs were obtained by PCR using Vent DNA polymerase (New England Biolabs) and the following primers: Itch HECT, 5′-GAGAATCGATAAGTCGACCCTATGTTCGGGACTTCAAAG-3′ (forward) and 5′-GAGAGGTACCGCGGCCGCTTACTCTTGTCCAAATCCTTCTG-3′ (reverse); Itch ΔHECT, 5′-GAGAATCGATAAGTCGACCAATGGGTAGCCTCACCATG-3′ (forward) and 5′-GAGAGGTACCGCGGCCGCTTACCGAACATAGGCTATCTGAG-3′ (reverse); Itch WW, 5′-GAGAATCGATAAGTCGACTCAATGGTTCACCATCTGCC-3′ (forward) and 5′-GAGAGGTACCGCGGCCGCTTACCGAACATAGGCTATCTGAG-3′ (reverse); Itch C2/PRD, 5′-GAGAATCGATAAGTCGACCAATGGGTAGCCTCACCATG-3′ (forward) and 5′-GAGAGGTACCGCGGCCGCTTAGGCAGATGGTGAACCATTG-3′ (reverse); Itch PRD/WW, 5′-GAGAATCGATAAGTCGACCTGAAGATGCAGGAGCTG-3′ (forward) and 5′-GAGAGGTACCGCGGCCGCTTACCGAACATAGGCTATCTGAG-3′ (reverse). The resulting PCR products were subcloned into pGEX-4T-1 (Amersham Biosciences) to add a GST tag. Itch ΔWW was produced by deleting the 5′ end of Itch full-length in pGEX-4T-1 with EcoRI digestion and subcloning in its place the PCR product obtained from primers forward 5′-GAGAGAATTCTCAATGGGTAGCCTCACCATG-3′, reverse 5′-GAGAGAATTCTAGGGCCTGAGCCTCCAG-3′. His6-Myc-ubiquitin was described previously (24Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1123) Google Scholar). hemagglutinin-ubiquitin expression vector was a kind gift from Ted Dawson (John Hopkins University) and FAM siRNA vectors were a kind gift from René Bernards (Netherlands Cancer Institute) (25Dirac A.M.G. Nijman S.M.B. Brummelkamp T.R. Bernards R. Raymond J.D. Methods in Enzymology. 2005; 398: 554-567Crossref PubMed Scopus (7) Google Scholar). All GST fusion proteins were expressed in DH5α and BL21 strains of Escherichia coli and purified with glutathione-Sepharose beads according to the manufacturer's instructions (Amersham Biosciences). Antibodies—Monoclonal antibodies against the FLAG epitope and β-actin were purchased from Sigma. Monoclonal antibody against Myc and hemagglutinin epitopes were obtained from Santa Cruz Biotechnology and US Biologicals, respectively. Polyclonal antibody against GFP was purchased from Molecular Probes and Pan-Endophilin polyclonal antibody was purchased from Zymed Laboratories Inc.. Affinity-purified polyclonal antibodies against GST (26McPherson P.S. Czernik A.J. Chilcote T.J. Onofri F. Benfenati F. Greengard P. Schlessinger J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6486-6490Crossref PubMed Scopus (148) Google Scholar) and FAM (27Murray R.Z. Jolly L.A. Wood S.A. Mol. Biol. Cell. 2004; 15: 1591-1599Crossref PubMed Scopus (68) Google Scholar) were described previously. Immunofluorescence Analysis—COS-7 cells were plated on poly-l-lysine-coated coverslips and transfected using FuGENE reagent (Roche Diagnostics). Approximately 15 h post-transfection, cells were fixed with 2% paraformaldehyde in phosphate-buffered saline (20 mm NaH2PO4, 0.9% NaCl, pH 7.4) for 20 min, permeabilized with 0.2% Triton X-100/phosphate-buffered saline for 4 min and processed with appropriate primary and secondary antibodies. Immunoprecipitation and Pulldown Assays—For cultured cells, 100-mm dishes of transfected HEK-293T cells were washed in phosphate-buffered saline and resuspended in 1 ml/dish buffer A (20 mm HEPES, pH 7.4, 150 mm NaCl, protease inhibitors). The cells were sonicated and Triton X-100 was added to a final concentration of 1%. Extracts were incubated for 20 min at 4 °C and centrifuged at 13,000 rpm in a microcentrifuge. For brain extracts, adult rat brains were homogenized in buffer A and centrifuged at 1,000 × g for 10 min. Triton X-100 was added to the supernatant to a final concentration of 1%, and the extract was incubated for 30 min at 4 °C. The sample was then centrifuged at 150,000 × g for 30 min. For immunoprecipitation assays, extracts of transfected cells