Glycogen Synthase Kinase 3β Interacts with and Phosphorylates the Spindle-associated Protein Astrin

Abstract

Emerging evidence shows that glycogen synthase kinase 3β (GSK3β) is involved in mitotic division and that inhibiting of GSK3β kinase activity causes defects in spindle microtubule length and chromosome alignment. However, the purpose of GSK3β involvement in spindle microtubule assembly and accurate chromosome segregation remains obscure. Here, we report that GSK3β interacts with the spindle-associated protein Astrin both in vitro and in vivo. Additionally, Astrin acts as a substrate for GSK3β and is phosphorylated at Thr-111, Thr-937 ((S/T)P motif) and Ser-974/Thr-978 ((S/T)XXX(S/T)-p motif; p is a phosphorylatable residue). Inhibition of GSK3β impairs spindle and kinetochore accumulation of Astrin and spindle formation at mitosis, suggesting that Astrin association with the spindle microtubule and kinetochore may be dependent on phosphorylation by GSK3β. Conversely, depletion of Astrin by small interfering RNA has no detectable influence on the localization of GSK3β. Interestingly, in vitro assays demonstrated that Astrin enhances GSK3β-mediated phosphorylation of other substrates. Moreover, we showed that coexpression of Astrin and GSK3β differentially increases GSK3β-mediated Tau phosphorylation on an unprimed site. Collectively, these data indicate that GSK3β interacts with and phosphorylates the spindle-associated protein Astrin, resulting in targeting Astrin to the spindle microtubules and kinetochores. In turn, the GSK3β-Astrin complex may also facilitate further physiological and pathological phosphorylation. Emerging evidence shows that glycogen synthase kinase 3β (GSK3β) is involved in mitotic division and that inhibiting of GSK3β kinase activity causes defects in spindle microtubule length and chromosome alignment. However, the purpose of GSK3β involvement in spindle microtubule assembly and accurate chromosome segregation remains obscure. Here, we report that GSK3β interacts with the spindle-associated protein Astrin both in vitro and in vivo. Additionally, Astrin acts as a substrate for GSK3β and is phosphorylated at Thr-111, Thr-937 ((S/T)P motif) and Ser-974/Thr-978 ((S/T)XXX(S/T)-p motif; p is a phosphorylatable residue). Inhibition of GSK3β impairs spindle and kinetochore accumulation of Astrin and spindle formation at mitosis, suggesting that Astrin association with the spindle microtubule and kinetochore may be dependent on phosphorylation by GSK3β. Conversely, depletion of Astrin by small interfering RNA has no detectable influence on the localization of GSK3β. Interestingly, in vitro assays demonstrated that Astrin enhances GSK3β-mediated phosphorylation of other substrates. Moreover, we showed that coexpression of Astrin and GSK3β differentially increases GSK3β-mediated Tau phosphorylation on an unprimed site. Collectively, these data indicate that GSK3β interacts with and phosphorylates the spindle-associated protein Astrin, resulting in targeting Astrin to the spindle microtubules and kinetochores. In turn, the GSK3β-Astrin complex may also facilitate further physiological and pathological phosphorylation. Glycogen synthase kinase 3 (GSK3), 2The abbreviations used are: GSK3glycogen synthase kinase 3MTmicrotubulesiRNAsmall interfering RNAaaamino acidGSTglutathione S-transferaseGFPgreen fluorescent protein. a serine/threonine kinase active in several signaling pathways, is involved in the regulation of cell fate, including Wnt and Hedgehog signal transduction, protein synthesis, glycogen metabolism, mitosis and apoptosis (1Ali A. Hoeflich K.P. Woodgett J.R. Chem. Rev. 2001; 101: 2527-2540Crossref PubMed Scopus (348) Google Scholar, 2Cohen P. Frame S. Nat. Rev. Mol. Cell Biol. 2001; 2: 769-776Crossref PubMed