Nuclear Export and Centrosome Targeting of the Protein Phosphatase 2A Subunit B56α

Abstract

Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B′/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56α, -β, and -ϵ isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56α subcellular localization. We detected B56α in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56α. Conversely, CRM1 overexpression shifted B56α to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451–469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56α. Active NESs were identified at similar positions in the cytoplasmic B56-β and ϵ isoforms, but not in the nuclear-localized B56-δ or γ isoforms. The transient expression of B56α induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56α co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56α-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56α can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations. Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B′/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56α, -β, and -ϵ isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56α subcellular localization. We detected B56α in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56α. Conversely, CRM1 overexpression shifted B56α to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451–469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56α. Active NESs were identified at similar positions in the cytoplasmic B56-β and ϵ isoforms, but not in the nuclear-localized B56-δ or γ isoforms. The transient expression of B56α induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56α co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56α-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56α can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations. IntroductionReversible protein phosphorylation is a key mechanism regulating a myriad of cellular processes. A delicate balance between the opposing effects of protein kinases and phosphatases determines the functional state of many proteins. Protein phosphatase 2A (PP2A) 3The abbreviations used are: PP2Aprotein phosphatase 2AAPCadenomatous polyposis coliFRAPfluorescence recovery after photobleachingGFPgreen fluorescent proteinNLSnuclear localization signalNESnuclear export sequencePBSphosphate-buffered salineYFPyellow fluorescent proteinHAhemagglutininLMBLeptomycin BmAbmonoclonal antibody. refers to a major family of heterotrimeric serine-threonine phosphatase enzymes in the cell (1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1511) Google Scholar, 2Eichhorn P.J. Creyghton M.P. Bernards R. Biochim. Biophys. Acta. 2009; 1795: 1-15Crossref PubMed Scopus (407) Google Scholar, 3Virshup D.M. Shenolikar S. Mol. Cell. 2009; 33: 537-545Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). The core enzyme is a dimer consisting of a 36-kDa catalytic C-subunit and a 65-kDa structural regulatory A-subunit, which acts as a scaffold to bring into proximity the C-subunit and protein substrates bound by the diverse regulatory B-subunits. There are four B-subunit gene families each with multiple genes that encode a range of splice variant peptides. The diverse nature of PP2A is inherent in its composition, which is potentially comprised of over 200 distinct protein complexes each containing different combinations of the A-, B-, and C-subunits, and hence allowing for variability and subtle regulation in phosphatase action (3Virshup D.M. Shenolikar S. Mol. Cell. 2009; 33: 537-545Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar).The B-subunits are postulated to regulate PP2A activity in different ways: (a) by targeting the holoenzyme to specific subcellular locations (e.g. B55α directs PP2A to microtubules (4Sontag E. Nunbhakdi-Craig V. Bloom G.S. Mumby M.C. J. Cell Biol. 1995; 128: 1131-1144Crossref PubMed Scopus (296) Google Scholar)), (b) determining substrate specificity (e.g. PP2A complexes containing B55 or B72 B-subunits cause the activation or inhibition of SV40 DNA replication, respectively, because of differences in substrate recognition sites on the SV40 large T antigen (5Cegielska A. Shaffer S. Derua R. Goris J. Virshup D.M. Mol. Cell. Biol. 1994; 14: 4616-4623Crossref PubMed Scopus (100) Google Scholar, 6Virshup D.M. Kauffman M.G. Kelly T.J. EMBO J. 1989; 8: 3891-3898Crossref PubMed Scopus (70) Google Scholar), and (c) by affecting the timing and tissue specificity of phosphatase activity because of differences in their expression.In contrast to the A- and C-subunits, which are each encoded by two tightly related families, the B-subunits derive from four disparate families that lack any sequence similarity. These gene families are known as the B/PR55, B′/PR61/B56 (hitherto referred to as the B56 family), B″/PR72 and B′ ″/PR93/PR110 families (reviewed in Refs. 