The Regulation of Glycogen Synthase Kinase-3 Nuclear Export by Frat/GBP

Abstract

Previous studies have shown that nuclear levels of glycogen synthase kinase-3 (GSK-3) are dynamically regulated and may affect access of GSK-3 to its substrates. In this study we show that the GSK-3-binding protein Frat/GBP regulates the nuclear export of GSK-3. We show that Frat/GBP contains a nuclear export sequence that promotes its own nuclear export and that of associated GSK-3. Treating cells with leptomycin B increased nuclear levels of endogenous GSK-3 suggesting that an endogenous process targets GSK-3 for nuclear export. To investigate this further, we used two approaches to disrupt the interaction between GSK-3 and endogenous Frat. First we isolated mutants of GSK-3 that selectively interfered with Frat binding and found that these mutants were poorly exported. Second we expressed a peptide that competes with Frat for GSK-3 binding and found that it caused endogenous GSK-3 to accumulate in the nucleus. Together these data suggest that Frat may be the endogenous factor that targets GSK-3 for nuclear export. The dynamic expression patterns of Frat mRNAs together with the role of Frat in mediating GSK-3 nuclear export have important implications for the control of the substrate access of GSK-3 in several signaling pathways. Previous studies have shown that nuclear levels of glycogen synthase kinase-3 (GSK-3) are dynamically regulated and may affect access of GSK-3 to its substrates. In this study we show that the GSK-3-binding protein Frat/GBP regulates the nuclear export of GSK-3. We show that Frat/GBP contains a nuclear export sequence that promotes its own nuclear export and that of associated GSK-3. Treating cells with leptomycin B increased nuclear levels of endogenous GSK-3 suggesting that an endogenous process targets GSK-3 for nuclear export. To investigate this further, we used two approaches to disrupt the interaction between GSK-3 and endogenous Frat. First we isolated mutants of GSK-3 that selectively interfered with Frat binding and found that these mutants were poorly exported. Second we expressed a peptide that competes with Frat for GSK-3 binding and found that it caused endogenous GSK-3 to accumulate in the nucleus. Together these data suggest that Frat may be the endogenous factor that targets GSK-3 for nuclear export. The dynamic expression patterns of Frat mRNAs together with the role of Frat in mediating GSK-3 nuclear export have important implications for the control of the substrate access of GSK-3 in several signaling pathways. The serine/threonine protein kinase glycogen synthase kinase-3 (GSK-3) 1The abbreviations used are: GSK-3, glycogen synthase kinase-3; Dsh, Dishevelled; Frat, frequently rearranged in T-cell lymphomas; GBP, GSK-3-binding protein; NES, nuclear export sequence; LMB, leptomycin B; GST, glutathione S-transferase; GFP, green fluorescent protein; HA, hemagglutinin; FITC, fluorescein isothiocyanate; MDCK, Madin-Darby canine kidney; KE/AQ, K208A/E209Q; mFrat, mouse Frat 1The abbreviations used are: GSK-3, glycogen synthase kinase-3; Dsh, Dishevelled; Frat, frequently rearranged in T-cell lymphomas; GBP, GSK-3-binding protein; NES, nuclear export sequence; LMB, leptomycin B; GST, glutathione S-transferase; GFP, green fluorescent protein; HA, hemagglutinin; FITC, fluorescein isothiocyanate; MDCK, Madin-Darby canine kidney; KE/AQ, K208A/E209Q; mFrat, mouse Fratis an important regulator of several cellular processes including transcription, cell cycle progression, cell survival, and cytoskeletal organization (1Harwood A.J. Cell. 2001; 105: 821-824Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Two highly conserved isoforms have been characterized in mammals, α and β. Regulation of GSK-3 is primarily achieved through the inhibition of its activity in response to multiple signaling pathways, including the Wnt and insulin pathways (2Woodgett J.R. Science's STKE. 2001; (http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/100/re12)PubMed Google Scholar). In the Wnt pathway, GSK-3 phosphorylates β-catenin as part of a multiprotein complex containing the binding proteins Axin and APC (adenomatous polyposis coli), which targets β-catenin for degradation. Activation of the Wnt pathway, which involves proteins of the Dishevelled (Dsh) family, inhibits β-catenin phosphorylation and consequently allows it to accumulate and act as a co-transcription factor (3Peifer M. Polakis P. Science. 2000; 287: 1606-1609Crossref PubMed Scopus (1137) Google Scholar). Deregulation of the Wnt pathway has been implicated in several human cancers. Alteration of GSK-3 function has also been implicated in diabetes, Alzheimer's disease, and clinical depression (4Grimes C.A. Jope R.S. Prog. Neurobiol. 