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

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 of the PubMed Google Scholar). experiments have identified the protein as one substrate of in the eye A. 2000; PubMed Google Scholar). mammalian a substrates have been identified for to and and of and of a factor substrate for FAM/USP9X is the of A. 2000; PubMed Google Scholar, H. S. S. A. 2003; PubMed Scopus Google Scholar). a binding partner for protein functions as an in and have in growth factor receptor and Chen H. J. A. 2002; PubMed Scopus Google Scholar). we the interaction between Itch and to HECT domain ubiquitin Itch activity leads to its ubiquitylation M. T. Y. Fang D. Liu Y.C. Karin M. 2004; PubMed Scopus Google Scholar, Joazeiro J.L. Hunter T. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). We show that auto-ubiquitylation activity leads to proteasomal degradation of Itch. between Itch and FAM Itch auto-ubiquitylation and the ligase from proteasomal degradation. with of FAM levels by overexpression or on endogenous Itch levels. Accordingly, the expression levels of both proteins are in a of and cell results that FAM the of expression of Itch, which suggests that cellular also be on FAM and Itch WT, Itch Itch Itch Itch PRD, and Itch were in (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). Itch were by and the Itch and Itch and Itch and Itch and Itch and The were into to a Itch was by the of Itch in with and in its the product from was previously S. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). expression was a from and FAM were a from in PubMed Scopus Google Scholar). proteins were expressed in and of and with to the the and were from and were from and was from and was from P.S. J. S. A. PubMed Scopus Google Scholar) and FAM Mol. Biol. Cell. 2004; PubMed Scopus Google Scholar) were cells were on and cells were with in for with for and with and and of cells were in and in protease The cells were and was to a of were for at and at in a For brain rat were in and at for was to the to a of and the was for at The was at for For extracts of cells were with protein or protein The were with the and protein or protein for at were with and for For pulldown extracts were with of the protein for at were in the and for ubiquitin was from and ubiquitin-activating enzyme and were from Itch proteins were as and from glutathione by of Itch ubiquitin ubiquitin-activating enzyme ubiquitin-conjugating enzyme and of proteins from or the for Itch, and in and The were for 90 at and by of and The were by to and to of FAM/USP9X as a Itch binding and proteins to were with rat brain were by and by (Fig. protein bands that associated with were identified and of the was to and the were and in an by as in B. S. M. A. L. D. McPherson P.S. S. A. 2004; PubMed Scopus Google were by to identify to the for of protein with a of or D.J. PubMed Scopus Google Scholar). of the bands isolated were with Itch which prevented the of Itch that region of the of the at in the to Itch was identified as an Endophilin binding partner in (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 was the to the ubiquitin specific protease USP9X/FAM (Fig. The mass of USP9X/FAM is FAM/USP9X with is a large protein and the a small of its is thus that the protease a complex with multiple specific binding domains to various substrates K. T. J. PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar). that FAM/USP9X and Itch form in vivo at we performed experiments of endogenous proteins in cell from cells. cells were levels of both Itch and FAM/USP9X (Fig. of endogenous Itch amounts of FAM/USP9X (Fig. Endophilin was also endogenous Itch is of with both FAM/USP9X and it is known that Endophilin with Itch, we of Itch with Endophilin also of endogenous Endophilin resulted in the of both FAM/USP9X and Itch (Fig. results that Itch can interact with both Endophilin and FAM/USP9X, in the complex in with of endogenous Itch or Endophilin proteins. from cells were with Itch or and were on a The was and with the FAM/USP9X the Itch and the Endophilin a protein were with the in the of or were performed on extracts from cells. of from cells was on The to was with or and with to The with the was into COS-7 and its localization was with that of endogenous The of Itch with FAM/USP9X is in the to the trans-Golgi network region was The to compartments both proteins are also We performed experiments to the interaction between Itch and FAM/USP9X is extracts from cells were by to and with or proteins were with an binding of Itch to FAM/USP9X was Itch to Endophilin (Fig. the Endophilin not interact with FAM/USP9X with several proteins to Itch, and proteins previously demonstrated to interact with Endophilin (Fig. (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, A.R. A. McPherson P.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Ramjaun A.R. K. McPherson P.