were precleared with either protein A-Sepharose or protein G-agarose beads. The precleared supernatants were then incubated with the indicated antibodies and protein A-Sepharose or protein G-agarose beads for 4-16 h at 4 °C. Beads were washed extensively with buffer A, 1% Triton X-100 and prepared for Western blot analysis. For pulldown assays, extracts were incubated with 10 μg of the appropriate GST fusion protein for 4-16 h at 4 °C. Beads were washed extensively in the same buffer and prepared for Western blot analysis. In Vitro Ubiquitylation Assays—Recombinant ubiquitin was purchased from Sigma, and ubiquitin-activating enzyme (E1) and UbcH7 (E2) were purchased from Affinity Bioreagents. Full-length Itch CA proteins were produced as GST fusions and eluted from glutathione beads by thrombin cleavage. Ubiquitylation reactions (25 μl) contained 10 μg of Itch CA, ubiquitin (25 μm), ubiquitin-activating enzyme E1 (100 nm), ubiquitin-conjugating enzyme UbcH7 E2 (0.5 μm), and 10 μg of total proteins extracted from HEK-293T cells, or the same extract immunodepleted for Itch, and ATP (4 mm) in 50 mm Tris-HCl, pH 7.5, 2.5 mm MgCl2, and 1 mm dithiothreitol. The reactions were incubated for 90 min at 25 °C and stopped by addition of SDS-PAGE loading buffer and boiling. The reaction mixtures were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to Western blot. Identification of FAM/USP9X as a Novel Itch-interacting Partner—To detect Itch binding patners, GST and GST-Itch fusion proteins precoupled to glutathione-Sepharose were then incubated with rat brain extract. Affinity-purified complexes were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue G (Fig. 1B). Several protein bands that specifically associated with GST-Itch were identified and cut out of the gel. Each band was individually processed to yield in-gel trypsin digestion products and the resulting peptide products were separated and analyzed in an automated system by nanoscale liquid chromatography quadrupole time-of-flight MS/MS as described in Ref. 28Blondeau F. Ritter B. Allaire P.D. Wasiak S. Girard M. Hussain N.K. Angers A. Legendre-Guillemin V. Roy L. Boismenu D. Kearney R.E. Bell A.W. Bergeron J.J. McPherson P.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3833-3838Crossref PubMed Scopus (244) Google Scholar. Spectra were analyzed by MASCOT software to identify tryptic peptide sequences matching to the National Center for Biotechnology Information of non-redundant protein data base with a confidence level of 95% or greater (29Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6661) Google Scholar). Six of the eight bands isolated were heavily contaminated with GST and/or Itch peptides, which prevented the identification of potential Itch interacting partners present within that region of the gel. One of the remaining bands, positioned at ∼40 kDa in the gel, contained 12 peptides assigned to Endophilin. Itch was identified as an Endophilin binding partner in earlier work (4Angers A. Ramjaun A.R. McPherson P.S. J. Biol. Chem. 2004; 279: 11471-11479Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The other band was positioned well above the 200 kDa marker. MS/MS analysis revealed 28 peptide matches to the ubiquitin specific protease USP9X/FAM (Fig. 1B). The calculated molecular mass of USP9X/FAM is ∼290 kDa. FAM/USP9X Directly Forms Complexes with Itch—FAM/USP9X is a large protein and the conserved enzymatically active site represents only a small fraction of its size. It is thus likely that the protease exhibits a complex structure, possibly with multiple specific binding domains to recognize various substrates (30Wood S.A. Pascoe W.S. Ru K. Yamada T. Hirchenhain J. Kemler R. Mattick J.S. Mech. Dev. 1997; 63: 29-38Crossref