Scopus (1304) Google Scholar, 3Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar, 4Jope R.S. Yuskaitis C.J. Beurel E. Neurochem. Res. 2007; 32: 577-595Crossref PubMed Scopus (642) Google Scholar). GSK3 has two structurally similar isoforms in mammals, GSK3α and GSK3β, that are ubiquitously expressed and differ in their N- and C-terminal regions (2Cohen P. Frame S. Nat. Rev. Mol. Cell Biol. 2001; 2: 769-776Crossref PubMed Scopus (1304) Google Scholar, 5Doble B.W. Woodgett J.R. J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1774) Google Scholar). Earlier reports indicated that not only are the developmental profiles of GSK3α and GSK3β expression different but also the regulation and functions of these two proteins are not always identical (6Wang Q.M. Park I.K. Fiol C.J. Roach P.J. DePaoli-Roach A.A. Biochemistry. 1994; 33: 143-147Crossref PubMed Scopus (66) Google Scholar, 7Phiel C.J. Wilson C.A. Lee V.M. Klein P.S. Nature. 2003; 423: 435-439Crossref PubMed Scopus (1101) Google Scholar, 8Liang M.H. Chuang D.M. J. Biol. Chem. 2007; 282: 3904-3917Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 9Liang M.H. Chuang D.M. J. Biol. Chem. 2006; 281: 30479-30484Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Factors known to influence the functions of GSK3β include the phosphorylation of GSK3β itself, the subcellular localization of GSK3β, the protein-protein interaction of GSK3β, and the phosphorylation state of GSK3β substrates (1Ali A. Hoeflich K.P. Woodgett J.R. Chem. Rev. 2001; 101: 2527-2540Crossref PubMed Scopus (348) Google Scholar, 3Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar, 4Jope R.S. Yuskaitis C.J. Beurel E. Neurochem. Res. 2007; 32: 577-595Crossref PubMed Scopus (642) Google Scholar, 10Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (758) Google Scholar). Insulin-mediated inhibition of GSK3 was mediated through a phosphorylation-dependent mechanism, with the phosphorylation at position Ser-21 and Ser-9 in GSK3α and GSK3β, respectively (11Cohen P. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1999; 354: 485-495Crossref PubMed Scopus (135) Google Scholar). glycogen synthase kinase 3 microtubule small interfering RNA amino acid glutathione S-transferase green fluorescent protein. GSK3β also has a preference for pre-phosphorylated substrates, recognizing the consensus sequence (S/T)XXX(S/T)-p. In this sequence the first S/T residue is the target for GSK3β phosphorylation, X is any amino acid, P denotes the phosphorylatable residue, and the S/T located at the C terminus is the phosphorylation priming site (12Fiol C.J. Mahrenholz A.M. Wang Y. Roeske R.W. Roach P.J. J. Biol. Chem. 1987; 262: 14042-14048Abstract Full Text PDF PubMed Google Scholar). On the other hand, a number of proteins, including Axin and Tau, are phosphorylated by GSK3β without pre-phosphorylation. Recombinant Tau is phosphorylated at Ser-396 by GSK3β directly at an (S/T)P motif (3Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar, 13Song J.S. Yang S.D. J. Protein Chem. 1995; 14: 95-105Crossref PubMed Scopus (58) Google Scholar, 14Cole A.R. Causeret F. Yaeirgi G. Hastie C.J. McLauchlan H. McManus E.J. Hernandez F. Eickholt B.J. Nikolic M. Sutherland C. J. Biol. Chem. 2006; 281: 16591-16598Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). In fact, GSK3β phosphorylates many microtubule-associated proteins, including Tau, MAP1B, MAP2, APC, CRMP-2, and neurofilament, to regulate microtubule growth and stability and reduce their binding ability with microtubules (2Cohen P. Frame S. Nat. Rev. Mol. Cell Biol. 2001; 2: 769-776Crossref PubMed Scopus (1304) Google Scholar, 3Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar, 15Goold R.G. Gordon-Weeks P.R. Mol. Cell. Neurosci. 