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). binding was proteins were with the interaction between Itch and FAM can in the of an However, results from the and that the association between FAM/USP9X and Endophilin can be via an by Itch. These also that FAM/USP9X in its direct interaction with the interaction between the we FAM/USP9X and Itch are localized to The localization of Itch and FAM/USP9X have been previously 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 with of the with significant with of the to Itch FAM/USP9X was associated with the and was found with of the endosomes or the Mol. Biol. Cell. 2004; PubMed Scopus Google Scholar). we COS-7 cells with and performed of endogenous FAM/USP9X a specific FAM Mol. Biol. Cell. 2004; PubMed Scopus Google Scholar) (Fig. to FAM was in a and that the cell. Similarly, the expression of Itch was as in the and the with our The proteins in a to the The of is in more peripheral that a of both proteins are in FAM/USP9X with Itch through the WW domain ligases of the family generally interact with their substrates through the WW domains, with recognition for the motif L. B. M. G. J. H. Protein 2003; PubMed Scopus Google Scholar). in we have shown that domain on Itch, the PRD, is also involved in protein 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). we to the specific region on Itch involved in binding to number of and C-terminal were by characterized domain or in (Fig. brain extracts were with the expressed as proteins to proteins were on and with a specific to a for Itch we also for proteins the WW domains were able to FAM/USP9X from the the WW domains were to FAM/USP9X (Fig. of the WW domains the of Itch to to FAM/USP9X (Fig. to Itch the domain to Endophilin (Fig. FAM/USP9X not to (Fig. or not These results demonstrate that FAM/USP9X is recognized by the WW domains of Itch. FAM/USP9X is a of the USP family of deubiquitylating enzymes characterized by a number of regions including the and in the of the The protease has been shown to a number of proteins as and the Itch various By of the direct interaction between Itch and FAM/USP9X, we that be a substrate for the that is that FAM/USP9X could Itch, and Itch could Itch can ubiquitylation cells were with and with or the cells were and ubiquitylated proteins were with an were by to and with or to the of Itch or FAM, in the ubiquitylated protein cells were with and ubiquitylated of Itch could be by the in the (Fig. the of FAM/USP9X, ubiquitylated was Interestingly, ubiquitylated FAM could not be in the cells and that Itch not FAM (Fig. of cell expression of in deubiquitylation of Itch, cells with and and with or were with and proteins were with to significant decrease in Itch ubiquitylation was FAM/USP9X overexpression by a reduction in the number of Itch from ubiquitin of protein (Fig. results demonstrate that FAM/USP9X is of ubiquitin chains from the ligase is ubiquitylated by and degraded in the proteasome. cells were with and or the ligase were and with or as and with or as Itch was a of ubiquitylated Itch could be to Itch could be ubiquitylated by in a we Itch from a protein expressed in with E1, E2, and in the of cell or for Itch at for 90 The was for with ubiquitylated Itch is The the and of Itch in and cell cells with and were with for or in were with and with to show ubiquitylation of to show cells were for with or in cell were for with and to show the of Itch and in the an interaction between Itch and FAM/USP9X, we to the of interaction at the cellular the association between ubiquitin ligases and ubiquitin proteases has been shown to the which auto-ubiquitylation by proteasome degradation L. L. C. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar, C. M. A. J. PubMed Scopus Google Scholar). This is with our observation that FAM/USP9X Itch. of an observation we the of Itch to we and in cells and to with with Itch can be as the of bands and a Itch the ubiquitin ligase activity (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) is also ubiquitylated (Fig. of the is that the is that in the of ligase activity is the endogenous This the that Itch be the substrate of ubiquitin ligases or that it is ubiquitylated in by endogenous Itch in cells. we performed in vitro ubiquitylation as a protein in and from with Itch was with mammalian ubiquitin-activating enzyme and in the of cell as a of E3 the ligase form of Itch was ubiquitylated the ligase activity was by Itch we the cell with a Itch and used the as a of E3 ligase Itch was form the cell in ubiquitylation of Itch could be in (Fig. The the of the by the cell extracts with an serves as a for degradation. ubiquitylated Itch proteasomal we cells with and with the proteasome inhibitor lactacystin. The of ubiquitylated Itch from cells was with that ubiquitylated Itch is degraded in the proteasome (Fig. that endogenous Itch was also degraded in cells were with and in the or of the proteasome inhibitor lactacystin. Cell were to endogenous Itch of endogenous Itch in cells with lactacystin. to results from our overexpression endogenous Itch is degraded in a (Fig. of