Nuclear-Cytoplasmic Shuttling of the Oncogenic Mouse UNP/USP4 Deubiquitylating Enzyme

Abstract

The oncogenic deubiquitylating enzyme (DUB) Unp/Usp4, which binds to the retinoblastoma family of tumor suppressor proteins, was originally described as a nuclear protein. However, more recent studies have shown it to be cytoplasmic. In addition, analysis of its subcellular localization has been complicated by the existence of the paralog Usp15. In this study, we resolved this controversy by investigating the localization of exogenously expressed Usp4 (using red fluorescent protein-Usp4) and of endogenous Usp4 (using highly specific antibodies that can distinguish Usp4 from Usp15). We found that by inhibiting nuclear export with leptomycin B, both exogenous and endogenous Usp4 accumulate in the nucleus. Further, using a Rev-green fluorescent protein-based export assay, we confirmed the existence of a nuclear export signal (133VEVYLLELKL142) in Usp4. In addition, a functional nuclear import signal (766QPQKKKK772) was also identified, which was specifically recognized by importin α/β. Finally, we show that the equilibrium of Usp4 subcellular localization varies between different cell types. Usp4 is thus the first DUB reported to have nucleocytoplasmic shuttling properties. The implications of this shuttling for its function as a DUB are discussed. The oncogenic deubiquitylating enzyme (DUB) Unp/Usp4, which binds to the retinoblastoma family of tumor suppressor proteins, was originally described as a nuclear protein. However, more recent studies have shown it to be cytoplasmic. In addition, analysis of its subcellular localization has been complicated by the existence of the paralog Usp15. In this study, we resolved this controversy by investigating the localization of exogenously expressed Usp4 (using red fluorescent protein-Usp4) and of endogenous Usp4 (using highly specific antibodies that can distinguish Usp4 from Usp15). We found that by inhibiting nuclear export with leptomycin B, both exogenous and endogenous Usp4 accumulate in the nucleus. Further, using a Rev-green fluorescent protein-based export assay, we confirmed the existence of a nuclear export signal (133VEVYLLELKL142) in Usp4. In addition, a functional nuclear import signal (766QPQKKKK772) was also identified, which was specifically recognized by importin α/β. Finally, we show that the equilibrium of Usp4 subcellular localization varies between different cell types. Usp4 is thus the first DUB reported to have nucleocytoplasmic shuttling properties. The implications of this shuttling for its function as a DUB are discussed. Ubiquitin is a 76-amino acid protein that serves as a multi-functional covalent “tag” for a wide variety of cellular proteins. Its best understood function is in marking proteins for degradation by the 26 S proteasome, but ubiquitin conjugation is also important in diverse pathways including DNA repair, receptor internalization, and signal transduction (1Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3275) Google Scholar, 2Hicke L. Nat. Rev. Mol. Cell. Biol. 2001; 2: 195-201Crossref PubMed Scopus (975) Google Scholar). Mutations affecting enzymes involved in the ubiquitin system have been implicated in several human diseases (1Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3275) Google Scholar, 3Miller R.J. Wilson S.M. Trends Pharmacol. Sci. 2003; 24: 18-23Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 4Sakamoto K.M. Mol. Genet. Metab. 2002; 77: 44-56Crossref PubMed Scopus (71) Google Scholar). Ubiquitin conjugation requires the action of several enzymes to activate and covalently attach ubiquitin to target proteins, with specificity being provided by the ubiquitin ligase/E3 enzymes, which are involved in substrate binding and have received much attention in recent years (5Jackson P.K. Eldridge A.G. Freed E. Furstenthal L. Hsu J.Y. Kaiser B.K. Reimann J.D. Trends Cell Biol. 2000; 10: 429-439Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). Conversely, deubiquitylating enzymes (DUBs), 1The abbreviations used are: DUB, deubiquitylating enzyme; CLSM, confocal laser-scanning microscopy; GFP, green fluorescent protein; GST, glutathione S-transferase; IMP, importin; LMB, leptomycin B; NES, nuclear export signal; pNES, putative leucine-rich NES; NLS, nuclear localization signal; pNLS, putative NLS; PVDF, polyvinylidene fluoride; RFP, red fluorescent protein; UBP, ubiquitin-specific protease; FBS, fetal bovine serum; MPEF, mouse primary embryonic fibroblast; PVDF, polyvinylidene difluoride; ELISA, enzyme-linked immunosorbent assay; T-ag, T-antigen. 