2005; 28: 524-534Crossref PubMed Scopus (87) Google Scholar, 16Zhou F.Q. Snider W.D. Science. 2005; 308: 211-214Crossref PubMed Scopus (133) Google Scholar). Therefore, establishing and dynamic balancing of the microtubules depends on spatial-temporal regulation by GSK3β activity. Microtubules (MTs) dynamics need to be controlled through the cell cycle, especially in spindle apparatus assembling for successfully segregating chromosomes. In mammals, GSK3β involvement has been reported in the polarized anchoring of MTs at the cell periphery (17Etienne-Manneville S. Hall A. Nature. 2003; 421: 753-756Crossref PubMed Scopus (712) Google Scholar). Furthermore, other recent studies found that GSK3 β interacts with spindle apparatus during mitosis and accumulates at centrosomes and inhibiting GSK3β causes defects in extended astral microtubules and chromosome misalignment during mitosis (18Wakefield J.G. Stephens D.J. Tavare J.M. J. Cell Sci. 2003; 116: 637-646Crossref PubMed Scopus (135) Google Scholar, 19Tighe A. Ray-Sinha A. Staples O.D. Taylor S.S. BMC Cell Biol. 2007; 8: 34Crossref PubMed Scopus (84) Google Scholar). Thus, identifying the interacting partners and candidate substrates for GSK3β in spindle microtubule assembly has great importance. Astrin is a non-motor spindle-associated protein essential for cell cycle progression (20Chang M.S. Huang C.J. Chen M.L. Chen S.T. Fan C.C. Chu J.M. Lin W.C. Yang Y.C. Biochem. Biophys. Res. Commun. 2001; 287: 116-121Crossref PubMed Scopus (46) Google Scholar, 21Mack G.J. Compton D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14434-14439Crossref PubMed Scopus (127) Google Scholar, 22Gruber J. Harborth J. Schnabel J. Weber K. Hatzfeld M. J. Cell Sci. 2002; 115: 4053-4059Crossref PubMed Scopus (68) Google Scholar). This protein has a large coiled-coil domain at its C terminus and forms dimers or high order structures under physiological conditions (22Gruber J. Harborth J. Schnabel J. Weber K. Hatzfeld M. J. Cell Sci. 2002; 115: 4053-4059Crossref PubMed Scopus (68) Google Scholar). Interestingly, and of the to mitosis and apoptosis (20Chang M.S. Huang C.J. Chen M.L. Chen S.T. Fan C.C. Chu J.M. Lin W.C. Yang Y.C. Biochem. Biophys. Res. Commun. 2001; 287: 116-121Crossref PubMed Scopus (46) Google Scholar, 22Gruber J. Harborth J. Schnabel J. Weber K. Hatzfeld M. J. Cell Sci. 2002; 115: 4053-4059Crossref PubMed Scopus (68) Google Scholar, Lin C.C. M.S. Lee Huang Cell Res. 2007; PubMed Scopus Google Scholar). recent also showed that in the of Astrin, are to and J. U. J. Cell Biol. 2007; PubMed Scopus Google Scholar). indicate that Astrin is to spindle apparatus and chromosome the for GSK3β that are involved in spindle microtubule assembly and accurate chromosome this used a to for proteins that with GSK3β. This a GSK3β and spindle-associated protein Astrin and shows that the association of Astrin with spindle MTs and kinetochore GSK3β In the GSK3β-Astrin complex increases GSK3β-mediated phosphorylation on unprimed suggesting that this may be a in spindle and by and and Astrin, to amino C to amino Astrin and Astrin in by Astrin the and and and and of and Astrin, Astrin and Astrin Astrin was the at the and GSK3β was to the domain by the a GSK3β protein. GSK3β, was by a GSK3β the of was by used for the S. Nature. PubMed Scopus Google Scholar, R. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Mol. Biol. Google Scholar). the Astrin with the binding domain in the GSK3β was to the domain by the was with the and on the ability of the to on and with of with a by was in 3 of at to the was to a of to the expression of for 3 by and in of a of and was at for at with a in a to the and the was under for at 3 with the to the E. Astrin was on for to binding a protein and Astrin proteins, including or with of was to the and proteins the by at for by and with and with was by of and of and to the the was a at for was to at for to the and the was with for at by and with binding to the by of and by with in different of the cell cycle and by the proteins and by and by the or by the Cell RNA and at in with and and and with or RNA mediated by of was on as Harborth J. A. Weber K. Nature. 