Itch and FAM/USP9X in and Cell Itch proteasomal degradation as a of its activity and that FAM can by deubiquitylating Itch, we that the of FAM be to levels of Itch We thus the amounts of both the protease and ligase by from rat tissue which the brain and a number of peripheral of FAM/USP9X was in the brain, and in the levels of the protease were in the and was in the and (Fig. Itch on the to be expressed in both the and peripheral However, as its levels were in in to FAM/USP9X to have a pattern of expression with the ligase levels of the ligase are associated with of the Similarly, endogenous Itch protein levels the amounts of FAM/USP9X in various cell which a more than tissue extracts (Fig. with FAM of the that FAM/USP9X Itch in we the of FAM/USP9X expression through experiments in cells. with various of two specific for FAM in PubMed Scopus Google Scholar) the levels of endogenous FAM/USP9X expression at and of (Fig. a with the a sequence not in of FAM/USP9X Accordingly, we a in endogenous Itch levels in cells with FAM/USP9X in (Fig. and The for our is that the decrease in Itch expression is to an increase in the ubiquitylated of the protein which as a for proteasomal degradation. that Itch is by FAM/USP9X in ubiquitylation of proteins is associated with their Nat. Rev. Mol. Cell. Biol. 2001; PubMed Scopus Google we the of proteasomal on Itch cells were with the two with the the cells were with or for the levels of Itch in the cell and proteasomal degradation (Fig. This is with that proteasome leads to the of ubiquitylated proteins A. S. C. J. S. Fang S. S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Liu Y.C. K.H. G. G. J. PubMed Scopus Google Scholar). of we that overexpression of FAM/USP9X in of endogenous Itch. cells were with with or Cell extracts were with and The of protease resulted in levels of Itch. These to be with the of FAM/USP9X expressed (Fig. These results that the of Itch is by the of in the cell. FAM/USP9X a role in Itch through has been previously that the c-Jun N-terminal can Itch and that is to an increase in Itch auto-ubiquitylation in cells M. T. Y. Fang D. Liu Y.C. Karin M. 2004; PubMed Scopus Google Scholar). was also shown by the that in the domain of Itch and on the of Itch to its proteins M. Liu Y.C. Karin M. S. A. 2006; PubMed Scopus Google Scholar). We therefore the of activity in the of Itch to to We in cells. of leads to its as shown by the of cell (Fig. is of endogenous Itch to FAM/USP9X (Fig. This is with the of Itch ubiquitylation in to by M. T. Y. Fang D. Liu Y.C. Karin M. 2004; PubMed Scopus Google Scholar). The ubiquitin ligase Itch in cellular by of its the we that Itch is a substrate for the protease proteins to and with the FAM/USP9X and Itch were shown to colocalize in compartments and to a in more peripheral a of interaction Itch is and from proteasomal degradation. to the interaction between FAM/USP9X and Itch is the of the in that the expression of the protease is the of intracellular Itch to degradation of the ligase in proteasomes. FAM/USP9X was the levels of Itch as we and have shown that of the to and ubiquitylation of Itch M. T. Y. Fang D. Liu Y.C. Karin M. 2004; PubMed Scopus Google Scholar, M. Liu Y.C. Karin M. S. A. 2006; PubMed Scopus Google Scholar). A. and A. we show that ubiquitylation of Itch leads to its proteasomal degradation and that degradation is in the of one role of FAM could be to the Itch ligase not at more after its in specific with levels of FAM activity could therefore to that Itch. By the endogenous levels of both Itch and FAM in various and cell it was that in was more FAM/USP9X, Itch levels were also have demonstrated a between FAM/USP9X and its the levels of the eye were in in a that the of protein A. 2000; PubMed Google Scholar, Zhang B. 2002; PubMed Scopus Google Scholar). The was gene was to Zhang B. 2002; PubMed Scopus Google Scholar). Similarly, the levels of FAM/USP9X substrates as and were also shown to be by FAM/USP9X in of protease overexpression Mol. Biol. 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Full Text Full Text PDF PubMed Scopus Google Scholar). from ubiquitylation and is one of receptor that have of whose are and thus to the EGF have been shown to have activity as a of EGF receptor and Accordingly, overexpression of leads to ubiquitylation and degradation of the EGF receptor A. S. C. J. S. Fang S. S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, K. 2002; PubMed Scopus Google Scholar, G. H. M. S. K. Y. A. S. Y. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The ubiquitin ligase Itch has been shown to to and in its degradation in proteasomes. Itch ubiquitylation and of the EGF receptor in an increase in EGF receptor levels A. S. C. J. S. Fang S. S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of complex is the protein Endophilin that at multiple in we have shown that Itch can also to and Endophilin (4Angers A. Ramjaun A.R. McPherson P.S. J. Biol. 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