1The abbreviations used are: DUB, deubiquitylating enzyme; CLSM, confocal laser-scanning microscopy; GFP, green fluorescent protein; GST, glutathione S-transferase; IMP, importin; LMB, leptomycin B; NES, nuclear export signal; pNES, putative leucine-rich NES; NLS, nuclear localization signal; pNLS, putative NLS; PVDF, polyvinylidene fluoride; RFP, red fluorescent protein; UBP, ubiquitin-specific protease; FBS, fetal bovine serum; MPEF, mouse primary embryonic fibroblast; PVDF, polyvinylidene difluoride; ELISA, enzyme-linked immunosorbent assay; T-ag, T-antigen. which can cleave branched ubiquitin conjugates and/or linear ubiquitin precursor proteins, have received much less attention. DUBs constitute large gene families in all eukaryotes and can be divided into the ubiquitin-specific protease (UBP) and ubiquitin C-terminal hydrolase families based on amino acid sequence (6Wilkinson K.D. FASEB J. 1997; 11: 1245-1256Crossref PubMed Scopus (500) Google Scholar, 7Baker R.T. Wolf D. Hilt W. Proteasomes: The World of Regulatory Proteolysis. Landes Bioscience Co., Austin, TX2000: 238-255Google Scholar, 8Soboleva T.A. Baker R.T. Curr. Protein Pept. Sci. 2004; 5: 191-200Crossref PubMed Scopus (49) Google Scholar). This multiplicity is not required merely for the cleavage of ubiquitin precursors but is indicative of other regulatory roles in the ubiquitin pathway. Clues to the function of some mammalian UBPs have come from their identification as growth regulators. These include the human tre-2/tre-17(USP6) proto-oncogene (9Papa F.R. Hochstrasser M. Nature. 1993; 366: 313-319Crossref PubMed Scopus (339) Google Scholar) and the mouse cytokine-inducible UBPs DUB-1 and DUB-2 (10Zhu Y. Lambert K. Corless C. Copeland N.G. Gilbert D.J. Jenkins N.A. D'Andrea A.D. J. Biol. Chem. 1997; 272: 51-57Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). We have studied the mouse Unp gene (Usp4 in the systematic nomenclature) (11Baker R.T. Wang X.W. Woollatt E. White J.A. Sutherland G.R. Genomics. 1999; 59: 264-274Crossref PubMed Scopus (62) Google Scholar), which was originally identified as a proto-oncogene related to tre-2/tre-17/USP6 (12Gupta K. Chevrette M. Gray D.A. Oncogene. 1994; 9: 1729-1731PubMed Google Scholar, 13Gupta K. Copeland N.G. Gilbert D.J. Jenkins N.A. Gray D.A. Oncogene. 1993; 8: 2307-2310PubMed Google Scholar). Unp/Usp4 was subsequently observed to contain the UBP Cys and His boxes (9Papa F.R. Hochstrasser M. Nature. 1993; 366: 313-319Crossref PubMed Scopus (339) Google Scholar, 14Gray D.A. Inazawa J. Gupta K. Wong A. Ueda R. Takahashi T. Oncogene. 1995; 10: 2179-2183PubMed Google Scholar) and since shown to have DUB activity (15Gilchrist C.A. Baker R.T. Biochim. Biophys. Acta. 2000; 1481: 297-309Crossref PubMed Scopus (21) Google Scholar, 16Gilchrist C.A. Gray D.A. Baker R.T. J. Biol. Chem. 1997; 272: 32280-32285Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In a study of primary human lung tumor tissue, Gray et al. (14Gray D.A. Inazawa J. Gupta K. Wong A. Ueda R. Takahashi T. Oncogene. 1995; 10: 2179-2183PubMed Google Scholar) observed that the human homolog of Usp4, USP4 (UNP; also termed Unph), had consistently elevated gene expression levels in small cell tumors and adenocarcinomas of the lung, suggesting a possible causative role for this UBP in neoplasia. In a separate study using cell lines rather than primary tissue, USP4 protein levels were shown to be slightly but consistently reduced in cell lines derived from small cell tumors, leading to the suggestion that USP4 may be a candidate tumor suppressor gene (17Frederick A. Rolfe M. Chiu M.I. Oncogene. 