2001; PubMed Scopus Google Scholar, J. K. Weber K. J. Cell Sci. 2001; PubMed Google Scholar). Astrin and (22Gruber J. Harborth J. Schnabel J. Weber K. Hatzfeld M. J. Cell Sci. 2002; 115: 4053-4059Crossref PubMed Scopus (68) Google was used as the at a of in a with and RNA was the with and with in for at by at for with and was with cell an with data during the to to for and number for by spindle and that for number was by the by of Cell and cell in cell and at for was by at for in an Protein the of used for further or a the to and a was with with the at the of to at for with at for 3 with for at by with for 3 and to the Astrin proteins with GSK3β in the kinase with or without at for by protein phosphorylation but not other the was used to this of Astrin as a the functions of GSK3β in this GSK3β and Astrin a complex in the spindle was with GSK3β and binding Astrin, showed that GSK3β interacts with Astrin Additionally, the was with Astrin and that an coiled-coil with the GSK3β On the other hand, no interaction was a protein similar to GSK3β, and that Astrin binding to GSK3β is the interaction GSK3β and Astrin in GSK3β was on and with Astrin or of Astrin, amino or Astrin or Astrin was by GSK3β in vitro GSK3β and Astrin with other in physiological at or mitotic of the cell cycle, the expression of as an that both GSK3β and Astrin by but not by that these two proteins in vivo. the subcellular localization of these two proteins was on that during GSK3β and Astrin in the In mitotic to accumulation of GSK3β and Astrin at the spindle apparatus was the cell the GSK3β and Astrin not at the Astrin at the This that complex formation GSK3β and Astrin is in the of spindle and GSK3β and at the spindle during to of the cell at the indicated cell cycle by and with Astrin and GSK3β with of Astrin a of Astrin is a substrate of GSK3β, in vitro kinase assays Astrin, Astrin Astrin or Astrin as substrates with GSK3β in the of showed that Astrin, Astrin and Astrin phosphorylated by GSK3β. of the Astrin was data that Astrin is phosphorylated by GSK3β in vitro in two regions in Astrin, amino to and to recent of the mitotic spindle also the of phosphorylation on Astrin, the phosphorylation of is the as in the at and site at C-terminal in the of two GSK3β phosphorylation M. G. R. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google and two and to the (S/T)P is also reported that GSK3β phosphorylation site of a priming (S/T)XXX(S/T)-p. on this motif of a of two and that GSK3β phosphorylated these Astrin or with candidate serine/threonine to with Astrin, the phosphorylation by GSK3β of the was evidence for phosphorylation of Astrin by GSK3β. of these on GSK3β phosphorylation of Astrin and expressed as as in suggesting that GSK3β phosphorylates Astrin at these further the of these phosphorylation with Astrin or Astrin of to the by with Astrin at the spindle with Astrin was at the spindle the cell an spindle the phosphorylation of Astrin be also in vivo. at mitosis with Astrin protein mitotic in an that and On the other hand, the protein the mitotic cell as as that the cell the protein was with 3 and that Astrin was phosphorylated in mitotic that Astrin is phosphorylated by GSK3β during the mitotic we in a that or A.A. D.A. G.J. P.S. R.W. Chem. Biol. Full Text Full Text PDF PubMed Scopus Google to the of GSK3β and the of data showed that mitotic Astrin in was by the with GSK3β that Astrin was phosphorylated by GSK3β at the inhibition of