1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1511) Google Scholar, 2Eichhorn P.J. Creyghton M.P. Bernards R. Biochim. Biophys. Acta. 2009; 1795: 1-15Crossref PubMed Scopus (407) Google Scholar). Five primary members of the B56 family (α, β, γ, δ, and ϵ) have been identified to date, and are encoded by distinct genes. B56 subunits share a highly conserved central region of 80% identity (which comprise two A-subunit binding domains), with variable N- and C-terminal regions postulated to direct the subcellular localization and substrate binding specificity of these proteins. It was proposed that B56α, B56β, and B56ϵ target PP2A activity to the cytoplasm, whereas B56δ and B56γ3 target it to the nucleus (7McCright B. Rivers A.M. Audlin S. Virshup D.M. J. Biol. Chem. 1996; 271: 22081-22089Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 8Gigena M.S. Ito A. Nojima H. Rogers T.B. Am. J. Physiol. Heart Circ. Physiol. 2005; 289: H285-H294Crossref PubMed Scopus (30) Google Scholar). Surprisingly, after more than a decade the intracellular targeting mechanisms responsible for nuclear and cytoplasmic positioning of the B56 subunits have not yet been defined.The PP2A B56 subunits play integral roles in cancer, through their regulation of c-Myc protein stability and expression (9Arnold H.K. Sears R.C. Mol. Cell. Biol. 2006; 26: 2832-2844Crossref PubMed Scopus (194) Google Scholar), Bcl-2 phosphorylation (10Ruvolo P.P. Clark W. Mumby M. Gao F. May W.S. J. Biol. Chem. 2002; 277: 22847-22852Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), and the Wnt signaling pathway (11Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (363) Google Scholar, 12Li X. Yost H.J. Virshup D.M. Seeling J.M. EMBO J. 2001; 20: 4122-4131Crossref PubMed Scopus (131) Google Scholar, 13Patturajan M. Nomoto S. Sommer M. Fomenkov A. Hibi K. Zangen R. Poliak N. Califano J. Trink B. Ratovitski E. Sidransky D. Cancer Cell. 2002; 1: 369-379Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 14Creyghton M.P. Roël G. Eichhorn P.J. Hijmans E.M. Maurer I. Destrée O. Bernards R. Genes Dev. 2005; 19: 376-386Crossref PubMed Scopus (61) Google Scholar). The key mediator of the Wnt signaling pathway is the multifunctional protein β-catenin, which when overexpressed causes transactivation of genes that directly promote cell transformation and cancer. B56α has been shown to bind proteins that regulate β-catenin stability, including APC (11Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (363) Google Scholar, 15Galea M.A. Eleftheriou A. Henderson B.R. J. Biol. Chem. 2001; 276: 45833-45839Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and p63 (13Patturajan M. Nomoto S. Sommer M. Fomenkov A. Hibi K. Zangen R. Poliak N. Califano J. Trink B. Ratovitski E. Sidransky D. Cancer Cell. 2002; 1: 369-379Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The B56β and B56γ subunits bind to axin (16Yamamoto H. Hinoi T. Michiue T. Fukui A. Usui H. Janssens V. Van Hoof C. Goris J. Asashima M. Kikuchi A. J. Biol. Chem. 2001; 276: 26875-26882Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), which is also a key component of the β-catenin and c-Myc degradation complexes. B56α has been shown to negatively regulate the Wnt signaling pathway by reducing β-catenin levels and inhibiting the transcription of β-catenin target genes through an APC-dependent mechanism (11Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (363) Google Scholar, 12Li X. Yost H.J. Virshup D.M. Seeling J.M. EMBO J. 2001; 20: 4122-4131Crossref PubMed Scopus (131) Google Scholar, 16Yamamoto H. Hinoi T. Michiue T. Fukui A. Usui H. Janssens V. Van Hoof C. Goris J. Asashima M. Kikuchi A. J. Biol. Chem. 2001; 276: 26875-26882Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar).We previously reported that B56α binds to the first four repeats of the Armadillo repeat domain of APC and promotes its nuclear accumulation (15Galea M.A. Eleftheriou A. Henderson B.R. J. Biol. Chem. 2001; 276: 45833-45839Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Given that B56α is also a key component of the β-catenin degradation complex, a more complete understanding of its subcellular location is important for understanding its functional roles. Therefore, in this study we used a range of biochemical and cell assays to detect B56α and characterize its nuclear transport and centrosomal localization in fixed cells and live cells. Its cytoplasmic distribution was regulated by nuclear export, and we identify a functional nuclear export sequence (NES) in the C terminus of B56α, and show that this sequence in conserved only in those B56 subunits (β and ϵ) known to locate predominantly to the cytoplasm. Functional implications including a role for B56α in nuclear export of the PP2A C-subunit are discussed.DISCUSSIONThe regulatory B56 subunits of PP2A play