1998; 16: 153-165Crossref PubMed Scopus (50) Google Scholar). Recently, both mouse and human Usp4 have been shown to interact with the Rb family of tumor suppressor proteins, a possible mechanism for Usp4-mediated cell transformation (18Blanchette P. Gilchrist C.A. Baker R.T. Gray D.A. Oncogene. 2001; 20: 5533-5537Crossref PubMed Scopus Google Scholar, E. D. E. A. Baker R.T. M. M. Oncogene. 2001; 20: PubMed Scopus Google Scholar). is controversy the cellular of described it as a nuclear protein as by cell K. Copeland N.G. Gilbert D.J. Jenkins N.A. Gray D.A. Oncogene. 1993; 8: 2307-2310PubMed Google Scholar). In cell studies by et al. (17Frederick A. Rolfe M. Chiu M.I. Oncogene. 1998; 16: 153-165Crossref PubMed Scopus (50) Google Scholar) using a different it to be cytoplasmic. of studies are by the identification of a DUB termed (11Baker R.T. Wang X.W. Woollatt E. White J.A. Sutherland G.R. Genomics. 1999; 59: 264-274Crossref PubMed Scopus (62) Google Scholar, C. Wang X.W. Copeland N.G. Jenkins N.A. Baker R.T. 2003; PubMed Scopus Google Scholar), which is to the by et al. (17Frederick A. Rolfe M. Chiu M.I. Oncogene. 1998; 16: 153-165Crossref PubMed Scopus (50) Google Scholar) was a that is in the subsequently identified it that both USP4 and were being USP4 and contain putative nuclear localization with that of USP4 being to that of (11Baker R.T. Wang X.W. Woollatt E. White J.A. Sutherland G.R. Genomics. 1999; 59: 264-274Crossref PubMed Scopus (62) Google Scholar, 13Gupta K. Copeland N.G. Gilbert D.J. Jenkins N.A. Gray D.A. Oncogene. 1993; 8: 2307-2310PubMed Google Scholar). of USP4 in (17Frederick A. Rolfe M. Chiu M.I. Oncogene. 1998; 16: 153-165Crossref PubMed Scopus (50) Google Scholar). However, the of USP4 in its the of has not been the described of T. K. J. 2000; PubMed Google Scholar), both nuclear and of of putative in in from the of T. K. J. 2000; PubMed Google Scholar), was than are into the mechanism D.A. Rev. 1998; PubMed Scopus Google Scholar). import is by of the importin that Conversely, nuclear export is by that nuclear export which are leucine-rich D.A. M.H. 2000; PubMed Scopus Google Scholar). The best studied is M. M. M. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). that and can between nuclear and in to that the of the cellular localization of USP4 to different cell of the cell rather than to different In to this and to a of their we antibodies that between Usp4 and and used to study their subcellular localization in a variety of cell types. We also a of the red fluorescent protein to Usp4 and used this to functional and Usp4 is thus shown to be a nucleocytoplasmic shuttling with different of nuclear and localization on the cell This is the first of nucleocytoplasmic shuttling for a DNA and was by using enzyme to the were a mouse Usp4 (15Gilchrist C.A. Baker R.T. Biochim. Biophys. Acta. 2000; 1481: 297-309Crossref PubMed Scopus (21) Google Scholar) and a mouse C. Wang X.W. Copeland N.G. Jenkins N.A. Baker R.T. 2003; PubMed Scopus Google Scholar). proteins were by the Usp4 gene to amino into using and Usp4 and proteins were using the system and the The was also using the system in the which was by the into the of to it The was provided by A. Cell 2000; PubMed Scopus Google Scholar, P. J. Mol. Biol. 1997; PubMed Scopus Google Scholar). putative Usp4 were between the and of this The was by Usp4 into of D.A. R. 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Cell and were in fetal bovine and were in FBS, and of of of primary embryonic were in FBS, were in FBS, amino amino were in FBS, were in FBS, were in and with were in and with of DNA and of of as described by the proteins, were in for in and on for protein expression analysis and were in for in for with primary antibodies to in with with for with and on using were by confocal laser-scanning using a The of in the to the was derived by analysis of confocal as A. D.