Identification of Novel Glycogen Synthase Kinase-3β Substrate-interacting Residues Suggests a Common Mechanism for Substrate Recognition
2006; Elsevier BV; Volume: 281; Issue: 41 Linguagem: Inglês
10.1074/jbc.m604633200
ISSN1083-351X
AutoresRonit Ilouz, Noga Kowalsman, Miriam Eisenstein, Hagit Eldar-Finkelman,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoSubstrate recognition and specificity are essential for the reliability and fidelity of protein kinase function. GSK-3 has a unique substrate specificity that requires prior phosphorylation of its substrates. However, how the enzyme selects its phosphorylated substrates is unknown. Here, we combined in silico modeling with mutagenesis and biological studies to identify GSK-3-substrate interaction sites located within its binding cleft. Protein-protein docking of GSK-3β and the phosphorylated cAMP responsive element binding protein (pCREB) (using the available experimentally determined structures), identified Phe67, Gln89, and Asn95 of GSK-3β as putative binding sites interacting with the CREB phosphorylation motif. Mutations of these residues to alanine impaired GSK-3β phosphorylation of several substrates, without abrogating its autocatalytic activity. Subsequently, expression of the GSK-3β mutants in cells resulted in decreased phosphorylation of substrates CREB, IRS-1, and β-catenin, and prevented their suppression of glycogen synthase activity as compared with cells expressing the wild-type GSK-3β. Our studies provide important additional understanding of how GSK-3β recognizes its substrates: In addition to prior phosphorylation typically required in GSK-3 substrates, substrate recognition involves interactions with GSK-3β residues: Phe67, Gln89, and Asn95, which confer a common basis for substrate binding and selectivity, yet allow for substrate diversity. Substrate recognition and specificity are essential for the reliability and fidelity of protein kinase function. GSK-3 has a unique substrate specificity that requires prior phosphorylation of its substrates. However, how the enzyme selects its phosphorylated substrates is unknown. Here, we combined in silico modeling with mutagenesis and biological studies to identify GSK-3-substrate interaction sites located within its binding cleft. Protein-protein docking of GSK-3β and the phosphorylated cAMP responsive element binding protein (pCREB) (using the available experimentally determined structures), identified Phe67, Gln89, and Asn95 of GSK-3β as putative binding sites interacting with the CREB phosphorylation motif. Mutations of these residues to alanine impaired GSK-3β phosphorylation of several substrates, without abrogating its autocatalytic activity. Subsequently, expression of the GSK-3β mutants in cells resulted in decreased phosphorylation of substrates CREB, IRS-1, and β-catenin, and prevented their suppression of glycogen synthase activity as compared with cells expressing the wild-type GSK-3β. Our studies provide important additional understanding of how GSK-3β recognizes its substrates: In addition to prior phosphorylation typically required in GSK-3 substrates, substrate recognition involves interactions with GSK-3β residues: Phe67, Gln89, and Asn95, which confer a common basis for substrate binding and selectivity, yet allow for substrate diversity. Glycogen synthase kinase 3 (GSK-3) 4The abbreviations used are: GSK-3, glycogen synthase kinase-3; PKB, protein kinase B; p90RSK, S6 ribosomal protein kinase; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; CREB, cAMP response element-binding protein; CBP, CREB-binding protein; EGFP, enhanced green fluorescence protein; IRS-1, insulin receptor substrate-1; PDB, Protein Data Bank; WT, wild type; UDPG, uridine 5-diphosphate [14C]glucose. is a ubiquitous serine/threonine kinase expressed as two isoforms (α and β) (1Woodgett J.R. 