AMSH Interacts with ESCRT-0 to Regulate the Stability and Trafficking of CXCR4
2010; Elsevier BV; Volume: 285; Issue: 18 Linguagem: Inglês
10.1074/jbc.m109.061309
ISSN1083-351X
AutoresMaria I. Sierra, Michelle H. Wright, Piers Nash,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoReversible ubiquitination is essential for the endocytic sorting and down-regulation of G protein-coupled receptors, such as the chemokine receptor CXCR4. The deubiquitinating enzyme AMSH has been implicated in the endocytic sorting of both G protein-coupled receptors and receptor-tyrosine kinases. Herein, we examine the role of AMSH in the regulation of CXCR4 stability and trafficking and characterize protein-protein interactions critical for this function. Loss of AMSH catalytic activity or depletion by RNAi results in increased steady-state levels of CXCR4 under basal conditions. Analysis of truncation and point mutation of AMSH reveal the importance of an RXXK motif for CXCR4 degradation. The RXXK motif of AMSH interacts with the SH3 domains of the STAM and Grb2 families of adaptor proteins with high affinity. Cells expressing a catalytically inactive mutant of AMSH show basal hyperubiquitination, but not increased degradation, of the ESCRT-0 components STAM1 and Hrs. This is dependent on the RXXK motif of AMSH. Ubiquitination of endocytic machinery modulates their activity, suggesting that AMSH may directly regulate endocytic adaptor protein function. This is reflected in CXCR4 trafficking and provides a mechanism by which AMSH specifies the fate of endocytosed receptors. Taken together, these studies implicate AMSH as a key modulator of receptor fate determination through its action on components of the endocytic machinery. Reversible ubiquitination is essential for the endocytic sorting and down-regulation of G protein-coupled receptors, such as the chemokine receptor CXCR4. The deubiquitinating enzyme AMSH has been implicated in the endocytic sorting of both G protein-coupled receptors and receptor-tyrosine kinases. Herein, we examine the role of AMSH in the regulation of CXCR4 stability and trafficking and characterize protein-protein interactions critical for this function. Loss of AMSH catalytic activity or depletion by RNAi results in increased steady-state levels of CXCR4 under basal conditions. Analysis of truncation and point mutation of AMSH reveal the importance of an RXXK motif for CXCR4 degradation. The RXXK motif of AMSH interacts with the SH3 domains of the STAM and Grb2 families of adaptor proteins with high affinity. Cells expressing a catalytically inactive mutant of AMSH show basal hyperubiquitination, but not increased degradation, of the ESCRT-0 components STAM1 and Hrs. This is dependent on the RXXK motif of AMSH. Ubiquitination of endocytic machinery modulates their activity, suggesting that AMSH may directly regulate endocytic adaptor protein function. This is reflected in CXCR4 trafficking and provides a mechanism by which AMSH specifies the fate of endocytosed receptors. Taken together, these studies implicate AMSH as a key modulator of receptor fate determination through its action on components of the endocytic machinery. IntroductionThe sorting and trafficking of cell surface receptors through endosomal compartments is a highly regulated process that is essential for maintaining cellular homeostasis and generating adaptive and coordinated responses to external stimuli. To avoid prolonged receptor activation and signaling, receptor-ligand complexes are endocytosed and either recycled back to the plasma membrane or sorted to lysosomes for degradation (1von Zastrow M. Life Sci. 2003; 74: 217-224Crossref PubMed Scopus (153) Google Scholar, 2Sorkin A. Von Zastrow M. Nat. Rev. Mol. Cell Biol. 2002; 3: 600-614Crossref PubMed Scopus (696) Google Scholar, 3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 4Tsao P. Cao T. von Zastrow M. Trends Pharmacol Sci. 2001; 22: 91-96Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). This process is mediated by reversible ubiquitination. 