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• W2012172588 abstract "Ribosomal S6 kinase 1 (S6K1), as a key regulator of mRNA translation, plays an important role in cell cycle progression through the G1 phase of proliferating cells and in the synaptic plasticity of terminally differentiated neurons. Activation of S6K1 involves the phosphorylation of its multiple Ser/Thr residues, including the proline-directed sites (Ser-411, Ser-418, Thr-421, and Ser-424) in the autoinhibitory domain near the C terminus. Phosphorylation at Thr-389 is also a crucial event in S6K1 activation. Here, we report that S6K1 phosphorylation at Ser-411 is required for the rapamycin-sensitive phosphorylation of Thr-389 and the subsequent activation of S6K1. Mutation of Ser-411 to Ala ablated insulin-induced Thr-389 phosphorylation and S6K1 activation, whereas mutation mimicking Ser-411 phosphorylation did not show any effect. Furthermore, phosphomimetic mutation of Thr-389 overcame the inhibitory effect of the mutation S411A. Thus, Ser-411 phosphorylation regulates S6K1 activation via the control of Thr-389 phosphorylation. In nervous system neurons, Cdk5-p35 kinase associates with S6K1 via the direct interaction between p35 and S6K1 and catalyzes S6K1 phosphorylation specifically at Ser-411. Inhibition of the Cdk5 activity or suppression of Cdk5 expression blocked S6K1 phosphorylation at Ser-411 and Thr-389, resulting in S6K1 inactivation. Similar results were obtained by treating asynchronous populations of proliferating cells with the CDK inhibitor compound roscovitine. Altogether, our findings suggest a novel mechanism by which the CDK-mediated phosphorylation regulates the activation of S6K1. Ribosomal S6 kinase 1 (S6K1), as a key regulator of mRNA translation, plays an important role in cell cycle progression through the G1 phase of proliferating cells and in the synaptic plasticity of terminally differentiated neurons. Activation of S6K1 involves the phosphorylation of its multiple Ser/Thr residues, including the proline-directed sites (Ser-411, Ser-418, Thr-421, and Ser-424) in the autoinhibitory domain near the C terminus. Phosphorylation at Thr-389 is also a crucial event in S6K1 activation. Here, we report that S6K1 phosphorylation at Ser-411 is required for the rapamycin-sensitive phosphorylation of Thr-389 and the subsequent activation of S6K1. Mutation of Ser-411 to Ala ablated insulin-induced Thr-389 phosphorylation and S6K1 activation, whereas mutation mimicking Ser-411 phosphorylation did not show any effect. Furthermore, phosphomimetic mutation of Thr-389 overcame the inhibitory effect of the mutation S411A. Thus, Ser-411 phosphorylation regulates S6K1 activation via the control of Thr-389 phosphorylation. In nervous system neurons, Cdk5-p35 kinase associates with S6K1 via the direct interaction between p35 and S6K1 and catalyzes S6K1 phosphorylation specifically at Ser-411. Inhibition of the Cdk5 activity or suppression of Cdk5 expression blocked S6K1 phosphorylation at Ser-411 and Thr-389, resulting in S6K1 inactivation. Similar results were obtained by treating asynchronous populations of proliferating cells with the CDK inhibitor compound roscovitine. Altogether, our findings suggest a novel mechanism by which the CDK-mediated phosphorylation regulates the activation of S6K1. The 40 S ribosomal protein S6 kinases (S6Ks) 2The abbreviations used are: S6K, S6 kinase; rpS6, ribosomal protein S6; TOS, TOR signaling; HA, hemagglutinin; GST, glutathione S-transferase; MOPS, 4-morpholinepropanesulfonic acid; siRNA, small interfering RNA. 2The abbreviations used are: S6K, S6 kinase; rpS6, ribosomal protein S6; TOS, TOR signaling; HA, hemagglutinin; GST, glutathione S-transferase; MOPS, 4-morpholinepropanesulfonic acid; siRNA, small interfering RNA. p70 (α2 isoform) and the alternative translation variant p85 (α1 isoform), collectively termed S6K1, play an important role in the control of cell growth. p70 S6K1 is identical to p85 S6K1 in the protein sequence except that it lacks the N-terminal 23 amino acids, which contains a nuclear localization signal. Thus, p85 S6K1 localizes in the nucleus, while p70 S6K1 appears predominantly in the cytoplasm (1Reinhard C. Fernandez A. Lamb N.J. Thomas G. EMBO J. 1994; 13: 1557-1565Crossref PubMed Scopus (179) Google Scholar, 2Coffer P.J. Woodgett J.R. Biochem. Biophys. Res. Commun. 