important functional roles in different signaling pathways however little attention has been given to their regulation. In this study we addressed the intracellular transport and distribution of the B56α subunit (outlined in Fig. 9), and show that both endogenous and ectopic forms of B56α are predominantly cytoplasmic and shift to the nucleus after inactivation or silencing of the CRM1 export receptor. Consistent with a regulatory role, the nuclear localization and export activities were contained within the N-terminal and C-terminal ends of B56α, respectively, thereby flanking the central PP2A active subunit binding site. We identified a functional NES at the C terminus of B56α and this sequence was required for B56α-mediated nuclear export of the PP2A catalytic subunit. Nuclear export signals were also identified at similar positions in two other B56 subunits known to locate in the cytoplasm. We further detected B56α at the centrosome where it displayed a slow turnover in vivo, indicative of a stable retention and into the role of B56α as a PP2A and have implications for a role of CRM1-mediated nuclear export in the of other PP2A B regulatory of B56α a activity within the (13Patturajan M. Nomoto S. Sommer M. Fomenkov A. Hibi K. Zangen R. Poliak N. Califano J. Trink B. Ratovitski E. Sidransky D. Cancer Cell. 2002; 1: 369-379Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) that B56α forms a with and the of p63 The protein was to bind to the terminus of B56α acids and its overexpression caused a nuclear accumulation of B56α in cells. In B56α bound to and in the nucleus, to the that the overexpression of in cells activation of and β-catenin stability and nuclear expression which are in cells. these were that to the role of the terminus of B56α in nuclear The nuclear accumulation of the B56α N-terminal sequence is of an active it is that binding as also B56α nuclear or We previously reported that B56α bound to the APC and its accumulation in the nucleus (15Galea M.A. Eleftheriou A. Henderson B.R. J. Biol. Chem. 2001; 276: 45833-45839Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). study is required to the nuclear activities and of of B56α as a Nuclear study on the that localization of PP2A complexes are regulated by is to with tissue and cell to the large of forms of PP2A that can only a reported regulation of PP2A subunits by nuclear The first a direct between the active A-subunit and members of the and postulated that the A-subunit regulate the transport or it to between nucleus and cytoplasm Biochem. Biophys. PubMed Scopus Google Scholar). A. E. M. M. 26: PubMed Scopus Google Scholar) reported that the catalytic C-subunit of PP2A in the nucleus in to of cells after identified a NES in the PP2A C-subunit between amino acids and the sequence was not directly for export In this study we the of endogenous C-subunit and that B56α was as as CRM1 in nuclear export of the C-subunit a amino (L461A) that blocked nuclear export of B56α, also blocked the regulation of These that B56α is of the PP2A C-subunit of the is that other B-subunits also regulate PP2A localization. J. A. W. S. J. J. Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar) identified a nuclear localization activity in the N-terminal amino acids of the B56ϵ subunit, and that a which the terminus was more an for B56ϵ and proposed that it contained an nuclear export signal within the region amino acids that to cytoplasmic localization. of a highly NES acids at the C terminus of B56ϵ to that study and the N-terminal of B56ϵ was more cytoplasmic than full-length B56ϵ J. A. W. S. J. J. Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). We that cells and as mechanisms to different or forms of the PP2A B56α, -β, and -ϵ regulatory subunits in different and cell thereby a of phosphatase and phosphatase activity regulates including cell and P.J. Creyghton M.P. Bernards R. Biochim. Biophys. Acta. 2009; 1795: 1-15Crossref PubMed Scopus (407) Google Scholar, 3Virshup D.M. Shenolikar S. Mol. Cell. 2009; 33: 537-545Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 2009; PubMed Scopus Google Scholar). The of B56α was previously to localization (10Ruvolo P.P. Clark W. Mumby M. Gao F. May W.S. J. Biol. Chem. 2002; 277: 22847-22852Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), however detected B56α at the Fig. The first cytoplasmic we was at the where B56α was at the nuclear envelope with into the cytoplasm. It to this localization a functional role nuclear as in nuclear or activity has also been detected at and at the in cells T. M.C. Cell 2006; PubMed Scopus Google Scholar) and in M. A. S. N. I. A. J.R. T. A. K. Cell. 128: Full Text Full Text PDF PubMed Scopus Google Scholar), with roles in and stability M. A. S. N. I. A. J.R. T. A. K. Cell. 128: Full Text Full Text PDF PubMed Scopus Google