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Chem. 1999; 274: 19129-19135Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and later characterized as a Zn2+-dependent ubiquitin isopeptidase with a substrate preference for Lys-63-linked polyubiquitin chains (37McCullough J. Clague M.J. Urbé S. J. Cell Biol. 2004; 166: 487-492Crossref PubMed Scopus (302) Google Scholar, 45McCullough J. Row P.E. Lorenzo O. Doherty M. Beynon R. Clague M.J. Urbé S. Curr. Biol. 2006; 16: 160-165Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). AMSH is composed of multiple protein-protein interaction domains that have been shown to control its recruitment and localization (Fig. 3A). At the N terminus, AMSH contains a nuclear localization signal and a microtubule-interacting and transport (MIT) domain, shown to interact with several chromatin-modifying proteins (CHMPs) that are components of the endosomal sorting complex required for transport (ESCRT) III complex (38Kyuuma M. Kikuchi K. Kojima K. Sugawara Y. 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AMSH has a clathrin binding domain that interacts with the terminal domain of the clathrin heavy chain and is required for AMSH localization to endosomes (45McCullough J. Row P.E. Lorenzo O. Doherty M. Beynon R. Clague M.J. Urbé S. Curr. Biol. 2006; 16: 160-165Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 50Nakamura M. Tanaka N. Kitamura N. Komada M. Genes Cells. 2006; 11: 593-606Crossref PubMed Scopus (32) Google Scholar). AMSH contains a STAM-interacting motif (PPVVDRSLKPGA) that serves as a binding site for the SH3 domain of the STAM family of adaptor proteins (37McCullough J. Clague M.J. Urbé S. J. Cell Biol. 2004; 166: 487-492Crossref PubMed Scopus (302) Google Scholar, 44Tanaka N. Kaneko K. Asao H. Kasai H. Endo Y. Fujita T. Takeshita T. Sugamura K. J. Biol. Chem. 1999; 274: 19129-19135Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 51Kikuchi K. Ishii N. Asao H. Sugamura K. Biochem. Biophys. Res. Commun. 2003; 306: 637-643Crossref PubMed Scopus (25) Google Scholar, 52Kim M.S. Kim J.A. Song H.K. Jeon H. Biochem. Biophys. Res. Commun. 2006; 351: 612-618Crossref PubMed Scopus (24) Google Scholar). STAM SH3 domain binding to this region has been shown to stimulate the deubiquitinating activity of AMSH in vitro (45McCullough J. Row P.E. Lorenzo O. Doherty M. Beynon R. Clague M.J. Urbé S. Curr. Biol. 2006; 16: 160-165Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 52Kim M.S. Kim J.A. Song H.K. Jeon H. Biochem. Biophys. Res. Commun. 2006; 351: 612-618Crossref PubMed Scopus (24) Google Scholar); however, a clear role for this interaction in a cellular context had not been previously demonstrated.The substrate preferences of AMSH for Lys-63-linked polyubiquitin chains taken together with its known interaction partners implicate AMSH in the regulation of the ubiquitination status of proteins during endocytic trafficking. AMSH has been implicated in the endocytic trafficking of various receptors (37McCullough J. Clague M.J. Urbé S. J. Cell Biol. 2004; 166: 487-492Crossref PubMed Scopus (302) Google Scholar, 38Kyuuma M. Kikuchi K. Kojima K. Sugawara Y. Sato M. Mano N. Goto J. Takeshita T. Yamamoto A. Sugamura K. Tanaka N. Cell Struct. Funct. 2007; 31: 159-172Crossref PubMed Scopus (48) Google Scholar, 39Ma Y.M. Boucrot E. Villén J. Affar el B. Gygi S.P. Göttlinger H.G. Kirchhausen T. J. Biol. Chem. 2007; 282: 9805-9812Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 40Herrera-Vigenor F. Hernández-García R. Valadez-Sánchez M. Vázquez-Prado J. Reyes-Cruz G. Biochem. Biophys. Res. Commun. 