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This inhibition is achieved through direct phosphorylation of N-terminal serine residues (Ser21 or Ser9 in α, β, respectively) by several protein kinases, such as PKB, p90RSK, PKA, and PKC (2Bardwell A.J. Flatauer L.J. Matsukuma K. Thorner J. Bardwell L. J. Biol. Chem. 2001; 276: 10374-10386Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 3Doble B.W. Woodgett J.R. J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1774) Google Scholar, 4Grimes C.A. Jope R.S. Prog. Neurobiol. 2001; 65: 391-426Crossref PubMed Scopus (1319) Google Scholar). GSK-3 also may be phosphorylated on Tyr216 located in the activation loop (5Hughes K. Nicolakaki E. Plyte S.E. Totty N.F. Woodgett J.R. EMBO J. 1993; 12: 803-808Crossref PubMed Scopus (525) Google Scholar). This phosphorylation is an autophosphorylation event as demonstrated by in vitro and in vivo cell systems (5Hughes K. Nicolakaki E. Plyte S.E. Totty N.F. Woodgett J.R. 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Previous studies formulated consensus sequences for optimal phosphorylation motifs of protein kinases using oriented peptide libraries, bioinformatics, and computational molecular modeling (15Kemp B.E. Pearson R.B. Trends Biochem. Sci. 1990; 15: 342-346Abstract Full Text PDF PubMed Scopus (807) Google Scholar, 16Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar, 17Brinkworth R.I. Breinl R.A. Kobe B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 74-79Crossref PubMed Scopus (154) Google Scholar, 18Yaffe M.B. Methods Mol. Biol. 2004; 250: 237-250PubMed Google Scholar, 19Alto N.M. Soderling S.H. Hoshi N. Langeberg L.K. Fayos R. Jennings P.A. Scott J.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4445-4450Crossref PubMed Scopus (146) Google Scholar). The increasing number of three-dimensional structures of protein kinases complexed with substrates had provided an important basis for understanding the mechanism of molecular recognition (20Taylor S.S. Radzio-Andzelm E. Hunter T. FASEB J. 1995; 9: 1255-1266Crossref PubMed Scopus (159) Google Scholar, 21Padilla A. Hauer J.A. Tsigelny I. Parello J. Taylor S.S. J. Pept. Res. 1997; 49: 210-220Crossref PubMed Scopus (7) Google Scholar, 22Narayana N. Cox S. Shaltiel S. Taylor S.S. Xuong N. Biochemistry. 1997; 36: 4438-4448Crossref PubMed Scopus (103) Google Scholar, 23Yang J. Cron P. Good V.M. Thompson V. Hemmings B.A. Barford D. Nat. Struct. Biol. 2002; 9: 940-944Crossref PubMed Scopus (436) Google Scholar). Still, our knowledge of the mechanisms by which protein kinases recognize their substrates is rather limited. GSK-3 has a unique substrate specificity that requires prior phosphorylation of its substrates in the context motif SXXS(p), where S(p) is the phosphorylated "priming" site (24Woodgett J.R. Cohen P. Biochim. Biophys. Acta. 1984; 788: 339-347Crossref PubMed Scopus (154) Google Scholar, 25Fiol 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, 26Roach P.J. J. Biol. Chem. 1991; 266: 14139-14142Abstract Full Text PDF PubMed Google Scholar). The three-dimensional structure of GSK-3β showed that three basic residues within the catalytic core, Arg96, Arg180, and Lys205, form a positive pocket that most likely serves as the docking site for the phosphorylated moiety of GSK-3 substrates (27ter Haar E. Coll J.T. Austen D.A. Hsiao H.M. Swenson L. Jain J. Nat. Struct. Biol. 2001; 8: 593-596Crossref PubMed Scopus (329) Google Scholar, 28Dajani R. Fraser E. Roe S.M. Young N. Good V. Dale T.C. Pearl L.H. Cell. 2001; 105: 721-732Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 29Bax B. Carter P.S. Lewis C. Guy A.R. Bridges A. Tanner R. Pettman G. Mannix C. Culbert A.A. Brown M.J. Smith D.G. Reith A.D. Structure. 