14-3-3 Binding to Ataxin-1(ATXN1) Regulates Its Dephosphorylation at Ser-776 and Transport to the Nucleus
2011; Elsevier BV; Volume: 286; Issue: 40 Linguagem: Inglês
10.1074/jbc.m111.238527
ISSN1083-351X
AutoresShaojuan Lai, Brennon O’Callaghan, Huda Y. Zoghbi, Harry T. Orr,
Tópico(s)Ubiquitin and proteasome pathways
ResumoSpinocerebellar ataxia type 1 (SCA1) is a lethal neurodegenerative disorder caused by expansion of a polyglutamine tract in ATXN1. A prominent site of pathology in SCA1 is cerebellar Purkinje neurons where mutant ATXN1 must enter the nucleus to cause disease. In SCA1, phosphorylation of ATXN1 at Ser-776 modulates disease. Interestingly, Ser-776 is located within a region of ATXN1 that harbors several functional motifs including binding sites for 14-3-3, and splicing factors RBM17 and U2AF65. The interaction of ATXN1 with these proteins is thought to be regulated by the phosphorylation status of Ser-776. In addition, Ser-776 is adjacent to the NLS in ATXN1. Although pS776-ATXN1 is enriched in nuclear extracts of cerebellar cells, the vast majority of 14-3-3 is in the cytoplasmic fraction. We found that dephosphorylation of cytoplasmic pS776-ATXN1 is blocked by virtue of it being in a complex with 14-3-3. In addition, data suggest that binding of 14-3-3 to cytoplasmic ATXN1 impeded its transport to the nucleus, suggesting that 14-3-3 must disassociate from ATXN1 for transport of ATXN1 to the nucleus. Consistent with this hypothesis is the observation that once in the nucleus pS776 is able to be dephosphorylated. Evidence is presented that PP2A is the pS776-ATXN1 phosphatase in the mammalian cerebellum. In the nucleus, we propose that dephosphorylation of pS776-ATXN1 by PP2A regulates the interaction of ATXN1 with the splicing factors RBM17 and U2AF65. Spinocerebellar ataxia type 1 (SCA1) is a lethal neurodegenerative disorder caused by expansion of a polyglutamine tract in ATXN1. A prominent site of pathology in SCA1 is cerebellar Purkinje neurons where mutant ATXN1 must enter the nucleus to cause disease. In SCA1, phosphorylation of ATXN1 at Ser-776 modulates disease. Interestingly, Ser-776 is located within a region of ATXN1 that harbors several functional motifs including binding sites for 14-3-3, and splicing factors RBM17 and U2AF65. The interaction of ATXN1 with these proteins is thought to be regulated by the phosphorylation status of Ser-776. In addition, Ser-776 is adjacent to the NLS in ATXN1. Although pS776-ATXN1 is enriched in nuclear extracts of cerebellar cells, the vast majority of 14-3-3 is in the cytoplasmic fraction. We found that dephosphorylation of cytoplasmic pS776-ATXN1 is blocked by virtue of it being in a complex with 14-3-3. In addition, data suggest that binding of 14-3-3 to cytoplasmic ATXN1 impeded its transport to the nucleus, suggesting that 14-3-3 must disassociate from ATXN1 for transport of ATXN1 to the nucleus. Consistent with this hypothesis is the observation that once in the nucleus pS776 is able to be dephosphorylated. Evidence is presented that PP2A is the pS776-ATXN1 phosphatase in the mammalian cerebellum. In the nucleus, we propose that dephosphorylation of pS776-ATXN1 by PP2A regulates the interaction of ATXN1 with the splicing factors RBM17 and U2AF65. IntroductionExpansion of a CAG trinucleotide repeat that encodes a polyglutamine tract within the protein ATXN1 3The abbreviations used are: ATXN1/Atxn1human Ataxin-1/mouse Ataxin-1SCA1spinocerebellar ataxia type 1NLSnuclear localization signalOAokadaic acid. causes SCA1, a fatal progressive neurodegenerative disease (1Orr H.T. Chung M.Y. Banfi S. Kwiatkowski Jr., T.J. Servadio A. Beaudet A.L. McCall A.E. Duvick L.A. Ranum L.P. Zoghbi H.Y. Nat. Genet. 1993; 4: 221-226Crossref PubMed Scopus (1460) Google Scholar). Other neurodegenerative disorders that are also caused by expansions of polyglutamine tracts include Huntington disease, spinobulbar muscular atrophy (Kennedy disease), dentatorubral-pallidoluysian atrophy, and SCAs 2, 3 (Machado-Joseph disease), 6, 7, and 17 (2Orr H.T. Zoghbi H.Y. Annu. Rev. Neurosci. 