2006; 347: 924-930Crossref PubMed Scopus (22) Google Scholar, 41Reyes-Ibarra A.P. García-Regalado A. Ramírez-Rangel I. Esparza-Silva A.L. Valadez-Sánchez M. Vázquez-Prado J. Reyes-Cruz G. Mol. Endocrinol. 2007; 21: 1394-1407Crossref PubMed Scopus (39) Google Scholar, 42Hislop J.N. Henry A.G. Marchese A. von Zastrow M. J. Biol. Chem. 2009; 284: 19361-19370Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar); however, a role for AMSH in the trafficking of CXCR4 has not been previously described. Endosomal sorting and degradation of CXCR4 are dependent on the adaptor protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), arrestin-2, and on the AAA-type ATPase Vps4 (24Marchese A. Raiborg C. Santini F. Keen J.H. Stenmark H. Benovic J.L. Dev. Cell. 2003; 5: 709-722Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 25Bhandari D. Trejo J. Benovic J.L. Marchese A. J. Biol. Chem. 2007; 282: 36971-36979Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Loss of both Hrs and arrestin-2 results in the accumulation of CXCR4 on enlarged early endosomes after activation with CXCL12. Vps4 has been implicated in the later-stage deubiquitination of CXCR4 and Hrs through what is assumed to be an indirect mechanism (24Marchese A. Raiborg C. Santini F. Keen J.H. Stenmark H. Benovic J.L. Dev. Cell. 2003; 5: 709-722Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 25Bhandari D. Trejo J. Benovic J.L. Marchese A. J. Biol. Chem. 2007; 282: 36971-36979Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar).Herein, we report a critical role for AMSH in the regulation of basal CXCR4 trafficking and steady-state homeostasis. We report that both AMSH depletion and overexpression of a catalytically inactive form of AMSH results in increased levels of CXCR4 protein. We identify conserved interactions between the RXXK motif in AMSH and the non-canonical SH3 domains of adaptor and scaffold proteins, including those of the STAM family with affinity approximating that of an optimal ligand. Recruitment of AMSH to the Hrs·STAM complex on sorting endosomes requires an intact RXXK, motif and our results suggest that AMSH functions at the sorting endosome to regulate the ubiquitination status of ESCRT-0 proteins. Because ubiquitination of endocytic machinery modulates their activity, we propose that AMSH acts to modulate ESCRT-0 function and subsequently affects the fate of endocytosed cargo as reflected in CXCR4 trafficking. Taken together, our studies on CXCR4 trafficking implicate AMSH as a key modulator of receptor fate through its action on the components of the endocytic machinery.DISCUSSIONReversible ubiquitination has been proposed as a key mechanism in regulating the trafficking of endocytic cargo (8Hanyaloglu A.C. von Zastrow M. Annu. Rev. Pharmacol. Toxicol. 2008; 48: 537-568Crossref PubMed Scopus (465) Google Scholar, 32Clague M.J. Urbé S. Trends Cell Biol. 2006; 16: 551-559Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 33Williams R.L. Urbé S. Nat. Rev. Mol. Cell Biol. 2007; 8: 355-368Crossref PubMed Scopus (551) Google Scholar, 34Komada M. Curr. Drug Discov. Technol. 2008; 5: 78-84Crossref PubMed Scopus (49) Google Scholar, 35Mukhopadhyay D. Riezman H. Science. 2007; 315: 201-205Crossref PubMed Scopus (938) Google Scholar, 64Komada M. Kitamura N. J Biochem. 