GSK3β the phosphorylation of Astrin, in the Astrin was as a also that or in and suggesting that inhibiting GSK3β activity cell in GSK3β for the of Astrin phosphorylation by GSK3β is for localization at the spindle we used GSK3β and the mitotic Astrin and used to the spindle MTs and at by and with or GSK3β and at the with mitotic two spindle and localization of Astrin or proteins and a spindle as spindle with the site of spindle and also found that inhibiting the GSK3β kinase activity the assembly of Astrin on the spindle MTs and Astrin the conditions the localization of was spindle association of mitotic kinase was not by inhibition of GSK3β activity and of and GSK3β kinase was spindle was a in and was a to On the other hand, showed that and of signal of Astrin on spindle apparatus to suggesting that the defects in spindle MTs may not with spindle anchoring and but may spindle recent also showed that a of Astrin is with the and in the of Astrin, are J. U. J. Cell Biol. 2007; PubMed Scopus Google Scholar). Thus, we the of Astrin In Astrin with the mitotic kinase and protein many and and Astrin was in and at the of was at that GSK3β activity is for localization of Astrin in further the of the interaction GSK3β and Astrin, was the to the expression of Astrin in showed spindle In GSK3β with Astrin at the spindle apparatus Furthermore, in was no detectable with the localization of GSK3β these indicate that the GSK3β kinase activity is essential for the spindle assembly of Conversely, the spindle association of GSK3β not on the of Astrin GSK3β-mediated Astrin interacts with GSK3β, the of the GSK3β-Astrin complex on the activity of GSK3β was recent showed that is a and the phosphorylation in are located in the C-terminal M.H. Huang Biochem. Biophys. Res. Commun. 2003; 308: PubMed Scopus Google Scholar, P. A.M. 1999; PubMed Scopus Google with the Astrin binding Lin C.C. M.S. Lee Huang Cell Res. 2007; PubMed Scopus Google Scholar). Therefore, Astrin may GSK3β-mediated phosphorylation. the of Astrin on GSK3β-mediated phosphorylation, assays as a In a GSK3β was found to and Interestingly, in the of Astrin, the phosphorylation of was with and that Astrin enhances the kinase activity of GSK3β. Therefore, we also the of Astrin to GSK3β-mediated phosphorylation of Axin and Tau and in the of Astrin, phosphorylation at both Axin and Tau On the other hand, was used under the was no in activity in the or of Astrin the of the interaction domain in Astrin for the kinase activity of GSK3β. Astrin and Astrin the GSK3β-mediated phosphorylation of data that Astrin enhances phosphorylation mediated by GSK3β. Astrin protein to GSK3β and GSK3β activity in we the of Astrin on GSK3β-mediated phosphorylation in with Tau and GSK3β in the or of of Tau was by with expression of Astrin in phosphorylation at the unprimed In Astrin was GSK3β phosphorylation of Tau at the was not a of GSK3β-mediated Tau phosphorylation by with phosphorylation of was of Astrin on GSK3β phosphorylation of Tau at the unprimed site of and the site to the of GSK3β, and expressed as of the that Astrin the GSK3β-mediated Tau site phosphorylation by these data that Astrin GSK3β phosphorylation of an unprimed in Tau not the phosphorylation of a that Astrin enhances GSK3β activity through both and these as in both Astrin and amino of Astrin to facilitate the GSK3β-mediated phosphorylation of Tau with was with GSK3β However, amino at this was not a interaction protein are in these indicate that Astrin directly enhances GSK3β activity. In this report we the of the spindle-associated protein Astrin as an interaction and candidate substrate for GSK3β. In we that the association of Astrin with spindle and kinetochore GSK3β kinase activity. data also that Astrin GSK3β-mediated phosphorylation. that GSK3β and Astrin are at the spindle apparatus during GSK3β-Astrin on and Hall (17Etienne-Manneville S. Hall A. Nature. 