2001; 65: 391-426Crossref PubMed Scopus (1304) Google Scholar).Recent studies have indicated that GSK-3 localization is dynamically regulated. Although GSK-3 is found predominantly in the cytoplasm of most cells, it has been found to enter the nucleus during S phase of the cell cycle and during apoptosis (5Bijur G.N., De Sarno P. Jope R.S. J. Biol. Chem. 2000; 275: 7583-7590Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 6Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (450) Google Scholar, 7Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1851) Google Scholar, 8Bijur G.N. Jope R.S. J. Biol. Chem. 2001; 276: 37436-37442Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). By contrast, GSK-3 has been shown to exit the nucleus of cardiac myocytes upon stimulation with isoproterenol (9Morisco C. Seta K. Hardt S.E. Lee Y. Vatner S.F. Sadoshima J. J. Biol. Chem. 2001; 276: 28586-28597Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Significantly, GSK-3 nuclear accumulation correlates with increased phosphorylation of nuclear substrates. For example, the increased levels of nuclear GSK-3 in S phase result in the increased phosphorylation of cyclin D1 (6Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (450) Google Scholar). The nuclear accumulation of GSK-3 during apoptosis suggests that the phosphorylation of other substrates may be involved in apoptotic signaling. This is supported by evidence indicating that GSK-3 inhibition promotes cell survival, whereas increased GSK-3 activity potentiates apoptosis (5Bijur G.N., De Sarno P. Jope R.S. J. Biol. Chem. 2000; 275: 7583-7590Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar,10Crowder R.J. Freeman R.S. J. Biol. Chem. 2000; 275: 34266-34271Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). GSK-3 also plays a role in controlling the localization of several substrates. Phosphorylation of cyclin D1, GATA-4, Dd-STATa (Dictyostelium STAT (signal transducers and activators of transcription) protein), TIMELESS, and NF-ATc (nuclear factor of activated T-cells) by GSK-3 alters their nuclear/cytoplasmic distribution (6Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (450) Google Scholar, 9Morisco C. Seta K. Hardt S.E. Lee Y. Vatner S.F. Sadoshima J. J. Biol. Chem. 2001; 276: 28586-28597Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 11Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Science. 1997; 275: 1930-1933Crossref PubMed Scopus (633) Google Scholar, 12Ginger R.S. Dalton E.C. Ryves W.J. Fukuzawa M. Williams J.G. Harwood A.J. EMBO J. 2000; 19: 5483-5491Crossref PubMed Scopus (58) Google Scholar, 13Martinek S. Inonog S. Manoukian A.S. Young M.W. Cell. 2001; 105: 769-779Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar).The mechanisms by which GSK-3 localization is controlled remain unclear. Analysis of the amino acid sequence does not reveal the presence of any recognizable nuclear import or export sequences. Thus GSK-3 localization may be regulated indirectly through associations with binding proteins. Frat/GBP (frequentlyrearranged in T-cell lymphomas/GSK-3-binding protein) is a recently identified GSK-3-binding protein (14Jonkers J. Korswagen H.C. Acton D. Breuer M. Berns A. EMBO J. 1997; 16: 441-450Crossref PubMed Scopus (115) Google Scholar, 15Yost C. Farr III, G.H. Pierce S.B. Ferkey D.M. Mingzi Chen M. Kimelman D. Cell. 1998; 93: 1031-1041Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Previous studies have suggested a possible role in Wnt signaling because of its interaction with the Wnt signaling protein Dsh (16Li L. Yuan H. Weaver C.D. Mao J. Farr III, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (356) Google Scholar).In this study we investigated the possibility that Frat may play a role in regulating GSK-3 localization. Consistent with this hypothesis, we found that Frat contained a nuclear export sequence (NES), and we show that this activity allows Frat to direct the nuclear export of exogenous GSK-3. In addition we also provide evidence that an endogenous Frat-like function is involved in regulating GSK-3 localization since inhibition of nuclear export or the disruption of the Frat-GSK-3 interaction resulted in the nuclear accumulation of GSK-3. The serine/threonine protein kinase glycogen synthase kinase-3 (GSK-3) 1The abbreviations used are: GSK-3, glycogen synthase kinase-3; Dsh, Dishevelled; Frat, frequently rearranged in T-cell lymphomas; GBP, GSK-3-binding protein; NES, nuclear export sequence; LMB, leptomycin B; GST, glutathione S-transferase; GFP, green fluorescent protein; HA, hemagglutinin; FITC, fluorescein isothiocyanate; MDCK, Madin-Darby canine kidney; KE/AQ, K208A/E209Q; mFrat, mouse Frat 1The abbreviations used are: GSK-3, glycogen synthase kinase-3; Dsh, Dishevelled; Frat, frequently rearranged in T-cell lymphomas; GBP, GSK-3-binding protein; NES, nuclear export sequence; LMB, leptomycin B; GST, glutathione S-transferase; GFP, green fluorescent protein; HA, hemagglutinin; FITC, fluorescein isothiocyanate; MDCK, Madin-Darby canine kidney; KE/AQ, K208A/E209Q; mFrat, mouse Fratis an important regulator of several cellular processes including transcription, cell cycle progression, cell survival, and cytoskeletal organization (1Harwood A.J. Cell. 2001; 105: 821-824Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Two highly conserved isoforms have been characterized in mammals, α and β. Regulation of GSK-3 is primarily achieved through the inhibition of its activity in response to multiple signaling pathways, including the Wnt and insulin pathways (2Woodgett J.R. Science's STKE. 2001; (http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/100/re12)PubMed Google Scholar). In the Wnt pathway, GSK-3 phosphorylates β-catenin as part of a multiprotein complex containing the binding proteins Axin and APC (adenomatous polyposis coli), which targets β-catenin for degradation. Activation of the Wnt pathway, which involves proteins of the Dishevelled (Dsh) family, inhibits β-catenin phosphorylation and consequently allows it to accumulate and act as a co-transcription factor (3Peifer M. Polakis P. Science. 