2005; 137: 1-8Crossref PubMed Scopus (70) Google Scholar), yet relatively little is known regarding the deubiquitinating enzymes involved or the mechanisms governing this process. The current study provides further evidence for a global regulatory role of AMSH in the endocytosis of surface receptors through modulation of endocytic adaptor proteins and describes a specific role for AMSH in the stability and trafficking of the chemokine receptor CXCR4.We report that loss of AMSH catalytic activity by overexpression of a catalytic mutant results in a dramatic increase in CXCR4 basal levels and a decrease in CXCL12-mediated degradation (Fig. 1). AMSH depletion by RNAi-mediated knockdown also results in an increase in steady-state levels of CXCR4 (Fig. 2), further supporting a role for AMSH deubiquitinating activity in the regulation of receptor turnover. This is analogous to the general effect of AMSH on epidermal growth factor receptor, the δ-opioid receptor, and the protease-activated receptor 2 (37McCullough J. Clague M.J. Urbé S. J. Cell Biol. 2004; 166: 487-492Crossref PubMed Scopus (302) Google Scholar, 38Kyuuma M. Kikuchi K. Kojima K. Sugawara Y. Sato M. Mano N. Goto J. Takeshita T. Yamamoto A. Sugamura K. Tanaka N. Cell Struct. Funct. 2007; 31: 159-172Crossref PubMed Scopus (48) Google Scholar, 39Ma Y.M. Boucrot E. Villén J. Affar el B. Gygi S.P. Göttlinger H.G. Kirchhausen T. J. Biol. Chem. 2007; 282: 9805-9812Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 42Hislop J.N. Henry A.G. Marchese A. von Zastrow M. J. Biol. Chem. 2009; 284: 19361-19370Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Although CXCR4 trafficking and degradation after CXCL12 activation has been the focus of numerous studies, less is known regarding the constitutive or basal endocytosis of CXCR4. CXCR4 is known to be constitutively endocytosed and recycled to the plasma membrane in a ligand-independent manner (28Zhang Y. Foudi A. Geay J.F. Berthebaud M. Buet D. Jarrier P. Jalil A. Vainchenker W. Louache F. Stem Cells. 2004; 22: 1015-1029Crossref PubMed Scopus (92) Google Scholar). This process is clathrin-dependent, with CXCR4 colocalizing with markers for early and recycling endosomes (28Zhang Y. Foudi A. Geay J.F. Berthebaud M. Buet D. Jarrier P. Jalil A. Vainchenker W. Louache F. Stem Cells. 2004; 22: 1015-1029Crossref PubMed Scopus (92) Google Scholar). Constitutive endocytosis and recycling of CXCR4 are important mechanisms for maintaining homeostatic control of cell surface levels to sense ligand. It is crucial for a cell to be able to rapidly up- and down-regulate cell surface levels of receptor in response to changing signals. Our results demonstrate that AMSH plays a role in modulating CXCR4 steady-state levels and that AMSH catalytic activity is critical for this regulatory mechanism.A number of studies suggest that AMSH may be recruited to the endocytic pathway via multiple mechanisms. Specifically, AMSH can interact with clathrin through its clathrin binding domain (Fig. 3A), resulting in localization of AMSH to clathrin-coated pits and early endosomes (45McCullough J. Row P.E. Lorenzo O. Doherty M. Beynon R. Clague M.J. Urbé S. Curr. Biol. 2006; 16: 160-165Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 50Nakamura M. Tanaka N. Kitamura N. Komada M. Genes Cells. 2006; 11: 593-606Crossref PubMed Scopus (32) Google Scholar). Furthermore, AMSH binds to the SH3 domain of STAM1 through an integral STAM binding motif enabling the recruitment of AMSH to early/sorting endosomes (Fig. 5) (37McCullough J. Clague M.J. Urbé S. J. Cell Biol. 2004; 166: 487-492Crossref PubMed Scopus (302) Google Scholar, 44Tanaka N. Kaneko K. Asao H. Kasai H. Endo Y. Fujita T. Takeshita T. Sugamura K. J. Biol. Chem. 1999; 274: 19129-19135Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 45McCullough J. Row P.E. Lorenzo O. Doherty M. Beynon R. Clague M.J. Urbé S. Curr. Biol. 2006; 16: 160-165Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 51Kikuchi K. Ishii N. Asao H. Sugamura K. Biochem. Biophys. Res. Commun. 