1994; 198: 780-786Crossref PubMed Scopus (39) Google Scholar). Recently, S6K2 was identified with a high homology to S6K1, and partially compensated for the loss of the S6K1 function (3Gout I. Minami T. Hara K. Tsujishita Y. Filonenko V. Waterfield M.D. Yonezawa K. J. Biol. Chem. 1998; 273: 30061-30064Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 4Lee-Fruman K.K. Kuo C.J. Lippincott J. Terada N. Blenis J. Oncogene. 1999; 18: 5108-5114Crossref PubMed Scopus (117) Google Scholar, 5Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar). It was proposed that S6K1 and S6K2 catalyze ribosomal protein S6 (rpS6) phosphorylation to initiate the selective translation of mRNAs containing a 5′-terminal oligopyrimidine-rich tract and also primarily encoding ribosomal proteins and translational elongation factors of the protein translation apparatus (6Jefferies H.B. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (803) Google Scholar). However, this model has been questioned in recent studies (7Pende M. Um S.H. Mieulet V. Sticker M. Goss V.L. Mestan J. Mueller M. Fumagalli S. Kozma S.C. Thomas G. Mol. Cell. Biol. 2004; 24: 3112-3124Crossref PubMed Scopus (604) Google Scholar, 8Tang H. Hornstein E. Stolovich M. Levy G. Livingstone M. Templeton D. Avruch J. Meyuhas O. Mol. Cell. Biol. 2001; 21: 8671-8683Crossref PubMed Scopus (251) Google Scholar). Nevertheless, S6K1 but not S6K2 is involved in cell growth control and the regulation of animal body size (7Pende M. Um S.H. Mieulet V. Sticker M. Goss V.L. Mestan J. Mueller M. Fumagalli S. Kozma S.C. Thomas G. Mol. Cell. Biol. 2004; 24: 3112-3124Crossref PubMed Scopus (604) Google Scholar). S6K1 has a highly acidic N terminus, followed by the Ser/Thr kinase catalytic domain and a regulatory C-terminal tail. Activation of S6K1 is controlled through a complex mechanism that involves the phosphorylation of at least eight Ser/Thr residues, including Thr-229, Ser-371, Thr-389, Ser-404, Ser-411, Ser-418, Thr-421, and Ser-424, which are located at the catalytic domain and the C terminus (9Dufner A. Thomas G. Exp. Cell Res. 1999; 253: 100-109Crossref PubMed Scopus (596) Google Scholar, 10Martin K.A. Blenis J. Adv. Cancer Res. 2002; 86: 1-39Crossref PubMed Scopus (174) Google Scholar). Current understanding is that multiple phosphorylating events occur in a stepwise manner starting from the Ser/Thr-Pro sites (Ser-411, Ser-418, Thr-421, and Ser-424) located within the autoinhibitory domain adjacent to the C terminus. This step facilitates Thr-389 phosphorylation in the linker region, relieving the autoinhibition and also releasing the kinase from the translation preinitiation complex (11Dennis P.B. Pullen N. Pearson R.B. Kozma S.C. Thomas G. J. Biol. Chem. 1998; 273: 14845-14852Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 12Holz M.K. Ballif B.A. Gygi S.P. Blenis J. Cell. 2005; 123: 569-580Abstract Full Text Full Text PDF PubMed Scopus (868) Google Scholar, 13Pearson R.B. Dennis P.B. Han J.W. Williamson N.A. Kozma S.C. Wettenhall R.E. Thomas G. EMBO J. 1995; 14: 5279-5287Crossref PubMed Scopus (387) Google Scholar). Subsequently, Thr-389-phosphorylated S6K1 binds to and is phosphorylated by phosphoinositide-dependent protein kinase 1 (PDK1) at Thr-229 in the catalytic activation loop for activation (14Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 15Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (718) Google Scholar). Thr-389 phosphorylation, which is rapamycin-sensitive and is mediated by mTOR/raptor, appears to be a critical and rate-limiting step in the activation (16Weng Q.P. Kozlowski M. Belham C. Zhang A. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 17Nojima H. Tokunaga C. Eguchi S. Oshiro N. Hidayat S. Yoshino K. Hara K. Tanaka N. Avruch J. Yonezawa K. J. Biol. Chem. 2003; 278: 15461-15464Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 18Kim D.H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2286) Google Scholar, 19Dennis P.B. Pullen N. Kozma S.C. Thomas G. Mol. Cell. Biol. 1996; 16: 6242-6251Crossref PubMed Scopus (221) Google Scholar). Interestingly, two conserved segments of S6K1, the N-terminal TOR signaling (TOS) motif and the C-terminal RSPRR sequence, participate in regulating Thr-389 phosphorylation (20Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 21Schalm S.S. Tee A.R. Blenis J. J. Biol. Chem. 2005; 280: 11101-11106Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The TOS motif is required for the phosphorylation and is shown to mediate the binding of S6K1 to the mTOR partner raptor, while the RSPRR sequence suppresses the phosphorylation by an unknown mechanism (17Nojima H. Tokunaga C. Eguchi S. Oshiro N. Hidayat S. Yoshino K. Hara K. Tanaka N. Avruch J. Yonezawa K. J. Biol. Chem. 2003; 278: 15461-15464Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 20Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 21Schalm S.S. Tee A.R. Blenis J. J. Biol. Chem. 2005; 280: 11101-11106Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Ser-411 within the RSPRR sequence is one of the Ser/Thr-Pro sites situated in the autoinhibitory domain of S6K1. Protein kinases that have been implicated in Ser-411 phosphorylation include ERKs, p38 MAPK, and Cdk1-cyclin B (16Weng Q.P. Kozlowski M. Belham C. Zhang A. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 22Papst P.J. Sugiyama H. Nagasawa M. Lucas J.J. Maller J.L. Terada N. J. Biol. Chem. 1998; 273: 15077-15084Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 23Shah O.J. Ghosh S. Hunter T. J. Biol. Chem. 2003; 278: 16433-16442Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In this study, we show that Cdk5, in association with p35, phosphorylates S6K1 at Ser-411 in nervous system neurons. Also, Ser-411 phosphorylation is mediated by roscovitine-sensitive CDKs in asynchronous populations of proliferating cells. Although Cdk5 is widely expressed, it displays the kinase activity primarily in neurons of the central nervous system in conjunction with the neuron-specific protein p35 or p39 (24Dhavan R. Tsai L.H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 749-759Crossref PubMed Scopus (931) Google Scholar). By virtue of phosphorylating diverse substrates with the consensus sequence of Ser/Thr immediately upstream of a proline and a basic residue at the +3 position (S/T-P-X-R/K, X is preferably a basic or neutral residue), Cdk5-p35 plays an important role in the nervous system to mediate various functions including neuronal differentiation, cytoskeletal organization, cell signaling, synaptic plasticity, and cell survival (24Dhavan R. Tsai L.H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 749-759Crossref PubMed Scopus (931) Google Scholar, 25Lim A.C. Qu D. Qi R.Z. Neurosignals. 2003; 12: 230-238Crossref PubMed Scopus (29) Google Scholar). Neurotoxins induce the cleavage of p35 by calpain into an N-terminally trun-cated p25 form, which exhibits many cellular characteristics different from those of p35 (26Kusakawa G. Saito T. Onuki R. Ishiguro K. Kishimoto T. Hisanaga S. J. Biol. Chem. 2000; 275: 17166-17172Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 27Lee M.S. Kwon Y.T. Li M. Peng J. Friedlander R.M. Tsai L.H. Nature. 2000; 405: 360-364Crossref PubMed Scopus (896) Google Scholar, 28Patrick G.N. Zukerberg L. Nikolic M. de la M.S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1297) Google Scholar). Moreover, the aberrant regulation of Cdk5 by p25 causes neuronal death and is implicated in tau hyperphosphorylation from several neurodegenerative diseases including Alzheimer disease (24Dhavan R. Tsai L.H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 749-759Crossref PubMed Scopus (931) Google Scholar, 28Patrick G.N. Zukerberg L. Nikolic M. de la M.S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1297) Google Scholar). To date, the role of Ser-411 phosphorylation in the regulation of S6K1 activity remains elusive. In this report, we demonstrate that Ser-411 phosphorylation is required for Thr-389 phosphorylation and S6K1 activation. Furthermore, Ser-411 phosphorylation is mediated by the Cdk5-p35 kinase in nervous system neurons and is sensitive to CDK inhibition by roscovitine in proliferating cells. Therefore, Ser-411 phosphorylation, which is mediated by CDKs, is crucial for S6K1-controlled protein synthesis. Plasmid Constructs—Various S6K1 constructs used in the present report were derived from the rat p70 S6K1 sequence. The constructs HA-S6K1/pRK7 and S6K1(S424A) were obtained from Dr. John Blenis (Harvard Medical School) (29Cheatham L. Monfar M. Chou M.M. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11696-11700Crossref PubMed Scopus (114) Google Scholar). S6K1 mutations, S411A, S411D, S411A/S424A, T389E, and T389E/S411A, were created by site-directed mutagenesis. The constructs of Cdk5 and p35 were described previously (30Lim A.C. Hou Z. Goh C.P. Qi R.Z. J. Biol. Chem. 2004; 279: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 31Qu D. Li Q. Lim H.Y. Cheung N.S. Li R. Wang J.H. Qi R.Z. J. Biol. Chem. 2002; 277: 7324-7332Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Antibodies—Anti-S6K1 (C-18), anti-S6K1(phospho-Ser-411), anti-Cdk5 (C-8), anti-p35 (C-19), anti-HA (F-7 and Y-11), and anti-His6 were purchased from Santa Cruz Biotechnology. Anti-S6K1(phospho-Thr-389), anti-S6K1(phospho-Thr-421/Ser-424), anti-rpS6, and anti-rpS6(phospho-Ser-235/236) were from Cell Signaling Technology. Anti-GST, anti-α-tubulin, and anti-FLAG (M2) were from Sigma. Protein Binding Assay—Recombinant proteins tagged with GST or His6 were expressed in Escherichia coli BL21(DE3) and were purified as described in earlier reports (30Lim A.C. Hou Z. Goh C.P. Qi R.Z. J. Biol. Chem. 2004; 279: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 31Qu D. Li Q. Lim H.Y. Cheung N.S. Li R. Wang J.H. Qi R.Z. J. Biol. Chem. 2002; 277: 7324-7332Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). To test the binding of S6K1 to p35 fragments, 1 μg of GST-p35 fragments or GST was incubated with 0.5 μg of His6-S6K1 for 1 h at 4 °Cin 500 μl of 25 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm dithiothreitol, 100 mm NaCl, 0.2% Triton X-100, and the protease inhibitor mixture (Roche Applied Science). The GST proteins were then retrieved using GSH-Sepharose (GE Healthcare), released from the beads by boiling in the SDS-PAGE loading buffer, and analyzed by immunoblotting. S6K1 Protein Phosphorylation and Kinase Assay—The active Cdk5 kinase was reconstituted from GST-Cdk5 and His6-p35 (32Qi Z. Huang Q.Q. Lee K.Y. Lew J. Wang J.H. J. Biol. Chem. 1995; 270: 10847-10854Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Phosphorylation of His6-S6K1 and its mutants were carried out with the Cdk5 kinase at 30 °C for 30 min in a kinase buffer (20 mm MOPS, pH 7.4, 5 mm MgCl2, and 1 mm dithiothreitol) containing 100 μm [γ-32P]ATP (∼5000 dpm/pmol). The reactions were terminated by boiling the samples for 5 min in the SDS-PAGE sample loading buffer. Protein phosphorylation was visualized by autoradiography. To determine the stoichiometry of phosphorylation, the reactions were conducted as described above for 60 min. The band corresponding to each S6K1 protein was excised from the polyacrylamide gel for radioactivity determination by scintillation counting. The measured values were subtracted by blanks, which were obtained from measuring the radioactivity of the gel band corresponding to each S6K1 protein phosphorylated in the absence of the Cdk5 kinase. The amount of each S6K1 protein was determined by Coomassie Blue staining and densitometry using bovine serum albumin as standard. The resulting values were used to calculate stoichiometries of phosphorylation. To determine the kinase activity of immunoprecipitated S6K1, the immunoprecipitates were washed with the kinase buffer and were suspended in the buffer supplemented with 50 μm of the S6 kinase substrate peptide (RRRLSSLRA, Upstate Cell Signaling Solutions). The reactions started with the addition of 100 μm [γ-32P]ATP (∼500 dpm/pmol) and continued at 30 °C for 20 min. Following the addition of 10% acetic acid to stop the reactions, the assay mixtures were spotted onto P-81 phosphocellulose paper squares (Whatman). Phosphate incorporated into the peptide was quantified by scintillation counting. For