PubMed Scopus (73) Google Scholar, 31Chen X. Overstreet E. Wood S.A. Fischer J.A. Dev. Genes Evol. 2000; 210: 603-610Crossref PubMed Scopus (30) Google Scholar). To verify that FAM/USP9X and Itch form complexes in vivo at physiological concentrations, we performed coimmunoprecipitation experiments of endogenous proteins in cell lysates from HEK-293T cells. HEK-293T cells were selected because they express detectable levels of both Itch and FAM/USP9X (Fig. 6B). Immunoprecipitation of endogenous Itch coimmunoprecipitated substantial amounts of FAM/USP9X (Fig. 2A). Endophilin was also coimmunoprecipitated. Hence endogenous Itch is capable of interacting with both FAM/USP9X and Endophilin. Since it is known that Endophilin directly interacts with Itch, we next investigated whether coimmunoprecipitation of Itch with Endophilin would also pull down FAM/UXP9X. Indeed, immunoprecipitation of endogenous Endophilin resulted in the coimmunoprecipitation of both FAM/USP9X and Itch (Fig. 2A). Taken together, these results suggest that Itch can interact with both Endophilin and FAM/USP9X, in the same ternary complex in vivo.FIGURE 2Itch forms complexes with FAM/USP9X. A, coimmunoprecipitation (IP) of endogenous Itch (left panel) or Endophilin (right panel) proteins. CL from HEK-293T cells were immunoprecipated with antibodies against either Itch or Endophilin. CL (100 μg) and immunoprecipated complexes were separated on a 3-12% SDS-PAGE. The gel was cut horizontally and Western blotted with the FAM/USP9X antibody (top), the Itch antibody (middle), and the Endophilin antibody (bottom). As a control (C3), protein A-Sepharose beads were incubated with the CL in the absence of either antibody. IB, immunoblotted.B, overlay assay using GST, GST-Itch, or GST-Endophilin were performed on extracts from HEK-293T cells. 100 μg of CL from HEK-293T cells was separated on SDS-PAGE. The membrane corresponding to each lane was cut vertically, incubated with 50 pmol/ml GST, GST-Itch, or GST-Endophilin separately, and Western blotted with antibodies to GST. The first lane shows control blots with the indicated antibodies. C, GFP-Itch was transfected into COS-7 cells, and its localization was compared with that of endogenous FAM/USP9X. The colocalization of Itch (green) with FAM/USP9X (red) is revealed in the blended image (right panel). Open arrows point to the trans-Golgi network region where most colocalization was observed. The closed arrows point to vesicular compartments where both proteins are also present. n = nucleus. Scale bar represents 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next performed overlay experiments to determine whether the interaction between Itch and FAM/USP9X is direct. In these assays, extracts from HEK-293T cells were resolved by SDS-PAGE, transferred to nitrocellulose, and incubated with purified GST, GST-Itch, or GST-Endophilin. Bound proteins were subsequently detected with an anti-GST antibody. Significant binding of Itch to FAM/USP9X was observed. Additionally, Itch bound directly to Endophilin (Fig. 2B). On the other hand, Endophilin did not directly interact with FAM/USP9X under these conditions but with several other proteins migrating around 100 kDa, most likely corresponding to Itch, GLK, and Dynamin, proteins previously demonstrated to interact with Endophilin (Fig. 2B) (4Angers A. Ramjaun A.R. McPherson P.S. J. Biol. Chem. 2004; 279: 11471-11479Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 32Ramjaun A.R. Angers A. Legendre-Guillemin V. Tong X.K. McPherson P.S. J. Biol. Chem. 2001; 276: 28913-28919Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 33Bell A.W. de Heuvel E. Ramjaun A.R. Wong K. Sossin W.S. McPherson P.S. J. Biol. Chem. 1997; 272: 