2001; 9: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). GSK-3β-binding of Axin and APC was localized to a hydrophobic site in the C-terminal helical domain (29Bax B. Carter P.S. Lewis C. Guy A.R. Bridges A. Tanner R. Pettman G. Mannix C. Culbert A.A. Brown M.J. Smith D.G. Reith A.D. Structure. 2001; 9: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 30Dajani R. Fraser E. Roe S.M. Yeo M. Good V.M. Thompson V. Dale T.C. Pearl L.H. EMBO J. 2003; 22: 494-501Crossref PubMed Scopus (260) Google Scholar). This interaction site, however, is downstream from the catalyticsubstrate binding cleft and is not directly involved in the phosphorylation process (29Bax B. Carter P.S. Lewis C. Guy A.R. Bridges A. Tanner R. Pettman G. Mannix C. Culbert A.A. Brown M.J. Smith D.G. Reith A.D. Structure. 2001; 9: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Therefore, detailed knowledge of the interactions between the substrate amino acids near the priming phosphorylation site and GSK-3β is still desirable. In this study, we sought to determine the important substrate recognition sites in GSK-3β by combining in silico protein-protein docking of GSK-3β and the phosphorylated cAMP responsive element-binding protein (pCREB), based on the crystal structures of GSK-3β (28Dajani R. Fraser E. Roe S.M. Young N. Good V. Dale T.C. Pearl L.H. Cell. 2001; 105: 721-732Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 31Bertrand J.A. Thieffine S. Vulpetti A. Cristiani C. Valsasina B. Knapp S. Kalisz H.M. Flocco M. J. Mol. Biol. 2003; 333: 393-407Crossref PubMed Scopus (277) Google Scholar) and the NMR structure of pCREB (32Radhakrishnan I. Perez-Alvarado G.C. Parker D. Dyson H.J. Montminy M.R. Wright P.E. Cell. 1997; 91: 741-752Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar), with biological tools. We present a model structure of the ternary complex of GSK-3β, ATP, and the pCREB peptide. The docking model identified specific electrostatic and hydrophobic interactions between pCREB and three amino acids in GSK-3β. Mutagenesis of these sites impaired GSK-3 ability to phosphorylate CREB, confirming their importance for substrate recognition. Importantly, additional GSK-3 substrates were affected by the mutations as well. Hence, our studies identified novel GSK-3β sites involved in recognition of diverse substrates, and provide important data for rational drug design of compounds targeting GSK-3. Peptides and Materials—Peptides were synthesized by Genemed Synthesis Inc (San Francisco, USA). p9CREB, ILSRRP-S(p)YR, pIRS-1 RREGGMSRPAS(p)VDG, PGS-1 YRRAAVPP-SPSLSRHSSPSQS(p)EDEEE (where S(p) is a phosphoserine). The following antibodies were used in the study: anti GSK-3β (Transduction laboratory, Lexington, KY), anti-phospho-GSK-3 (Tyr216) and anti-phospho-CREB (Ser133) antibodies were obtained from Upstate Biotechnology (Lake Placid, NY), anti-phospho-CREB (Ser129/133) was obtained from BioSource International, Inc (Camarillo, CA), CREB antibody, anti-phospho-β-catenin, or β-catenin antibody were obtained from Cell Signaling Technology (Beverly, MA) and anti-phospho-IRS-1 (Ser332) were described before (33Liberman Z. Eldar-Finkelman H. J. Biol. Chem. 2005; 280: 4422-4428Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Radioactive materials were purchased from Amersham Biosciences. Molecular Modeling—The available x-ray structures of GSK-3β (28Dajani R. Fraser E. Roe S.M. Young N. Good V. Dale T.C. Pearl L.H. Cell. 2001; 105: 721-732Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 31Bertrand J.A. Thieffine S. Vulpetti A. Cristiani C. Valsasina B. Knapp S. Kalisz H.M. Flocco M. J. Mol. Biol. 2003; 333: 393-407Crossref PubMed Scopus (277) Google Scholar), one with phosphorylated Tyr216 (PDB (Ref. 34Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27935) Google Scholar) code 1o9u) and another with bound non-hydrolysable analog of ATP, ANP, (PDB