2003; 421: 753-756Crossref PubMed Scopus (712) Google demonstrated that GSK3β is involved in the polarized anchoring of microtubules at the cell studies also demonstrated that inhibition of GSK3β causes mitosis as extended astral microtubules and chromosome misalignment (18Wakefield J.G. Stephens D.J. Tavare J.M. J. Cell Sci. 2003; 116: 637-646Crossref PubMed Scopus (135) Google Scholar, 19Tighe A. Ray-Sinha A. Staples O.D. Taylor S.S. BMC Cell Biol. 2007; 8: 34Crossref PubMed Scopus (84) Google Scholar). no candidate substrate for GSK3β has to a GSK3β and spindle MTs Here, we that the of coiled-coil of Astrin interacts directly with the GSK3β the C-terminal of Astrin is the for targeting Astrin to G.J. Compton D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14434-14439Crossref PubMed Scopus (127) Google Scholar). that GSK3β and Astrin and the GSK3β-Astrin complex may be to the spindle apparatus through However, the that the association of the GSK3β-Astrin complex with may complex the dynamic interaction GSK3β and spindle studies showed that both GSK3β and Astrin are to the spindle apparatus (18Wakefield J.G. Stephens D.J. Tavare J.M. J. Cell Sci. 2003; 116: 637-646Crossref PubMed Scopus (135) Google Scholar, 19Tighe A. Ray-Sinha A. Staples O.D. Taylor S.S. BMC Cell Biol. 2007; 8: 34Crossref PubMed Scopus (84) Google Scholar, M.S. Huang C.J. Chen M.L. Chen S.T. Fan C.C. Chu J.M. Lin W.C. Yang Y.C. Biochem. Biophys. Res. Commun. 2001; 287: 116-121Crossref PubMed Scopus (46) Google Scholar, 21Mack G.J. Compton D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14434-14439Crossref PubMed Scopus (127) Google Scholar, Lin C.C. M.S. Lee Huang Cell Res. 2007; PubMed Scopus Google Scholar). In this we that GSK3β and Astrin are in mitotic on the spindle apparatus the to the be that the assembly of a mitotic spindle an regulation of microtubule in and Thus, the GSK3β-Astrin complex association with a in spindle of Astrin is its association with suggesting that Astrin is a GSK3β target with an in at the In the was for Astrin but by suggesting subcellular localization Therefore, Astrin may be by other GSK3β at the as and protein kinase Astrin the and the on by kinase for Astrin phosphorylation has not been that Astrin is a candidate substrate of GSK3β 3 and On the other hand, two of phosphorylation are by a motif for other is that kinase is involved in the phosphorylation, as the and protein In M. G. R. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google the of two in kinase phosphorylation and in kinase phosphorylation the terminus of these may also a in the phosphorylation of GSK3β-mediated phosphorylation with to the physiological of that the spindle and the kinetochore association of Astrin may be dependent on phosphorylation by GSK3β and However, is the Astrin not the spindle MTs but the spindle is to that these spindle assembling through regulation of the Astrin interaction with other spindle-associated other is that phosphorylation at may be not for regulation of Astrin In studies be to GSK3β phosphorylation Astrin and the phosphorylation state of site through the cell remains to be Astrin is phosphorylated at the spindle or to the spindle phosphorylation. that GSK3β and Astrin are not at the during the and GSK3β activity is for Astrin in the mitotic Inhibition of GSK3β activity during mitosis polarized anchoring of spindle MTs and their may be to the spindle by an GSK3β phosphorylation at the On the other we the that GSK3β the phosphorylation of Astrin at by as and spindle assembly high mitotic chromosome studies indicate that protein kinase and the proteins in the may be involved in the cycle and formation of the