2007; 30: 575-621Crossref PubMed Scopus (1074) Google Scholar). CAG repeats in mutant SCA1 alleles vary from 39 to 82, with age of onset (ranging from 4 to 74 years of age) and severity of disease being inversely correlated with length of the repeat (1Orr H.T. Chung M.Y. Banfi S. Kwiatkowski Jr., T.J. Servadio A. Beaudet A.L. McCall A.E. Duvick L.A. Ranum L.P. Zoghbi H.Y. Nat. Genet. 1993; 4: 221-226Crossref PubMed Scopus (1460) Google Scholar). A prominent site of neurodegeneration in SCA1 is Purkinje cells of the cerebellar cortex. In addition, subsets of neurons in the brainstem are affected as well (3Robitaille Y. Schut L. Kish S.J. Acta Neuropathol. 1995; 90: 572-581Crossref PubMed Scopus (105) Google Scholar).SCA1 is caused by an expansion of the glutamine tract in ATXN1. Yet, considerable data indicate that ATXN1 residues outside of the glutamine tract have a substantial impact on severity of disease (4Klement I.A. Skinner P.J. Kaytor M.D. Yi H. Hersch S.M. Clark H.B. Zoghbi H.Y. Orr H.T. Cell. 1998; 95: 41-53Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar, 5Emamian E.S. Kaytor M.D. Duvick L.A. Zu T. Tousey S.K. Zoghbi H.Y. Clark H.B. Orr H.T. Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 6Tsuda H. Jafar-Nejad H. Patel A.J. Sun Y. Chen H.K. Rose M.F. Venken K.J. Botas J. Orr H.T. Bellen H.J. Zoghbi H.Y. Cell. 2005; 122: 633-644Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 7Okazawa H. Rich T. Chang A. Lin X. Waragai M. Kajikawa M. Enokido Y. Komuro A. Kato S. Shibata M. Hatanaka H. Mouradian M.M. Sudol M. Kanazawa I. Neuron. 2002; 34: 701-713Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 8Tsai C.C. Kao H.Y. Mitzutani A. Banayo E. Rajan H. McKeown M. Evans R.M. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4047-4052Crossref PubMed Scopus (122) Google Scholar, 9Lam Y.C. Bowman A.B. Jafar-Nejad P. Lim J. Richman R. Fryer J.D. Hyun E.D. Duvick L.A. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2006; 127: 1335-1347Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 10Serra H.G. Duvick L. Zu T. Carlson K. Stevens S. Jorgensen N. Lysholm A. Burright E. Zoghbi H.Y. Clark H.B. Andresen J.M. Orr H.T. Cell. 2006; 127: 697-708Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 11Lim J. Crespo-Barreto J. Jafar-Nejad P. Bowman A.B. Richman R. Hill D.E. Orr H.T. Zoghbi H.Y. Nature. 2008; 452: 713-718Crossref PubMed Scopus (255) Google Scholar). Two highly conserved regions outside of the polyglutamine tract in ATXN1 that have such a role are the 120 residue ATXN1/HBP1 (AXH) domain, amino acids 570–689 (12de Chiara C. Giannini C. Adinolfi S. de Boer J. Guida S. Ramos A. Jodice C. Kioussis D. Pastore A. FEBS Lett. 2003; 551: 107-112Crossref PubMed Scopus (55) Google Scholar), and a short stretch of amino acids, residues 771–778, at the C terminus (13de Chiara C. Menon R.P. Strom M. Gibson T.J. Pastore A. PLoS One. 2009; 4: e8372Crossref PubMed Scopus (47) Google Scholar, 14Carlson K.M. Melcher L. Lai S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurogenetics. 2009; 23: 313-323Crossref PubMed Scopus (13) Google Scholar). While several transcription factors as well as RNA interact with ATXN1 via the AXH domain (6Tsuda H. Jafar-Nejad H. Patel A.J. Sun Y. Chen H.K. Rose M.F. Venken K.J. Botas J. Orr H.T. Bellen H.J. Zoghbi H.Y. Cell. 2005; 122: 633-644Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 9Lam Y.C. Bowman A.B. Jafar-Nejad P. Lim J. Richman R. Fryer J.D. Hyun E.D. Duvick L.A. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2006; 127: 1335-1347Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 15Yue S. Serra H.G. Zoghbi H.Y. Orr H.T. Hum. Mol. Genet. 2001; 10: 25-30Crossref PubMed Scopus (126) Google Scholar), the C-terminal region is of particular interest since the interaction of certain proteins with this region is impacted by length of the polyglutamine and/or phosphorylation of Ser-776 (11Lim J. Crespo-Barreto J. Jafar-Nejad P. Bowman A.B. Richman R. Hill D.E. Orr H.T. Zoghbi H.Y. Nature. 2008; 452: 713-718Crossref PubMed Scopus (255) Google Scholar, 14Carlson K.M. Melcher L. Lai S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurogenetics. 2009; 23: 313-323Crossref PubMed Scopus (13) Google Scholar). Moreover, data indicate that the phosphorylation status of Ser-776 has a critical role in regulating SCA1 pathogenesis (5Emamian E.S. Kaytor M.D. Duvick L.A. Zu T. Tousey S.K. Zoghbi H.Y. Clark H.B. Orr H.T. Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 16Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 17Duvick L. Barnes J. Ebner B. Agrawal S. Andresen M. Lim J. Giesler G.J. Zoghbi H.Y. Orr H.T. Neuron. 2010; 67: 929-935Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Transgenic mice expressing ATXN1[82Q] containing a phosphorylation resistant Ser to Ala (S776A) substitution fail to develop neurodegeneration (5Emamian E.S. Kaytor M.D. Duvick L.A. Zu T. Tousey S.K. Zoghbi H.Y. Clark H.B. Orr H.T. Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). In contrast, the potentially phospho-mimicking amino acid substitution S776D enhances pathogenesis induced by ATXN1[82Q] and converts wild type ATXN1[30Q] to a pathogenic protein (17Duvick L. Barnes J. Ebner B. Agrawal S. Andresen M. Lim J. Giesler G.J. Zoghbi H.Y. Orr H.T. Neuron. 2010; 67: 929-935Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar).Ser-776 is positioned within or immediately adjacent to three functional motifs in the C terminus of ATXN1; the NLS, amino acids 771–774 (4Klement I.A. Skinner P.J. Kaytor M.D. Yi H. Hersch S.M. Clark H.B. Zoghbi H.Y. Orr H.T. Cell. 1998; 95: 41-53Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar), the 14-3-3 binding site, amino acids 774–776 (14Carlson K.M. Melcher L. Lai S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurogenetics. 2009; 23: 313-323Crossref PubMed Scopus (13) Google Scholar, 16Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar), and the U2AF-homology ligand motif (ULM), amino acids 771–776 (14Carlson K.M. Melcher L. Lai S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurogenetics. 2009; 23: 313-323Crossref PubMed Scopus (13) Google Scholar). The ULM in ATXN1 binds to the U2AF-homology motifs (UHM) in RBM17 and U2AF65 (11Lim J. Crespo-Barreto J. Jafar-Nejad P. Bowman A.B. Richman R. Hill D.E. Orr H.T. Zoghbi H.Y. Nature. 2008; 452: 713-718Crossref PubMed Scopus (255) Google Scholar, 14Carlson K.M. Melcher L. Lai S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurogenetics. 2009; 23: 313-323Crossref PubMed Scopus (13) Google Scholar). Moreover, as seen for other ULM/UHM interactions (18Selenko P. Gregorovic G. Sprangers R. Stier G. Rhani Z. Krämer A. Sattler M. Mol. Cell. 2003; 11: 965-976Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), phosphorylation of Ser-776 in the ULM of ATXN1 seems to regulate its interaction with RBM17 (11Lim J. Crespo-Barreto J. Jafar-Nejad P. Bowman A.B. Richman R. Hill D.E. Orr H.T. Zoghbi H.Y. Nature. 2008; 452: 713-718Crossref PubMed Scopus (255) Google Scholar) and U2AF65 (14Carlson K.M. Melcher L. Lai S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurogenetics. 2009; 23: 313-323Crossref PubMed Scopus (13) Google Scholar). Thus, understanding how phosphorylation of Ser-776 is regulated is critical for understanding the biology of ATXN1.The phosphorylation state of a protein is a dynamic process dictated by both protein kinases and phosphatases. Ser-776 of ATXN1 lies within strong consensus phosphorylation sites for the kinases AKR mouse thymoma (Akt) and cyclic AMP-dependent protein kinase (PKA). While initial data using tissue culture cells and a Drosophila model of SCA1 indicated that Akt can phosphorylate Ser-776 (16Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar), more recent data suggests that PKA is the S776-ATXN1 kinase in the mammalian cerebellum (19Jorgensen N.D. Andresen J.M. Lagalwar S. Armstrong B. Stevens S. Byam C.E. Duvick L.A. Lai S. Jafar-Nejad P. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurochem. 2009; 110: 675-686Crossref PubMed Scopus (40) Google Scholar). In this study, we utilized a mouse cerebellar extract-based dephosphorylation assay to investigate the dephosphorylation of pS776-ATXN1. We present data indicating that the protein phosphatase 2A (PP2A) dephosphorylation of pS776-ATXN1 is restricted to the nucleus of cerebellar cells. In addition, data indicate that binding of 14-3-3 to pS776-ATXN1 is restricted to the cytoplasm and impedes the dephosphorylation of pS776 as well as entry of ATXN1 into nuclei of cerebellar cells. These data, along with pervious data (19Jorgensen N.D. Andresen J.M. Lagalwar S. Armstrong B. Stevens S. Byam C.E. Duvick L.A. Lai S. Jafar-Nejad P. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurochem. 2009; 110: 675-686Crossref PubMed Scopus (40) Google Scholar), support a model in which phosphorylation and dephosphorylation of ATXN1 at Ser-776 take place in separate subcellular compartments in the cerebellum. The binding of 14-3-3 to pS776-ATXN1 protects against dephosphorylation in the cytoplasm. Moreover, the pS776-ATXN1/14-3-3 complex must be disassociated in order for ATXN1 to be transported to the nucleus, implying that this disassociation is regulated.DISCUSSIONATXN1, the protein affected in the polyglutamine neurodegenerative disease SCA1, is phosphorylated at Ser-776 (5Emamian E.S. Kaytor M.D. Duvick L.A. Zu T. Tousey S.K. Zoghbi H.Y. Clark H.B. Orr H.T. Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 31Huttin E.L. Jedrychowski M.P. Elias J.E. Goswami T. Rad R. Ceausoleil S.A. Villén J. Haas W. Sowa M.E. Gygi S.P. Cell. 2010; 143: 1174-1189Abstract Full Text Full Text PDF PubMed Scopus (1188) Google Scholar). Phosphorylation of Ser-776 in ATXN1 occurs in the cytoplasm (19Jorgensen N.D. Andresen J.M. Lagalwar S. Armstrong B. Stevens S. Byam C.E. Duvick L.A. Lai S. Jafar-Nejad P. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurochem. 2009; 110: 675-686Crossref PubMed Scopus (40) Google Scholar), creating a binding site for members of a family of small acidic proteins collectively designated as the 14-3-3 proteins (13de Chiara C. Menon R.P. Strom M. Gibson T.J. Pastore A. PLoS One. 2009; 4: e8372Crossref PubMed Scopus (47) Google Scholar, 16Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). The functional consequence(s) of 14-3-3 binding to ATXN1 was previously unclear. Earlier work indicated that binding of 14-3-3 to pS776-ATXN1 stabilized ATXN1 (16Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar) with a subsequent study suggesting that 14-3-3 binding also regulates the interaction of ATXN1 with the splicing factors RBM17 and U2AF65 in the nucleus (13de Chiara C. Menon R.P. Strom M. Gibson T.J. Pastore A. PLoS One. 2009; 4: e8372Crossref PubMed Scopus (47) Google Scholar). Here we provide evidence for two functional consequences of 14-3-3 binding to pS776-ATXN1 that are likely to be restricted to the cytoplasm of cerebellar cells (Fig. 7). First, binding of 14-3-3 to cytoplasmic ATXN1 blocks dephosphorylation of pS776 by PP2A. Because unphosphorylated ATXN1 is more rapidly degraded than pS776-ATXN1, we propose that binding of 14-3-3 indirectly stabilizes ATXN1 by blocking pS776 dephosphorylation. In addition, binding of 14-3-3 to pS776-ATXN1 reduces the proportion of ATXN1 localized to the nucleus, presumably by masking the NLS in ATXN1 adjacent to Ser-776.A previous study revealed that 14-3-3 bound to both wild type and expanded mutant ATXN1 in transfected cells, promoting accumulation of nuclear ATXN1 in the absence of detectable 14-3-3 co-localizing with nuclear ATXN1 (16Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). The evidence presented here suggests that any increase in nuclear ATXN1 is an indirect effect of increasing the amount of cytoplasmic ATXN1 by 14-3-3. Even though pS776-ATXN1 was enriched in the nucleus, data indicate that binding of 14-3-3 to endogenous wild type ATXN1 in the cerebellum was largely restricted to the cytoplasm. First, the vast majority of 14-3-3 in the cerebellum localized to the cytoplasmic fraction. Because protection of a target protein from dephosphorylation is a previously described function of 14-3-3 (32Morrison D.K. Trends Cell Bio. 2008; 19: 16-23Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar), the inability of cytoplasmic ATXN1 to be dephosphorylated in an in vitro assay is consistent with the majority of it being in a complex with 14-3-3 protecting it from dephosphorylation. Furthermore, either disrupting the cytoplasmic 14-3-3/ATXN1 complex by adding the competing R18 peptide or reducing the level of 14-3-3 by siRNA promoted pS776-ATXN1 dephosphorylation.Earlier work shows that compared with S776-ATXN1 phospho-resistant A776-ATXN1 is relatively unstable in transfected cells (16Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar) and in the cerebellum of transgenic mice (19Jorgensen N.D. Andresen J.M. Lagalwar S. Armstrong B. Stevens S. Byam C.E. Duvick L.A. Lai S. Jafar-Nejad P. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurochem. 2009; 110: 675-686Crossref PubMed Scopus (40) Google Scholar). Here we showed that the stability of D776-ATXN1 in vivo, as assessed by relative ratio of protein to RNA, in the cerebellum of transgenic mice was comparable to the stability of S776-ATXN1. Because the majority of S776-ATXN1 is phosphorylated in the cerebellum (data not shown), these data argue that at least in terms of promoting the stability of ATXN1 D776 mimics pS776. Intriguingly, D776-ATXN1 is as stable as S776-ATXN1 even though it is unable to bind to 14-3-3 (13de Chiara C. Menon R.P. Strom M. Gibson T.J. Pastore A. PLoS One. 2009; 4: e8372Crossref PubMed Scopus (47) Google Scholar), suggesting that phosphorylation of Ser-776 is sufficient to stabilize ATXN1 and that 14-3-3 binding, by blocking dephosphorylation of pS776, protects ATXN1 from subsequent proteolysis in the cytoplasm.In contrast to the cytoplasm of cerebellar cells, we found that nuclear 14-3-3 levels were dramatically lower and that nuclear ATXN1 was readily dephosphorylated. Both of these results are consistent with little to no ATXN1 in a stable complex with 14-3-3 in the nucleus. Moreover, we found that in transgenic mice D776- and A776-ATXN1, forms of ATXN1 not able to bind to 14-3-3 (13de Chiara C. Menon R.P. Strom M. Gibson T.J. Pastore A. PLoS One. 2009; 4: e8372Crossref PubMed Scopus (47) Google Scholar, 16Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar), had a higher proportion of ATXN1 in the nuclear fraction than did mice expressing S776-ATXN1 that binds 14-3-3. In addition, siRNA-mediated knockdown of 14-3-3 in DAOY cells decreased the portion of endogenous ATXN1 in the cytoplasm and increased the fraction of ATXN1 in the nucleus. One should be cautious in concluding that the results obtained in DAOY cells accurately reflect the situation in Purkinje cells. DAOY cells are mitotic cells whereas the cerebellar neuronal cells are post-mitotic. Regardless, these data indicate that upon binding to pS776-ATXN1 14-3-3 masks the adjacent NLS and blocks transport of ATXN1 to the nucleus.The ability to affect subcellular location and function of a protein by concealing the NLS is known for several 14-3-3 targets (23Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1317) Google Scholar, 32Morrison D.K. Trends Cell Bio. 2008; 19: 16-23Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 33Tzivion, G., Dobson, M., Ramakrishnan, G. (2011) Biochim. Biophys. Acta, in press.Google Scholar). In order for these target proteins to be transported into the nucleus 14-3-3 must disassociate. For example, in the case of the FoxO transcription factors 14-3-3 dissociation is mediated by dephosphorylation of the target protein (31Huttin E.L. Jedrychowski M.P. Elias J.E. Goswami T. Rad R. Ceausoleil S.A. Villén J. Haas W. Sowa M.E. Gygi S.P. Cell. 2010; 143: 1174-1189Abstract Full Text Full Text PDF PubMed Scopus (1188) Google Scholar). Our observation that cytoplasmic ATXN1 was not dephosphorylated unless its interaction with 14-3-3 was first disrupted argues against pS776 dephosphorylation as a means by which the ATXN1/14-3-3 complex is dissociated. The mitotic activator Cdc25 is suppressed by phosphorylation at Ser-287 and docking with 14-3-3. In this case, phosphorylation of Cdc25 at a second site, Thr-138, reduces its affinity for 14-3-3 and is required for release of 14-3-3 (33Tzivion, G., Dobson, M., Ramakrishnan, G. (2011) Biochim. Biophys. Acta, in press.Google Scholar, 34Margolis S.S. Perry J.A. Forester C.M. Nutt L.K. Guo Y. Jardim M.J. Thomenius M.J. Freel C.D. Darbandi R. Ahn J.H. Arroyo J.D. Wang X.F. Shenolikar S. Nairn A.C. Dunphy W.G. Hahn W.C. Virshup D.M. Kornbluth S. Cell. 2006; 127: 759-773Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). We speculate that like Cdc25, a second post-translational modification of ATXN1 is required for its discharge from 14-3-3. Besides enabling ATXN1 to enter the nucleus, a potential advantage of a second post-translational mark on ATXN1 is that once in the nucleus its low affinity for 14-3-3 would presumably persist. de Chiara et al. (13de Chiara C. Menon R.P. Strom