code, 1pyx), were used to model the structure of a phosphorylated GSK-3·ATP complex (using the homology module of InsightII, Accelrys, San-Diego, CA). A model of the ternary complex GSK-3·ATP·CREB was obtained by protein-protein docking, using the program MolFit. The p9CREB fragments (residues 127–135) from the 17 NMR models of pCREB in complex with the co-activator CBP (32Radhakrishnan I. Perez-Alvarado G.C. Parker D. Dyson H.J. Montminy M.R. Wright P.E. Cell. 1997; 91: 741-752Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar) (PDB code, 1kdx) present some backbone variation in the turn region. We selected 3 variants of the p9CREB fragment, with different backbone and side chain conformations. In the first protein-protein docking step, we docked each fragment to the GSK-3·ATP model structure, employing the geometric (35Katchalski-Katzir E. Shariv I. Eisenstein M. Friesem A.A. Aflalo C. Vakser I.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2195-2199Crossref PubMed Scopus (867) Google Scholar), weighted-geometric (36Ben-Zeev E. Eisenstein M. Proteins. 2003; 52: 24-27Crossref PubMed Scopus (49) Google Scholar), geometric-electrostatic (37Heifetz A. Katchalski-Katzir E. Eisenstein M. Protein Sci. 2002; 11: 571-587Crossref PubMed Scopus (117) Google Scholar), and geometric-hydrophobic (38Berchanski A. Shapira B. Eisenstein M. Proteins. 2004; 351: 309-326Google Scholar) options in MolFit. Standard translation and rotation grid intervals were used (1.05 Å and 12°, respectively). The surface grid points that belong to the side chains of residues Arg96, Arg180, and Lys205 of GSK-3 (the primed phosphate binding site) were up-weighted in the weighted geometric docking search, thereby biasing the docking results to include more models in which these residues interact with any residue of p9CREB. The lists of solutions from the four individual docking scans were intersected. Thus, the final list of models included only models that appeared in all 4 docking searches, and each model was evaluated by a weighted-geometric-electrostatic-hydrophobic complementarity score, which is the sum of the (weighted-geometric) + (geometric-electrostatic – geometric) + (geometric-hydrophobic – geometric) scores. The first docking step clearly preferred one of the 3 conformers of p9CREB (see "Results" and "Discussion"). In a second docking step, the preferred conformer was extended by including also the N-terminal helix (N-pCREB; residues 119–135) and docked to GSK-3β·ATP. The best GSK-3β·ATP·N-pCREB model, which was very similar to the preferred GSK-3β·ATP·p9CREB model, was refined by testing small local relative rotations of the molecules (±2°, ±4°, and ±6°) and searching for the best shape and chemical complementarity. Next, we superposed each of the 17 NMR structures of pCREB onto the refined GSK-3β·ATP·N-pCREB model, using the common Cα atoms. This showed that although there is a large variation in the positions of the C-terminal helices of pCREB, only a few of them interact with GSK-3β. We selected a model with only few clashes with GSK-3β for the final modeling step, which consisted of 20 iterations of intermittent energy minimizations of the GSK-3β·ATP·pCREB ternary complex and dynamics simulations (10,000 steps of 1fs in each iteration). These computations also included a layer of water molecules around the complex (10-Å thick). We used the Discover-3 module in the InsightII package (Accelrys Inc., San Diego, CA) for these simulations, employing the CVFF force field. Plasmids and Mutants—We previously described His-tagged GSK-3β construct and GSK-3β in pCMV4 plasmid (6Eldar-Finkelman H. Agrast G.M. Foord O. Fischer E.H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10228-10233Crossref PubMed Scopus (132) Google Scholar). GSK-3β fused to an N-terminal FLAG-tag was initially