Scholar). In PP2A subunits are known to with at the centrosome T. M.C. Cell 2006; PubMed Scopus Google Scholar). PP2A was to at binding to the large centrosomal scaffold protein M. H. M. M. H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In this study we detected B56α at the centrosome by with of other PP2A B56 and at by T. M.C. Cell 2006; PubMed Scopus Google Scholar). The B56α was also detected at the which that targeting is of its NES and the targeting involved sequences that bind to the PP2A A-subunit, and B56α and the endogenous A-subunit were to at that B56α centrosomal localization is dependent on the PP2A than the other of B56α were at the centrosome with the that B56α is at the centrosome and an important functional to or centrosome B56α regulate the β-catenin given that other Wnt involved in of β-catenin localize to the These K. M. N. Kikuchi A. EMBO 2009; PubMed Scopus Google Scholar), APC S. V. T. J. Cell PubMed Scopus Google Scholar), and T. M. 26: PubMed Scopus Google Scholar). the centrosomal targeting of B56α to the role of PP2A in regulating an by that siRNA of B56α can in in cells B. M. S. S. E. C. R. C. G. Cancer 2009; PubMed Scopus Google we identified a nuclear export pathway for B56α and that this a mechanism for regulating nuclear accumulation of the PP2A catalytic subunit. The nuclear export signal we in B56α is not as we identified active NES sequences at similar positions in the related isoforms B56ϵ and B56β, which share sequence with B56α B. Virshup D.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, these have implications for regulation of cytoplasmic and nuclear PP2A phosphatase It is that or binding to proteins the NES and shift B56α to the B56α was detected at specific cytoplasmic the nuclear and the We further that a of cells displayed of B56α with at the the of cell Fig. The of B56α in PP2A of and a functional for IntroductionReversible protein phosphorylation is a key mechanism regulating a myriad of cellular processes. A delicate balance between the opposing effects of protein kinases and phosphatases determines the functional state of many proteins. Protein phosphatase 2A (PP2A) 3The abbreviations used are: PP2Aprotein phosphatase 2AAPCadenomatous polyposis coliFRAPfluorescence recovery after photobleachingGFPgreen fluorescent proteinNLSnuclear localization signalNESnuclear export sequencePBSphosphate-buffered salineYFPyellow fluorescent proteinHAhemagglutininLMBLeptomycin BmAbmonoclonal antibody. refers to a major family of heterotrimeric serine-threonine phosphatase enzymes in the cell (1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1511) Google Scholar, 2Eichhorn P.J. Creyghton M.P. Bernards R. Biochim. Biophys. Acta. 2009; 1795: 1-15Crossref PubMed Scopus (407) Google Scholar, 3Virshup D.M. Shenolikar S. Mol. Cell. 2009; 33: 537-545Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). The core enzyme is a dimer consisting of a 36-kDa catalytic C-subunit and a 65-kDa structural regulatory A-subunit, which acts as a scaffold to bring into proximity the C-subunit and protein substrates bound by the diverse regulatory B-subunits. There are four B-subunit gene families each with multiple genes that encode a range of splice variant peptides. The diverse nature of PP2A is inherent in its composition, which is potentially comprised of over 200 distinct protein complexes each containing different combinations of the A-, B-, and C-subunits, and hence allowing for variability and subtle regulation in phosphatase action (3Virshup D.M. Shenolikar S. Mol. Cell. 2009; 33: 537-545Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar).The B-subunits are postulated to regulate PP2A activity in different ways: (a) by targeting the holoenzyme to specific subcellular locations (e.g. B55α directs PP2A to microtubules (4Sontag E. Nunbhakdi-Craig V. Bloom G.S. Mumby M.C. J. Cell Biol. 1995; 128: 1131-1144Crossref PubMed Scopus (296) Google Scholar)), (b) determining substrate specificity (e.g. PP2A complexes containing B55 or B72 B-subunits cause the activation or inhibition of SV40 DNA replication, respectively, because of differences in substrate recognition sites on the SV40 large T antigen (5Cegielska A. Shaffer S. Derua R. Goris J. Virshup D.M. Mol. Cell. Biol. 1994; 14: 4616-4623Crossref PubMed Scopus (100) Google Scholar, 6Virshup D.M. Kauffman M.G. Kelly T.J. EMBO J. 1989; 8: 3891-3898Crossref PubMed Scopus (70) Google Scholar), and (c) by affecting the timing and tissue specificity of phosphatase activity because of differences in their expression.In contrast to the A- and C-subunits, which are each encoded by two tightly related families, the B-subunits derive from four disparate families that lack any sequence similarity. These gene families are known as the B/PR55, B′/PR61/B56 (hitherto referred to as the B56 