8710-8716Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). No binding was detected when resolved HEK-293T proteins were incubated with GST alone. Thus, the interaction between Itch and FAM can occur in the absence of an intermediate. However, results from the coimmunoprecipitation and overlay assays indicate that the association between FAM/USP9X and Endophilin can be explained via an indirect interaction, most likely mediated by Itch. These data also imply that FAM/USP9X exhibits specificity in its direct interaction with select substrates. To further confirm the interaction between the proteins, we investigated whether FAM/USP9X and Itch are localized to similar subcellular compartments. The subcellular localization of Itch and FAM/USP9X have been previously explored separately. In COS-7 cells Itch expression was found in the trans-Golgi network and endosomal compartments (4Angers A. Ramjaun A.R. McPherson P.S. J. Biol. Chem. 2004; 279: 11471-11479Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The perinuclear pool overlapped with markers of the Golgi with no significant overlap with those of the endoplasmic reticulum. Similar to Itch localization, FAM/USP9X staining was associated with the Golgi, multive-sicular bodies, and lysosomes. Little, if any, colocalization was found with markers of the early endosomes or the endoplasmic reticulum (27Murray R.Z. Jolly L.A. Wood S.A. Mol. Biol. Cell. 2004; 15: 1591-1599Crossref PubMed Scopus (68) Google Scholar). To explore this further, we transiently transfected COS-7 cells with GFP-tagged Itch-full length and performed immunofluorescent detection of endogenous FAM/USP9X using a specific FAM polyclonal antibody (27Murray R.Z. Jolly L.A. Wood S.A. Mol. Biol. Cell. 2004; 15: 1591-1599Crossref PubMed Scopus (68) Google Scholar) (Fig. 2C). Similar to previous reports, FAM staining was visualized predominantly in a perinuclear compartment and exhibited punctate staining that extended throughout the cell. Similarly, the expression of Itch was predominantly detected as punctate structures in the cytoplasm and around the nucleus, consistent with our previous observations. The proteins overlap in a perinuclear compartment most likely corresponding to the Golgi apparatus. The extent of colocalization is less apparent in more peripheral vesicular structures, suggesting that only a subset pool of both proteins are colocalized in vivo. FAM/USP9X Interacts with Itch through the WW Domain—HECT domain ubibiquitin ligases of the Nedd4 family generally interact with their substrates through the WW domains, with recognition preference for the motif PPXY(PY) (34Otte L. Wiedemann U. Schlegel B. Pires J.R. Beyermann M. Schmieder P. Krause G. Volkmer-Engert R. Schneider-Mergener J. Oschkinat H. Protein Sci. 2003; 12: 491-500Crossref PubMed Scopus (104) Google Scholar). Additionally, in earlier work we have shown that another domain on Itch, the PRD, is also involved in protein interaction, specifically to Endophilin (4Angers A. Ramjaun A.R. McPherson P.S. J. Biol. Chem. 2004; 279: 11471-11479Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Hence, we sought to map the specific region on Itch involved in binding to FAM/USP9X. A number of N- and C-terminal truncations were engineered by successively deleting each characterized domain separately or in combination (Fig. 3A). Rat brain extracts were mixed with the constructs expressed as GST fusion proteins precoupled to glutathione-Sepharose. Bound proteins were then resolved on SDS-PAGE and blotted with a polyclonal antibody specific to FAM/USP9X. As a control for Itch interaction, we also blotted for Endophilin. All GST-Itch fusion proteins containing the WW domains were able to pull down FAM/USP9X from the extract. Conversely