cloned into pCMV-Tag 2B (Stratagene) in ECoRV and BamHI1. These 3 expression vectors were used as templates for mutagenesis of GSK-3 by the QuikChange site-directed mutagenesis kit (Stratagene) to replace Phe67, Gln89, Asn95, Glu97, and Ser66 to alanine. All constructs were sequenced to confirm the presence of mutations. The sequences of mutagenic oligonucleotides are available from the authors upon request. pCREB-EGFP plasmid was purchased from BD Biosciences Clontech (Palo Alto, CA). pCMV4IRS-1 plasmid was described (33Liberman Z. Eldar-Finkelman H. J. Biol. Chem. 2005; 280: 4422-4428Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). GFP-β-catenin was kindly provided by Dr. Rina Abersfeld from Tel Aviv University. Protein Expression in Bacteria—GSK-3β was expressed as His-tagged proteins in BL21(DE3) (Novagen, Darmstadt, Germany), induced by 0.4 mm isopropyl 1-thio-β-d-galactopyranoside overnight at 16 °C. Cells were homogenized with a sonication buffer (50 mm NaH2PO4 at pH 8.0, 5% glycerol, 0.25% Tween 20, 0.01% β-mercaptoethanol, 0.1 mm o-vanadate, 500 mm NaCl, 25 μg/ml leupeptin, 25 μg/ml aprotinin, 1 μg/ml pepstatin). GSK-3 proteins were purified using TALON metal affinity resin according to the manufacturer's instructions (BD Biosciences Clontech). Cell Transfections and Protein Purification—HEK-293 cells were grown in Dulbecco's Modified Eagle's Medium, containing 25 mm glucose, 10% fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. HEK-293 cells were transiently transfected with indicated constructs (5 μg each), using calcium phosphate method as described (6Eldar-Finkelman H. Agrast G.M. Foord O. Fischer E.H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10228-10233Crossref PubMed Scopus (132) Google Scholar). Co-expression used 5 μg each, and expression of GSK-3β mutant proteins was verified by Western blot analysis, using GSK-3β antibody. Cells were lysed in ice-cold buffer G (20 mm Tris-HCl, pH 7.5, 10 mm β-glycerophosphate, 10% glycerol, 1 mm EGTA, 1 mm EDTA, 50 mm NaF, 5 mm NaPPi, 0.5 mm orthovanadate, 1 mm benzamidine, 10 μg/ml leupeptin, 5 μg/ml aprotinin, 1 μg/ml pepstatin, 500 nm microcystine LR, and 0.5% Triton X-100). Cell extracts were centrifuged for 20 min at 15,000 × g. Supernatants were collected, and equal amounts of proteins (20 μg) were boiled with SDS sample buffer and subjected to gel electrophoresis (7.5–12% polyacrylamide gel), transferred to nitrocellulose membranes, and immunoblotted with indicated antibodies. For partial purification, cells were lysed with buffer H (50 mm Tris-HCl, pH 7.3, 1 mm EGTA, 1 mm EDTA, 1 mm orthovanadate, leupeptin, aprotinin, and pepstatin A 25 μg/ml each, 500 nm microcystine LR, and 0.25% Triton X-100) and centrifuged at 15,000 × g. The resulting supernatants were passed through DE-52 (Whatman) mini-columns that were equilibrated with buffer H. Flow-through and one wash with buffer H containing 0.02 m NaCl were collected. The amount of each mutant was determined by Western blot analysis of DE-52 preparations with αGSK-3β antibody. In Vitro Kinase Assays—The GSK-3β mutant proteins purified from bacteria or prepared from HEK-293 cells were incubated with indicated substrates (200 μm) in a reaction mixture (50 mm Tris-HCl, pH 7.3, 10 mm Mg-Acetate, and 0.01% β-mercaptoethanol) together with [γ-32P]ATP (100 μm, 0.5 μCi/assay) for 20 min or as indicated in figure legends. Reactions were stopped, spotted on p81 paper (Whatman) washed with phosphoric acid, and counted for radioactivity, as described (6Eldar-Finkelman H. Agrast G.M. Foord O. Fischer E.H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10228-10233Crossref PubMed Scopus (132) Google Scholar). For CREB phosphorylation, CREB-EGFP was immunoprecipitated with anti-GFP antibody MBL (Woburn, MA) in complex with protein A-Sepharose. GSK-3 proteins