family), B″/PR72 and B′ ″/PR93/PR110 families (reviewed in Refs. 1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1511) Google Scholar, 2Eichhorn P.J. Creyghton M.P. Bernards R. Biochim. Biophys. Acta. 2009; 1795: 1-15Crossref PubMed Scopus (407) Google Scholar). Five primary members of the B56 family (α, β, γ, δ, and ϵ) have been identified to date, and are encoded by distinct genes. B56 subunits share a highly conserved central region of 80% identity (which comprise two A-subunit binding domains), with variable N- and C-terminal regions postulated to direct the subcellular localization and substrate binding specificity of these proteins. It was proposed that B56α, B56β, and B56ϵ target PP2A activity to the cytoplasm, whereas B56δ and B56γ3 target it to the nucleus (7McCright B. Rivers A.M. Audlin S. Virshup D.M. J. Biol. Chem. 1996; 271: 22081-22089Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 8Gigena M.S. Ito A. Nojima H. Rogers T.B. Am. J. Physiol. Heart Circ. Physiol. 2005; 289: H285-H294Crossref PubMed Scopus (30) Google Scholar). Surprisingly, after more than a decade the intracellular targeting mechanisms responsible for nuclear and cytoplasmic positioning of the B56 subunits have not yet been defined.The PP2A B56 subunits play integral roles in cancer, through their regulation of c-Myc protein stability and expression (9Arnold H.K. Sears R.C. Mol. Cell. Biol. 2006; 26: 2832-2844Crossref PubMed Scopus (194) Google Scholar), Bcl-2 phosphorylation (10Ruvolo P.P. Clark W. Mumby M. Gao F. May W.S. J. Biol. Chem. 2002; 277: 22847-22852Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), and the Wnt signaling pathway (11Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (363) Google Scholar, 12Li X. Yost H.J. Virshup D.M. Seeling J.M. EMBO J. 2001; 20: 4122-4131Crossref PubMed Scopus (131) Google Scholar, 13Patturajan M. Nomoto S. Sommer M. Fomenkov A. Hibi K. Zangen R. Poliak N. Califano J. Trink B. Ratovitski E. Sidransky D. Cancer Cell. 2002; 1: 369-379Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 14Creyghton M.P. Roël G. Eichhorn P.J. Hijmans E.M. Maurer I. Destrée O. Bernards R. Genes Dev. 2005; 19: 376-386Crossref PubMed Scopus (61) Google Scholar). The key mediator of the Wnt signaling pathway is the multifunctional protein β-catenin, which when overexpressed causes transactivation of genes that directly promote cell transformation and cancer. B56α has been shown to bind proteins that regulate β-catenin stability, including APC (11Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (363) Google Scholar, 15Galea M.A. Eleftheriou A. Henderson B.R. J. Biol. Chem. 2001; 276: 45833-45839Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and p63 (13Patturajan M. Nomoto S. Sommer M. Fomenkov A. Hibi K. Zangen R. Poliak N. Califano J. Trink B. Ratovitski E. Sidransky D. Cancer Cell. 2002; 1: 369-379Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The B56β and B56γ subunits bind to axin (16Yamamoto H. Hinoi T. Michiue T. Fukui A. Usui H. Janssens V. Van Hoof C. Goris J. Asashima M. Kikuchi A. J. Biol. Chem. 2001; 276: 26875-26882Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), which is also a key component of the β-catenin and c-Myc degradation complexes. B56α has been shown to negatively regulate the Wnt signaling pathway by reducing β-catenin levels and inhibiting the transcription of β-catenin target genes through an APC-dependent mechanism (11Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (363) Google Scholar, 12Li X. Yost H.J. Virshup D.M. Seeling J.M. EMBO J. 2001; 20: 4122-4131Crossref PubMed Scopus (131) Google Scholar, 16Yamamoto H. Hinoi T. Michiue T. Fukui A. Usui H. Janssens V. Van Hoof C. Goris J. Asashima M. Kikuchi A. J. Biol. Chem. 2001; 276: 26875-26882Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar).We previously reported that B56α binds to the first four repeats of the Armadillo repeat domain of APC and promotes its nuclear accumulation (15Galea M.A. Eleftheriou A. Henderson B.R. J. Biol. Chem. 2001; 276: 45833-45839Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Given that B56α is also a key component of the β-catenin degradation complex, a more complete understanding of its subcellular location is important for understanding its functional roles. Therefore, in this study we used a range of biochemical and cell assays to detect B56α and characterize its nuclear transport and centrosomal localization in fixed cells and live cells. Its cytoplasmic distribution was regulated by nuclear export, and we identify a functional nuclear export sequence (NES) in the C terminus of B56α, and show that this sequence in conserved only in those B56 subunits (β and ϵ) known to locate predominantly to the cytoplasm. Functional implications including a role for B56α in nuclear export of the PP2A C-subunit are