those lacking the WW domains were unable to bind FAM/USP9X (Fig. 3B). Deletion of the WW domains alone completely abolished the ability of Itch to bind to FAM/USP9X (Fig. 3B). Similar to previous reports, Itch required the PRD domain to recognize Endophilin (Fig. 3B). Additionally, FAM/USP9X did not bind to control GST (Fig. 3B) or GST-Endophilin (data not shown). These results demonstrate that FAM/USP9X is recognized by the WW domains of Itch. FAM/USP9X Can Deubiquitylate Itch—FAM/USP9X is a member of the USP family of deubiquitylating enzymes characterized by a number of conserved regions including the Cys and His boxes in the catalytic core of the enzyme. The protease has been shown to deubiquitylate a number of target proteins such as Epsin and AF-6. On the other hand, Itch itself ubiquitylates various substrates. By virtue of the direct interaction between Itch and FAM/USP9X, we reasoned that each might be a substrate for the other, that is that FAM/USP9X could deubiquitylate Itch, and Itch could ubiquitylate FAM/USP9X. Since Itch can undergo autocatalytic ubiquitylation (see below), HEK-293T cells were cotransfected with plasmids expressing Myc-ubiquitin and FLAG-Itch with or without V5-FAM. Forty-eight hours post-transfection, the cells were lysed, and ubiquitylated proteins were immunoprecipitated with an anti-Myc antibody. Proteins were then separated by SDS-PAGE, transferred to nitrocellulose, and blotted with anti-FLAG or anti-V5 antibody to assess the presence of Itch or FAM, respectively, in the ubiquitylated protein pool. When cells were transfected with FLAG-Itch and Myc-ubiquitin, ubiquitylated forms of Itch could be detected by the FLAG antibody in the immunoprecipitated fraction (Fig. 4A, lower panel). In the presence of FAM/USP9X, all ubiquitylated FLAG-Itch was lost. Interestingly, ubiquitylated FAM could not be detected in the cells coexpressing FLAG-Itch and V5-FAM, suggesting that Itch might not ubiquitylate FAM (Fig. 5A, upper panel). FLAG Western blots of cell lysates (CL) revealed equal expression of FLAG-Itch in all conditions. To further explore FAM-induced deubiquitylation of Itch, HEK-293T cells transfected with FLAG-Itch and Myc-ubiquitin and with or without V5-FAM were immunoprecipitated with anti-FLAG antibody, and immunoprecipated proteins were blotted with monoclonal anti-Myc antibody to detect ubiquitylation. A significant decrease in Itch ubiquitylation was observed upon FAM/USP9X overexpression represented by a reduction in the number of Itch migrating species resulting from ubiquitin modifications of this protein (Fig. 4B). Together these results demonstrate that FAM/USP9X is capable of cleaving ubiquitin chains from the ligase Itch.FIGURE 5Itch is ubiquitylated by itself and degraded in the proteasome. A, HEK-293T cells were transfected with Myc-ubiquitin and FLAG-Itch WT or the ligase dead mutant FLAG-Itch CA. 48 h post-transfection, cell-lysates were obtained and immunoprecipitated (IP) with either anti-FLAG or anti-Myc antibodies as indicated and immunoblotted with either anti-FLAG or anti-Myc antibodies" @default.
• W1991330040 created "2016-06-24" @default.
• W1991330040 creator A5012036880 @default.
• W1991330040 creator A5020832911 @default.
• W1991330040 creator A5033326808 @default.
• W1991330040 creator A5051152499 @default.
• W1991330040 creator A5071387334 @default.
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• W1991330040 date "2006-12-01" @default.
• W1991330040 modified "2023-10-12" @default.
• W1991330040 title "The Ubiquitin Ligase Itch Is Auto-ubiquitylated in Vivo and in Vitro but Is Protected from Degradation by Interacting with the Deubiquitylating Enzyme FAM/USP9X" @default.
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