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) using the the was for of in the with on used as the The was using the of was as was as and was as were using was also using and with as nuclear nuclear and were with protein Nature. PubMed Scopus Google Scholar), for and resolved by were to a which was in for with primary antibodies for with for and using the Protein binding of expressed mouse to Usp4 proteins to Usp4 was using binding as using the protein as a A. D.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). were to the is the of as A. D.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The the of of and have with Gupta et al. (12Gupta K. Chevrette M. Gray D.A. Oncogene. 1994; 9: 1729-1731PubMed Google Scholar, 13Gupta K. Copeland N.G. Gilbert D.J. Jenkins N.A. Gray D.A. Oncogene. 1993; 8: 2307-2310PubMed Google Scholar) which with a protein in cell of the for Usp4. et al. (17Frederick A. Rolfe M. Chiu M.I. Oncogene. 1998; 16: 153-165Crossref PubMed Scopus (50) Google Scholar) a a of USP4 to the subsequently identified K.M. Mol. Genet. Metab. 2002; 77: 44-56Crossref PubMed Scopus (71) Google Scholar) are Finally, we found that the by et al. E. D. E. A. Baker R.T. M. M. Oncogene. 2001; 20: PubMed Scopus Google Scholar) a C-terminal of human with In this we to specific antibodies to Usp4 and Usp15. The of mouse Usp4 and mouse were as since were with for the of the proteins. DNA was by and into The were used for expression of and proteins, which were and into for of from were using Usp4 and for their specificity by The antibodies recognized their proteins and not These antibodies also human USP4 specifically not also identified proteins of the in from mouse and of Usp4 have slightly than of C. Wang X.W. Copeland N.G. Jenkins N.A. Baker R.T. 2003; PubMed Scopus Google Scholar). with that the Usp4 a of than the with can be in of the In addition, a with a of Usp4 and antibodies that all with the separate antibodies was thus possible to that antibodies are specific for Usp4 and Usp15. of Usp4 and and are sequence This is also in the that both are ubiquitin-specific and can the family of tumor suppressor proteins (11Baker R.T. Wang X.W. Woollatt E. White J.A. Sutherland G.R. Genomics. 1999; 59: 264-274Crossref PubMed Scopus (62) Google Scholar, P. Gilchrist C.A. Baker R.T. Gray D.A. Oncogene. 2001; 20: 5533-5537Crossref PubMed Scopus Google Scholar, E. D. E. A. Baker R.T. M. M. Oncogene. 2001; 20: PubMed Scopus Google Scholar, C. Wang X.W. Copeland N.G. Jenkins N.A. Baker R.T. 2003; PubMed Scopus Google Scholar). and R. T. that the subcellular localization of endogenous USP4 and proteins in human is Usp4 was observed in the the was in the and the but not the and a with its protein in the of the specificity of the antibodies and of with other proteins in the cell These were confirmed in the mouse cell a of was Usp4 was found in the of of in the of of and the cell in of in not show being in the but rather the and some but not and Usp4 in Cell Usp4 localization in and cell lines was we the localization of endogenous Usp4 in a of other cell of and a of different endogenous USP4 USP4 was in the in being nuclear in of that USP4 was nuclear in of in of and the cell in of of and Usp4 and sequence of Usp4 and are shown as and with the sequence shown in and in Usp4 are shown with and shown in B, analysis with a cell from Usp4 were resolved by to a and with a from by a and The of the is analysis with Usp4 and and proteins were resolved by to a and with with as the by a and The of the is Usp4 expressed in proteins of different to and/or degradation (15Gilchrist C.A. Baker R.T. Biochim. Biophys. Acta. 2000; 1481: 297-309Crossref PubMed Scopus (21) Google Scholar). and mouse protein from mouse were resolved by to a and with Usp4 and antibodies proteins of different was resolved in a wide by and to a and the was into were with and the was to The were and with a of Usp4 and antibodies can be in of the subcellular localization of endogenous Usp4 and in and and and and were with and with and with and as described and several with were with on and by confocal localization of endogenous Usp4 in cell were and using antibodies as described in the to were by and for nuclear and were in the the of cell are shown the of subcellular localization of Usp4 in a variety of cell lines that Usp4 localization may be in in different cell types. nuclear localization of Usp4 in and and nuclear and localization in and that Usp4 may contain as into and of the nucleus. In to this we studied the of leptomycin on the localization of endogenous Usp4. Usp4 to is that nuclear export of proteins by the nuclear export which the T. Y. M. M. Cell 1998; PubMed Scopus Google Scholar, K. M. D. M. T. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). Usp4 localization in to was thus in In the of LMB, Usp4 was of in nuclear in of and the cell in of with for and of Usp4 in the from to The of subcellular localization of endogenous Usp4 to that Usp4 recognized by Usp4 to to of exogenous Usp4 to LMB, it was expressed in as protein. of was the cell by in the of a expression was observed in the in both in the of a small of This was in to which the with nuclear nuclear with the in the of of with in of the nuclear to for a of than that in of had on the These that exogenously expressed is to endogenous Usp4 in of of the Usp4 sequence putative leucine-rich putative including that described by Gupta et al. K. Copeland N.G. Gilbert D.J. Jenkins N.A. Gray D.A. Oncogene. 1993; 8: 2307-2310PubMed Google Scholar), were also identified Usp4 a in export was used to the of putative of Usp4. The is based on the of a functional to export the A. Cell 2000; PubMed Scopus Google Scholar, P. J. Mol. Biol. 1997; PubMed Scopus Google Scholar), from the nucleus. The protein and and is to between the and the of the cell in to export The a of with a and in the of of a functional into the nuclear export of and can as a of Usp4 and were into the to and was used as a and with was used as a The subcellular localization of all expressed proteins in was the was in the of with observed in of The the was in the of the of and the cell in the in to the that the of Usp4 is not to export the protein from the in the of the In the a different subcellular all some nuclear more than of had it can be that Usp4 a functional which is than the NES, but is to nuclear export of the to in in in protein expression in suggesting that are important for protein Usp4 a which of the Usp4 were was the of the expression and a was also The were as and The not show subcellular localization different from that of in analysis in the This that not as a functional is with in Usp4. In the of a in Usp4 of localization of the with in the of the protein. with the for the was than that for the protein with a of This that is required for Usp4 nuclear of both and for localization that were not different to for of with of a role in Usp4 nuclear the of Usp4 with the were with The this was that was for Usp4 nuclear the protein not accumulate in the of to the as the protein. in the of a small of had The on the other in the of of and of had in the analysis a in the between the and the that is the nuclear import signal in Usp4. Usp4 by that is functional to the mechanism of nuclear import of Usp4. The first of nuclear protein import by D.A. M.H. 2000; PubMed Scopus Google Scholar). The amino acid sequence of is to the as that of large tumor which is recognized by the Usp4 by binding was using different Usp4 Usp4 to and the Usp4 The first was used to by in and the was used to binding in the of the Usp4 protein. protein the to was used as a was used as a and were expressed as proteins as (11Baker R.T. Wang X.W. Woollatt E. White J.A. Sutherland G.R. Genomics. 1999; 59: 264-274Crossref PubMed Scopus (62) Google Scholar). The show that both proteins have a for the than for and the of for both proteins was and and not to the binding for proteins as A. D.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) and the family D. J. A. D.