2003; 306: 637-643Crossref PubMed Scopus (25) Google Scholar, 52Kim M.S. Kim J.A. Song H.K. Jeon H. Biochem. Biophys. Res. Commun. 2006; 351: 612-618Crossref PubMed Scopus (24) Google Scholar). In addition to the recruitment of AMSH to early endocytic compartments, AMSH may be recruited to the multivesicular body via interactions with the ESCRT-III protein CHMP3 (38Kyuuma M. Kikuchi K. Kojima K. Sugawara Y. Sato M. Mano N. Goto J. Takeshita T. Yamamoto A. Sugamura K. Tanaka N. Cell Struct. Funct. 2007; 31: 159-172Crossref PubMed Scopus (48) Google Scholar, 39Ma Y.M. Boucrot E. Villén J. Affar el B. Gygi S.P. Göttlinger H.G. Kirchhausen T. J. Biol. Chem. 2007; 282: 9805-9812Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 44Tanaka N. Kaneko K. Asao H. Kasai H. Endo Y. Fujita T. Takeshita T. Sugamura K. J. Biol. Chem. 1999; 274: 19129-19135Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 46Zamborlini A. Usami Y. Radoshitzky S.R. Popova E. Palu G. Göttlinger H. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19140-19145Crossref PubMed Scopus (143) Google Scholar, 47Tsang H.T. Connell J.W. Brown S.E. Thompson A. Reid E. Sanderson C.M. Genomics. 2006; 88: 333-346Crossref PubMed Scopus (127) Google Scholar, 48Row P.E. Prior I.A. McCullough J. Clague M.J. Urbé S. J. Biol. Chem. 2006; 281: 12618-12624Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The integrity of these protein-protein interactions is presumably crucial for the spatial and temporal localization of AMSH to different functional compartments within the endocytic pathway. To determine which of these interactions influence the endocytosis and turnover of CXCR4, we examined several truncation mutants of AMSH. Mutational analysis revealed the importance of the STAM binding motif of AMSH for CXCR4 stability (Figs. 3C and 8). A truncation mutant of AMSH containing the STAM binding motif and a catalytically inactive JAMM domain (RXXK-D348A) results in a similar increase in CXCR4 levels observed with overexpression of the full-length catalytically inactive AMSH. This implies that the RXXK-D348A fragment alone is able to displace endogenous wild type AMSH by effectively competing for relevant protein-protein interactions. As the previously described function for this fragment is recruitment by STAM (37McCullough J. Clague M.J. Urbé S. J. Cell Biol. 2004; 166: 487-492Crossref PubMed Scopus (302) Google Scholar, 44Tanaka N. Kaneko K. Asao H. Kasai H. Endo Y. Fujita T. Takeshita T. Sugamura K. J. Biol. Chem. 1999; 274: 19129-19135Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 45McCullough J. Row P.E. Lorenzo O. Doherty M. Beynon R. Clague M.J. Urbé S. Curr. Biol. 2006; 16: 160-165Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 51Kikuchi K. Ishii N. Asao H. Sugamura K. Biochem. Biophys. Res. Commun. 2003; 306: 637-643Crossref PubMed Scopus (25) Google Scholar, 52Kim M.S. Kim J.A. Song H.K. Jeon H. Biochem. Biophys. Res. Commun. 2006; 351: 612-618Crossref PubMed Scopus (24) Google Scholar), our findings suggest that the interaction between AMSH and STAM may be critical for appropriate trafficking and degradation of CXCR4. Indeed, our results show that mutation of the STAM binding RXXK motif of AMSH within this mutant abrogates its ability to interfere with CXCR4 levels (Fig. 8). AMSH recruitment by STAM may be an important mechanism of CXCR4 endocytosis and stability and, therefore, important for a number of cellular processes involving functional CXCR4 including human immunodeficiency virus infection and stem cell homing.Previous reports have established that the RXXK motif of AMSH binds a variant subfamily of SH3 domains found i
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