were added to the immunoprecipitates under conditions similar to those described above. In "hot" assays, the reactions were boiled with SDS sample buffer and subjected to gel electrophoresis. In cold assays, the reactions were boiled with SDS sample buffer and subjected to immunoblot analysis with αpCREB129/133. Similar experiments were performed in autophosphorylation assays except that the substrate was omitted, and ATP concentration was raised to 300 μm. Glycogen Synthase Activity—HEK-293 cells expressing GSK-3 mutants transiently were incubated with Dulbecco's modified Eagle's medium-low glucose containing 0.5% bovine serum albumin for 4 h. Cells were scraped into GS buffer (50 mm Tris-HCl, pH 7.8, 100 mm NaF, 10 mm EDTA, 5% glycerol, and protease inhibitors: 20 μg/ml leupeptin, 10 μg/ml aprotinin, 10 mg/ml pepstatin A, 1 mm benzamidine) and centrifuged at 8000 × g. Lysates were snap frozen in liquid nitrogen and stored at –80 °C. Glycogen synthase activity was assayed in the supernatants, according to the method of Thomas et al. (39Thomas J.A. Schlender K.K. Larner J. Anal. Biochem. 1968; 25: 486-499Crossref PubMed Scopus (949) Google Scholar) and based on the incorporation of uridine 5-diphosphate [14C]glucose (UDPG) into glycogen. Aliquots of tissue homogenate (15 μl) were incubated with 15 μl of reaction mixture (66.6 mm Tris-HCl, pH 7.8, 32.5 mm KF, 0.8 μCi/μl [14C]UDPG (400 μm), 13 mg/ml glycogen rabbit liver, Sigma) for 20 min at 30 °C, as described (6Eldar-Finkelman H. Agrast G.M. Foord O. Fischer E.H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10228-10233Crossref PubMed Scopus (132) Google Scholar). The reactions were then spotted on ET31 (Whatman) papers, washed with 66% ice-cold ethanol, and counted for radioactivity. Glycogen synthase assays were measured in the presence of 0.1 mm or 10 mm glucose-6-phosphate (G6P), and their ratio was calculated. CREB is a GSK-3 substrate phosphorylated directly at Ser129 by GSK-3 (40Fiol C.J. Williams J.S. Chou C.H. Wang Q.M. Roach P.J. Andrisani O.M. J. Biol. Chem. 1994; 269: 32187-32193Abstract Full Text PDF PubMed Google Scholar). Similar to other GSK-3 substrates, CREB requires pre-phosphorylation at Ser133, which is carried out by cAMP-dependent kinase (40Fiol C.J. Williams J.S. Chou C.H. Wang Q.M. Roach P.J. Andrisani O.M. J. Biol. Chem. 1994; 269: 32187-32193Abstract Full Text PDF PubMed Google Scholar). A minimal peptide sequence encompassing the CREB phosphorylation motif, termed p9CREB (ILSRRPS(p)YR), was shown in in vitro kinase assays to be a GSK-3 substrate (Fig. 1A). In addition, these experiments emphasized the importance of phosphorylation. Namely, replacement of phosphorylated serine by serine (CREB) glutamic acid (E9CREB, mimicking phosphorylation) or phosphorylated tyrosine (pY9CREB) practically abolished GSK-3 phosphorylation (Fig. 1A).FIGURE 1Phosphorylation of p9CREB and model structure of the ternary complex GSK-3β·ATP·pCREB. A, phosphorylation of p9CREB peptide variants by GSK-3. His-tagged purified GSK-3 was incubated with p9CREB peptide variants in the presence of [γ-32P]ATP, as described under "Experimental Procedures" for indicated time points. The reactions were spotted on p81 papers and counted for radioactivity. Phosphorylation is presented as a percentage of maximal phosphorylation obtained with p9CREB substrate. Shown is a mean of two independent experiments. Full circles represent p9CREB. Empty circle, filled triangle, and empty inverted triangle represent CREB, ECREB, and pYCREB, respectively. B, solvent accessible surface of GSK-3β is shown in gray. Residues Gln89 and Asn95 are emphasized in orange, residue Phe67 is colored in light green, Ser66 is shown (only in the inset) in black and the positive primed phosphate binding site is depicted in yellow. The ribbon diagram in the