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). was also recognized by but Usp4 proteins were recognized by with and The of binding to was that of the with the that both and contain the of the binding that the is to be the nuclear import receptor for Usp4. of the Protein in Usp4 Usp4 was shown to interact with the family (18Blanchette P. Gilchrist C.A. Baker R.T. Gray D.A. Oncogene. 2001; 20: 5533-5537Crossref PubMed Scopus Google Scholar, E. D. E. A. Baker R.T. M. M. Oncogene. 2001; 20: PubMed Scopus Google Scholar), we the that may be involved in Usp4 subcellular Usp4 localization was in and functional in endogenous Usp4 localization with being nuclear in both a for both cell lines and were with antibodies the of in and expression the cell with some nuclear in and in expression of not to Usp4 subcellular The of this was to the cellular localization of Usp4 and the of it is nuclear cytoplasmic. this is important in the function of Usp4. We show for the first that Usp4 is a shuttling protein functional for nuclear import and which are recognized by of the importin family of nuclear import and export The Usp4 NLS, of which Usp4 nuclear localization export is by LMB, is recognized specifically by as shown using protein binding The Usp4 NES, was to export a protein from the nucleus. the export activity of Usp4 to be the activity of some other cellular shuttling proteins, as and and and the protein was than that of Usp4 in the export A. Cell 2000; PubMed Scopus Google Scholar). This can be by the that the of cellular proteins, including Usp4, were the The of Usp4 to that is the Usp4 nuclear export the of Usp4 to between and the to be in to the action of its nuclear and on the the in the the subcellular localization of Usp4 can be by the of Usp4 to in between and that localization of endogenous Usp4 in a of cell lines varies from in to nuclear in and was in the as as the in is to Usp4 and of the cellular nuclear The of Gupta et al. (12Gupta K. Chevrette M. Gray D.A. Oncogene. 1994; 9: 1729-1731PubMed Google Scholar) and et al. (17Frederick A. Rolfe M. Chiu M.I. Oncogene. 1998; 16: 153-165Crossref PubMed Scopus (50) Google Scholar) are with Usp4 was in the of mouse (12Gupta K. Chevrette M. Gray D.A. Oncogene. 1994; 9: 1729-1731PubMed Google Scholar), the human of Usp4 was in the in human lung (17Frederick A. Rolfe M. Chiu M.I. Oncogene. 1998; 16: 153-165Crossref PubMed Scopus (50) Google Scholar). as cell of the cell of expression to the localization of a shuttling the equilibrium to it more nuclear more cytoplasmic. other shuttling proteins subcellular localization has been shown for the nuclear tumor suppressor which is from the in diverse M. J. Sci. A. 1995; PubMed Scopus Google Scholar, Sci. A. PubMed Scopus Google Scholar) and in embryonic M.I. M. R. Curr. Biol. 1998; 8: Full Text Full Text PDF PubMed Scopus Google Scholar). et al. J. M. A. J. 2000; PubMed Scopus Google Scholar) have shown that subcellular localization of on the of localization of a shuttling protein as Usp4 may between cell and different The of of Usp4 not its import into the nucleus. of endogenous exogenous Usp4 not Usp4 export from the nucleus. These that Usp4 may have nucleocytoplasmic which not export is that Usp4 is with proteins as of the The with endogenous Usp4 that is to be involved in Usp4 subcellular We have that the nuclear localization of Usp4 is in protein not which and for may be involved in Usp4 in this is the that between and with its nuclear export A. L. K. J.A. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar) and that the levels of and are also in and D. Sci. A. 2000; PubMed Scopus Google Scholar, M. L. Oncogene. 2000; PubMed Scopus Google Scholar), which show nuclear Usp4 studies are required to the role of family proteins in Usp4 can have The antibodies this that the subcellular localization of endogenous Usp4 and is These are since show that Usp4 and may to their roles in different cellular In addition, as to possible was in the of a for of which is to the as a of the M. A. Trends Cell Biol. 2000; 10: Full Text Full Text PDF PubMed Scopus Google Scholar). proteins are to be by the A. R. C. A. C. D. 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