mitotic spindle P. A.M. 1999; PubMed Scopus Google Scholar, P. R. 2004; 14: PubMed Scopus Google Scholar, S. K. Trends Cell Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. J. Cell Sci. 2007; PubMed Scopus Google Scholar, M. M. 2007; PubMed Scopus Google Scholar). of these by or inhibition in spindle chromosome and progression in GSK3β is the kinase involved in the Wnt and is the substrate glycogen proteins, including a and microtubule-associated proteins (3Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar, S. J. PubMed Scopus Google Scholar, R.G. R. Gordon-Weeks P.R. J. Cell Sci. 1999; PubMed Google Scholar, C. M. J. J. Cell Biol. PubMed Scopus Google Scholar). on inhibition of GSK3β in mitosis that GSK3β may be a spindle that protein spindle MTs and MTs and This is with several studies a for GSK3β in and microtubule at the (18Wakefield J.G. Stephens D.J. Tavare J.M. J. Cell Sci. 2003; 116: 637-646Crossref PubMed Scopus (135) Google Scholar, 19Tighe A. Ray-Sinha A. Staples O.D. Taylor S.S. BMC Cell Biol. 2007; 8: 34Crossref PubMed Scopus (84) Google Scholar, P. M. 2007; PubMed Scopus Google Scholar, Chen Wang S. W.D. Biophys. PubMed Scopus Google Scholar, C.C. Cell 2006; PubMed Scopus Google Scholar). that GSK3β kinase activity is as an in mitotic spindle segregation is not only mediated by spindle microtubules but also S.T. Trends Cell Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. W.C. J. Cell Sci. 2004; PubMed Scopus Google Scholar). In at with the is with by A. Ray-Sinha A. Staples O.D. Taylor S.S. BMC Cell Biol. 2007; 8: 34Crossref PubMed Scopus (84) Google Scholar). also found that Astrin and at with the that many with the the kinetochore J. U. J. Cell Biol. 2007; PubMed Scopus Google Scholar). that GSK3β kinase activity is involved in kinetochore or Astrin for Astrin and GSK3β on studies that GSK3β activity be by its interaction with Frame S. M. P. Cohen P. 1999; PubMed Scopus Google protein K. P.J. Johnson G.V. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google and Lin C.C. Wang C. Lee P.J. Huang Biochemistry. 2006; PubMed Scopus Google directly with GSK3β, in an of GSK3 activity. In this we found that GSK3β-mediated phosphorylation of substrates, as and Tau, is in the of Astrin in suggesting that Astrin GSK3β activity This is the first report to that a protein to GSK3β and is phosphorylated by GSK3β. Tau is a substrate of GSK3β both in and in has been demonstrated that Tau is by GSK3β, including both and unprimed (S/T)P (3Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar). phosphorylation state of Tau is directly to its ability to and of phosphorylation at a as the site is in Johnson G.V. J. Neurochem. 2004; PubMed Scopus Google Scholar). the phosphorylation of unprimed GSK3β in Tau, as the site may be pathological A. R.W. J. J. Cell Sci. PubMed Google Scholar). data that Astrin the GSK3β-mediated phosphorylation of Tau at unprimed that Astrin may a in the Tau pathological phosphorylation by GSK3β in In mitotic regulation of phosphorylation This of Tau phosphorylation, to as Tau has been B. R. G. Klein 1994; PubMed Scopus Google Scholar, U. F. S. Mol. Biol. Cell. 1995; PubMed Scopus Google Scholar, M. P. J. Cell Biol. PubMed Scopus Google Scholar, P. S. M. C. S. A. J. Neurochem. 2002; PubMed Scopus Google and Tau may also to spindle data indicate that Astrin may both physiological and pathological phosphorylation by GSK3β. In we the spindle-associated protein Astrin as a interaction and candidate substrate of GSK3β. data that GSK3β acts as a on spindle MTs and In we that the association of Astrin with spindle MTs and may be dependent on GSK3β kinase activity. Moreover, Astrin the GSK3β-Astrin complex increases GSK3β-mediated phosphorylation on unprimed suggesting that this is for spindle for and