2000; 287: 1606-1609Crossref PubMed Scopus (1137) Google Scholar). Deregulation of the Wnt pathway has been implicated in several human cancers. Alteration of GSK-3 function has also been implicated in diabetes, Alzheimer's disease, and clinical depression (4Grimes C.A. Jope R.S. Prog. Neurobiol. 2001; 65: 391-426Crossref PubMed Scopus (1304) Google Scholar). Recent studies have indicated that GSK-3 localization is dynamically regulated. Although GSK-3 is found predominantly in the cytoplasm of most cells, it has been found to enter the nucleus during S phase of the cell cycle and during apoptosis (5Bijur G.N., De Sarno P. Jope R.S. J. Biol. Chem. 2000; 275: 7583-7590Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 6Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (450) Google Scholar, 7Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1851) Google Scholar, 8Bijur G.N. Jope R.S. J. Biol. Chem. 2001; 276: 37436-37442Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). By contrast, GSK-3 has been shown to exit the nucleus of cardiac myocytes upon stimulation with isoproterenol (9Morisco C. Seta K. Hardt S.E. Lee Y. Vatner S.F. Sadoshima J. J. Biol. Chem. 2001; 276: 28586-28597Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Significantly, GSK-3 nuclear accumulation correlates with increased phosphorylation of nuclear substrates. For example, the increased levels of nuclear GSK-3 in S phase result in the increased phosphorylation of cyclin D1 (6Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (450) Google Scholar). The nuclear accumulation of GSK-3 during apoptosis suggests that the phosphorylation of other substrates may be involved in apoptotic signaling. This is supported by evidence indicating that GSK-3 inhibition promotes cell survival, whereas increased GSK-3 activity potentiates apoptosis (5Bijur G.N., De Sarno P. Jope R.S. J. Biol. Chem. 2000; 275: 7583-7590Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar,10Crowder R.J. Freeman R.S. J. Biol. Chem. 2000; 275: 34266-34271Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). GSK-3 also plays a role in controlling the localization of several substrates. Phosphorylation of cyclin D1, GATA-4, Dd-STATa (Dictyostelium STAT (signal transducers and activators of transcription) protein), TIMELESS, and NF-ATc (nuclear factor of activated T-cells) by GSK-3 alters their nuclear/cytoplasmic distribution (6Alt J.R. Cleveland J.L. Hannink M. Diehl J.A. Genes Dev. 2000; 14: 3102-3114Crossref PubMed Scopus (450) Google Scholar, 9Morisco C. Seta K. Hardt S.E. Lee Y. Vatner S.F. Sadoshima J. J. Biol. Chem. 2001; 276: 28586-28597Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 11Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Science. 1997; 275: 1930-1933Crossref PubMed Scopus (633) Google Scholar, 12Ginger R.S. Dalton E.C. Ryves W.J. Fukuzawa M. Williams J.G. Harwood A.J. EMBO J. 2000; 19: 5483-5491Crossref PubMed Scopus (58) Google Scholar, 13Martinek S. Inonog S. Manoukian A.S. Young M.W. Cell. 2001; 105: 769-779Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). The mechanisms by which GSK-3 localization is controlled remain unclear. Analysis of the amino acid sequence does not reveal the presence of any recognizable nuclear import or export sequences. Thus GSK-3 localization may be regulated indirectly through associations with binding proteins. Frat/GBP (frequentlyrearranged in T-cell lymphomas/GSK-3-binding protein) is a recently identified GSK-3-binding protein (14Jonkers J. Korswagen H.C. Acton D. Breuer M. Berns A. EMBO J. 1997; 16: 441-450Crossref PubMed Scopus (115) Google Scholar, 15Yost C. Farr III, G.H. Pierce S.B. Ferkey D.M. Mingzi Chen M. Kimelman D. Cell. 1998; 93: 1031-1041Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Previous studies have suggested a possible role in Wnt signaling because of its interaction with the Wnt signaling protein Dsh (16Li L. Yuan H. Weaver C.D. Mao J. Farr III, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (356) Google Scholar). In this study we investigated the possibility that Frat may play a role in regulating GSK-3 localization. Consistent with this hypothesis, we found that Frat contained a nuclear export sequence (NES), and we show that this activity allows Frat to direct the nuclear export of exogenous GSK-3. In addition we also provide evidence that an endogenous Frat-like function is involved in regulating GSK-3 localization since inhibition of nuclear export or the disruption of the Frat-GSK-3 interaction resulted in the nuclear accumulation of GSK-3. We particularly thank Jos Jonkers, Renée van Amerongen, Anton Berns, Mike Jones, Laurence Pearl, Rana Dajani, Chris Marshall, and Hugh Paterson for useful comments and help.