M. Gibson T.J. Pastore A. PLoS One. 2009; 4: e8372Crossref PubMed Scopus (47) Google Scholar) recently reported, using Iso-Thermal Calorimetry, that the Kd of a pS776-ATXN1 peptide for 14-3-3 (0.4 nm) was two orders of magnitude less than for U2AF65 and RBM17 (35.8 and 40.0 μm, respectively). Thus, it is likely to be critical for ATXN1's affinity for 14-3-3 to remain reduced for it to effectively interact with splicing factors RBM17 and U2AF65 in the nucleus, even in the presence of small amounts of 14-3-3.Last, several lines of evidence presented here support PP2A as the phosphatase that dephosphorylates pS776-ATXN1. In vitro PP2A was able to readily dephosphorylate pS776-ATXN1 and selective siRNA knockdown of PP2A increased the amount of pS776-ATXN1 in transfected HeLa cells. Notably, immunodepletion of PP2A decreased the ability of a cerebellar nuclear extract to dephosphorylate endogenous pS776-ATXN1. PP2A is a major brain serine/threonine phosphatase that participates in a variety of signaling pathways that modulate neuronal function and activity (23Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1317) Google Scholar). Of note is the reported role of PP2A in the second step of pre-RNA splicing (36Shi Y. Reddy B. Manley J.L. Mol. Cell. 2006; 23: 819-829Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) given that ATXN1 interacts with RBM17, which is also implicated in regulating the second step of splicing (37Lallena M.J. Chalmers K.J. Llamazares S. Lamond A.I. Valcárcel J. Cell. 2002; 109: 285-296Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). An important feature of PP2A activity, as well as that of PPPs in general, is the critical role regulatory subunits have in determining substrate specificity and cellular pattern of activity (23Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1317) Google Scholar). In the brain, the subcellular localization and cellular expression of the variable B regulatory subunits of PP2A is known to vary (38Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Crossref PubMed Scopus (148) Google Scholar). Of note, while Purkinje cells express both Bα and Bβ isoforms, cerebellar stellate and basket cells also express Bα with Bβ expression being restricted to Purkinje cells. In addition, Bα is found in the soma and nuclei of Purkinje cells while Bβ is not detected by immunohistochemistry in Purkinje cell nuclei.In summary, we found that 14-3-3 binding to cytoplasmic pS776-ATXN1 regulates the dephosphorylation and proteolytic degradation of ATXN1 as well as nuclear entry of pS776-ATXN1, thus, providing mechanistic insight into how 14-3-3 modulates the function of ATXN1. IntroductionExpansion of a CAG trinucleotide repeat that encodes a polyglutamine tract within the protein ATXN1 3The abbreviations used are: ATXN1/Atxn1human Ataxin-1/mouse Ataxin-1SCA1spinocerebellar ataxia type 1NLSnuclear localization signalOAokadaic acid. causes SCA1, a fatal progressive neurodegenerative disease (1Orr H.T. Chung M.Y. Banfi S. Kwiatkowski Jr., T.J. Servadio A. Beaudet A.L. McCall A.E. Duvick L.A. Ranum L.P. Zoghbi H.Y. Nat. Genet. 1993; 4: 221-226Crossref PubMed Scopus (1460) Google Scholar). Other neurodegenerative disorders that are also caused by expansions of polyglutamine tracts include Huntington disease, spinobulbar muscular atrophy (Kennedy disease), dentatorubral-pallidoluysian atrophy, and SCAs 2, 3 (Machado-Joseph disease), 6, 7, and 17 (2Orr H.T. Zoghbi H.Y. Annu. Rev. Neurosci. 2007; 30: 575-621Crossref PubMed Scopus (1074) Google Scholar). CAG repeats in mutant SCA1 alleles vary from 39 to 82, with age of onset (ranging from 4 to 74 years of age) and severity of disease being inversely correlated with length of the repeat (1Orr H.T. Chung M.Y. Banfi S. Kwiatkowski Jr., T.J. Servadio A. Beaudet A.L. McCall A.E. Duvick L.A. Ranum L.P. Zoghbi H.Y. Nat. Genet. 1993; 4: 221-226Crossref PubMed Scopus (1460) Google Scholar). A prominent site of neurodegeneration in SCA1 is Purkinje cells of the cerebellar cortex. In addition, subsets of neurons in the brainstem are affected as well (3Robitaille Y. Schut L. Kish S.J. Acta Neuropathol. 