insert (stereo pair) represents pCREB. The p9CREB fragment is colored in dark blue and the N- and C-terminal helices are colored in cyan and blue, respectively. The side chains of residues Ser129 (phosphorylation site), Ser133(p) (primed phosphorylation site), Tyr134 and Arg135, (which interact with Phe67 or Gln89 and Asn95, respectively) are shown. ATP is shown as brown ball and stick model, and the Mg2+ ion is depicted by the magenta sphere. C, amino acid sequence alignment of the catalytic domain of GSK-3β in various species (as produced with the program ClustalW) and alignment of GSK-3β paralogs (CDK-2 and MAPK) and PKA. The latter sequence alignment is based on manual structural alignments (using the program InsightII) of the relevant structures (PDB codes, 1o9u, 1jst, 2erk, and 2cpk, respectively). The numbering corresponds to that of GSK-3. Potential residues for substrate recognition are denoted by yellow or green (identical residues) background. It shows that Gln89 and Asn95 are preferentially conserved in GSK-3. Conserved residues in all sequences are denoted by red letters; residues conserved in two or three sequences are denoted by blue letters. The residues mutated in this study are marked with an asterisk. D, ability of GSK-3 to phosphorylate p9CREB variants, was examined in assay conditions similar to those described in A (and under "Experimental Procedures"). Results are presented as the percentage of the phosphorylation obtained with p9CREB as a substrate that was set to 100%, and are mean of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In Silico Molecular Modeling of the Ternary Complex GSK-3·ATP·pCREB—The structure of the ternary complex GSK-3β·ATP·CREB was modeled as described under "Experimental Procedures." We used the structures of the non-ATP-bound GSK-3β (28Dajani R. Fraser E. Roe S.M. Young N. Good V. Dale T.C. Pearl L.H. Cell. 2001; 105: 721-732Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar) and the ANP-bound GSK-3β (31Bertrand J.A. Thieffine S. Vulpetti A. Cristiani C. Valsasina B. Knapp S. Kalisz H.M. Flocco M. J. Mol. Biol. 2003; 333: 393-407Crossref PubMed Scopus (277) Google Scholar) to model the complex between phosphorylated GSK-3β and ATP. The two structures differ only slightly in the position of the P-loop, but not in the substrate binding cavity. Hence, the phosphorylated GSK-3β·ATP model was constructed by combining these two structures; the conformation of the P-loop backbone and side chains was as in the ANP-bound GSK-3β, and the conformation of Tyr216(p) was as in the phosphorylated GSK-3β. Currently, there is only one available structure of pCREB (32Radhakrishnan I. Perez-Alvarado G.C. Parker D. Dyson H.J. Montminy M.R. Wright P.E. Cell. 1997; 91: 741-752Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar) bound to the KIX domain of CBP. pCREB consists of two helices (N- and C-terminal) joined by a loop, and the 17 NMR models of pCREB present considerable variability in the relative position of the N- and C-terminal helices. Our protein-protein docking program MolFit treats the docked molecules as rigid bodies and cannot cope with such structural flexibility. Therefore, we docked pCREB to GSK-3β in steps. First, we docked three representing conformers of p9CREB (residues 127–135) to GSK-3·ATP employing a weighting scheme that emphasized contacts involving the primed binding site of GSK-3β, but did not specify the binding partner (see "Experimental Procedures"). Thus, it was rewarding to find that the first docking step clearly preferred one of the three conformers of p9CREB, and ranked near the top a model in which the primed binding site was occupied by CREB-Ser133(p) (rank 31 out of 9,415 models obtained after intersection; see "Experimental Procedures"). The s
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