1995; 90: 572-581Crossref PubMed Scopus (105) Google Scholar).SCA1 is caused by an expansion of the glutamine tract in ATXN1. Yet, considerable data indicate that ATXN1 residues outside of the glutamine tract have a substantial impact on severity of disease (4Klement I.A. Skinner P.J. Kaytor M.D. Yi H. Hersch S.M. Clark H.B. Zoghbi H.Y. Orr H.T. Cell. 1998; 95: 41-53Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar, 5Emamian E.S. Kaytor M.D. Duvick L.A. Zu T. Tousey S.K. Zoghbi H.Y. Clark H.B. Orr H.T. Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 6Tsuda H. Jafar-Nejad H. Patel A.J. Sun Y. Chen H.K. Rose M.F. Venken K.J. Botas J. Orr H.T. Bellen H.J. Zoghbi H.Y. Cell. 2005; 122: 633-644Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 7Okazawa H. Rich T. Chang A. Lin X. Waragai M. Kajikawa M. Enokido Y. Komuro A. Kato S. Shibata M. Hatanaka H. Mouradian M.M. Sudol M. Kanazawa I. Neuron. 2002; 34: 701-713Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 8Tsai C.C. Kao H.Y. Mitzutani A. Banayo E. Rajan H. McKeown M. Evans R.M. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4047-4052Crossref PubMed Scopus (122) Google Scholar, 9Lam Y.C. Bowman A.B. Jafar-Nejad P. Lim J. Richman R. Fryer J.D. Hyun E.D. Duvick L.A. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2006; 127: 1335-1347Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 10Serra H.G. Duvick L. Zu T. Carlson K. Stevens S. Jorgensen N. Lysholm A. Burright E. Zoghbi H.Y. Clark H.B. Andresen J.M. Orr H.T. Cell. 2006; 127: 697-708Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 11Lim J. Crespo-Barreto J. Jafar-Nejad P. Bowman A.B. Richman R. Hill D.E. Orr H.T. Zoghbi H.Y. Nature. 2008; 452: 713-718Crossref PubMed Scopus (255) Google Scholar). Two highly conserved regions outside of the polyglutamine tract in ATXN1 that have such a role are the 120 residue ATXN1/HBP1 (AXH) domain, amino acids 570–689 (12de Chiara C. Giannini C. Adinolfi S. de Boer J. Guida S. Ramos A. Jodice C. Kioussis D. Pastore A. FEBS Lett. 2003; 551: 107-112Crossref PubMed Scopus (55) Google Scholar), and a short stretch of amino acids, residues 771–778, at the C terminus (13de Chiara C. Menon R.P. Strom M. Gibson T.J. Pastore A. PLoS One. 2009; 4: e8372Crossref PubMed Scopus (47) Google Scholar, 14Carlson K.M. Melcher L. Lai S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurogenetics. 2009; 23: 313-323Crossref PubMed Scopus (13) Google Scholar). While several transcription factors as well as RNA interact with ATXN1 via the AXH domain (6Tsuda H. Jafar-Nejad H. Patel A.J. Sun Y. Chen H.K. Rose M.F. Venken K.J. Botas J. Orr H.T. Bellen H.J. Zoghbi H.Y. Cell. 2005; 122: 633-644Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 9Lam Y.C. Bowman A.B. Jafar-Nejad P. Lim J. Richman R. Fryer J.D. Hyun E.D. Duvick L.A. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2006; 127: 1335-1347Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 15Yue S. Serra H.G. Zoghbi H.Y. Orr H.T. Hum. Mol. Genet. 2001; 10: 25-30Crossref PubMed Scopus (126) Google Scholar), the C-terminal region is of particular interest since the interaction of certain proteins with this region is impacted by length of the polyglutamine and/or phosphorylation of Ser-776 (11Lim J. Crespo-Barreto J. Jafar-Nejad P. Bowman A.B. Richman R. Hill D.E. Orr H.T. Zoghbi H.Y. Nature. 2008; 452: 713-718Crossref PubMed Scopus (255) Google Scholar, 14Carlson K.M. Melcher L. Lai S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurogenetics. 2009; 23: 313-323Crossref PubMed Scopus (13) Google Scholar). Moreover, data indicate that the phosphorylation status of Ser-776 has a critical role in regulating SCA1 pathogenesis (5Emamian E.S. Kaytor M.D. Duvick L.A. Zu T. Tousey S.K. Zoghbi H.Y. Clark H.B. Orr H.T. Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 16Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 17Duvick L. Barnes J. Ebner B. Agrawal S. Andresen M. Lim J. Giesler G.J. Zoghbi H.Y. Orr H.T. Neuron. 2010; 67: 929-935Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Transgenic mice expressing ATXN1[82Q] containing a phosphorylation resistant Ser to Ala (S776A) substitution fail to develop neurodegeneration (5Emamian E.S. Kaytor M.D. Duvick L.A. Zu T. Tousey S.K. Zoghbi H.Y. Clark H.B. Orr H.T. Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). In contrast, the potentially phospho-mimicking amino acid substitution S776D enhances pathogenesis induced by ATXN1[82Q] and converts wild type ATXN1[30Q] to a pathogenic protein (17Duvick L. Barnes J. Ebner B. Agrawal S. Andresen M. Lim J. Giesler G.J. Zoghbi H.Y. Orr H.T. Neuron. 2010; 67: 929-935Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar).Ser-776 is positioned within or immediately adjacent to three functional motifs in the C terminus of ATXN1; the NLS, amino acids 771–774 (4Klement I.A. Skinner P.J. Kaytor M.D. Yi H. Hersch S.M. Clark H.B. Zoghbi H.Y. Orr H.T. Cell. 1998; 95: 41-53Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar), the 14-3-3 binding site, amino acids 774–776 (14Carlson K.M. Melcher L. Lai S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurogenetics. 2009; 23: 313-323Crossref PubMed Scopus (13) Google Scholar, 16Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar), and the U2AF-homology ligand motif (ULM), amino acids 771–776 (14Carlson K.M. Melcher L. Lai S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurogenetics. 2009; 23: 313-323Crossref PubMed Scopus (13) Google Scholar). The ULM in ATXN1 binds to the U2AF-homology motifs (UHM) in RBM17 and U2AF65 (11Lim J. Crespo-Barreto J. Jafar-Nejad P. Bowman A.B. Richman R. Hill D.E. Orr H.T. Zoghbi H.Y. Nature. 2008; 452: 713-718Crossref PubMed Scopus (255) Google Scholar, 14Carlson K.M. Melcher L. Lai S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurogenetics. 2009; 23: 313-323Crossref PubMed Scopus (13) Google Scholar). Moreover, as seen for other ULM/UHM interactions (18Selenko P. Gregorovic G. Sprangers R. Stier G. Rhani Z. Krämer A. Sattler M. Mol. Cell. 2003; 11: 965-976Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), phosphorylation of Ser-776 in the ULM of ATXN1 seems to regulate its interaction with RBM17 (11Lim J. Crespo-Barreto J. Jafar-Nejad P. Bowman A.B. Richman R. Hill D.E. Orr H.T. Zoghbi H.Y. Nature. 2008; 452: 713-718Crossref PubMed Scopus (255) Google Scholar) and U2AF65 (14Carlson K.M. Melcher L. Lai S. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurogenetics. 2009; 23: 313-323Crossref PubMed Scopus (13) Google Scholar). Thus, understanding how phosphorylation of Ser-776 is regulated is critical for understanding the biology of ATXN1.The phosphorylation state of a protein is a dynamic process dictated by both protein kinases and phosphatases. Ser-776 of ATXN1 lies within strong consensus phosphorylation sites for the kinases AKR mouse thymoma (Akt) and cyclic AMP-dependent protein kinase (PKA). While initial data using tissue culture cells and a Drosophila model of SCA1 indicated that Akt can phosphorylate Ser-776 (16Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar), more recent data suggests that PKA is the S776-ATXN1 kinase in the mammalian cerebellum (19Jorgensen N.D. Andresen J.M. Lagalwar S. Armstrong B. Stevens S. Byam C.E. Duvick L.A. Lai S. Jafar-Nejad P. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurochem. 2009; 110: 675-686Crossref PubMed Scopus (40) Google Scholar). In this study, we utilized a mouse cerebellar extract-based dephosphorylation assay to investigate the dephosphorylation of pS776-ATXN1. We present data indicating that the protein phosphatase 2A (PP2A) dephosphorylation of pS776-ATXN1 is restricted to the nucleus of cerebellar cells. In addition, data indicate that binding of 14-3-3 to pS776-ATXN1 is restricted to the cytoplasm and impedes the dephosphorylation of pS776 as well as entry of ATXN1 into nuclei of cerebellar cells. These data, along with pervious data (19Jorgensen N.D. Andresen J.M. Lagalwar S. Armstrong B. Stevens S. Byam C.E. Duvick L.A. Lai S. Jafar-Nejad P. Zoghbi H.Y. Clark H.B. Orr H.T. J. Neurochem. 2009; 110: 675-686Crossref PubMed Scopus (40) Google Scholar), support a model in which phosphorylation and dephosphorylation of ATXN1 at Ser-776 take place in separate subcellular compartments in the cerebellum. The binding of 14-3-3 to pS776-ATXN1 protects against dephosphorylation in the cytoplasm. Moreover, the pS776-ATXN1/14-3-3 complex must be disassociated in order for ATXN1 to be transported to the nucleus, implying that this disassociation is regulated.
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