each experiment, blanks were taken from the reactions performed in the absence of the peptide. These blanks were subtracted from the above values. Cell Culture and Transfection—HEK293T, COS-7, and neuroblastoma Neuro 2A were grown at 37 °C in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum. Transfection of HEK293T and COS-7 was performed using Lipofectamine PLUS (Invitrogen). Prior to insulin stimulation, HEK293T cells were starved in serum-free Dulbecco's modified Eagle's medium for 20 h and were treated with rapamycin (20 ng/ml) or the solvent for 30 min. The cells were then stimulated with 100 nm insulin for 30 min. For CDK inhibition, roscovitine was used at 50 μm to treat COS-7 cells for 2 h. Cultures of cortical neurons were prepared from cortices of mouse embryos (embryonic day 16) or rat embryos (embryonic day 18) as reported (31Qu D. Li Q. Lim H.Y. Cheung N.S. Li R. Wang J.H. Qi R.Z. J. Biol. Chem. 2002; 277: 7324-7332Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). After 5 days in vitro, neurons were treated with 1 μm of the Cdk5 inhibitor GW8510 (4-([(7-oxo-6,7-dihydro-8H-(1, 3)thiazolo[5,4-e]indol-8-ylidene)methyl] amino)-N-(2-pyridinyl)benzenesulfonamide, Sigma) for various time periods. Two siRNAs were designed to target rat cdk5. The sequence 5′-CCAATGTACCCAGCTACAA-3′ was synthesized in sense and antisense directions with dTdT overhang at the 3′ terminus to generate a double-stranded siRNA (siRNA-1). A scrambled sequence was used as a control siRNA. A pSuper hairpin siRNA construct of cdk5 containing the sequence of 5′-TGCCACGGGGAGAGACCTG-3′ (siRNA-2) was obtained from Dr. Nancy Y. Ip (Hong Kong University of Science and Technology). Transfection of neurons was performed at the time of cell preparation using the Nucleofector™ apparatus (Amaxa). 2 μg of plasmids or 2.5 μg of siRNA duplexes was nucleofected according to the manufacturer's protocol. Immunoprecipitation—Lysates were prepared by extracting cells with lysis buffer (20 mm Tris-HCl, pH 7.5, 1 mm dithiothreitol, 2 mm EDTA, 2 mm EGTA, 25 mm NaF, 25 mm β-glycerophosphate, 0.1 mm Na3VO4, 0.5 mm phenylmethylsulfonyl fluoride, and 0.3% Nonidet P-40), and extracts were clarified by centrifugation (13,000 × g; 20 min). Anti-FLAG immunoprecipitation was performed using anti-FLAG M2-agarose (Sigma). Proteins expressed with an HA-tag were immunoprecipitated using the anti-HA antibody (Y-11) and protein A/G beads (Invitrogen). To coimmunoprecipitate endogenous p35 and S6K1, differentiation of Neuro 2A was induced with 10 μm retinoic acid in Dulbecco's modified Eagle's medium plus 1% fetal calf serum for 48 h. The Neuro 2A cells were then washed with phosphate-buffered saline and were treated with the thiol-cleavable cross-linker dithiobis(succinimidylpropionate) in phosphate-buffered saline for 20 min at room temperature. Following the treatment, the cells were washed with phosphate-buffered saline and were then lysed in RIPA buffer (20 mm Tris-HCl, pH 7.5, 0.1% SDS, 1% Nonidet P-40, and 0.5% deoxycholate) supplemented with the Roche mixture protease inhibitors. The lysates were clarified by centrifugation before anti-p35 immunoprecipitation was performed. Immunoprecipitates were washed in the lysis buffer supplemented with 250 mm NaCl and were then analyzed by immunoblotting. Cdk5-p35 Associates with S6K1—In the brain, Cdk5-p35 appears in various multiprotein complexes, whereas the deregulated form Cdk5-p25 is found mainly as a heterodimeric complex (25Lim A.C. Qu D. Qi R.Z. Neurosignals. 2003; 12: 230-238Crossref PubMed Scopus (29) Google Scholar). We carried out biochemical isolation of p35-binding proteins from rat brain (30Lim A.C. Hou Z. Goh C.P. Qi R.Z. J. Biol. Chem. 2004; 279: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 31Qu D. Li Q. Lim H.Y. Cheung N.S. Li R. Wang J.H. Qi R.Z. J. Biol. Chem. 2002; 277: 7324-7332Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). During the isolation, S6K1 was identified by using mass spectrometry (data not shown). To investigate the association of S6K1 with Cdk5-p35, HEK293T cells were double-transfected with S6K1 and p35 or p25 (amino acids 99–307) for immunoprecipitation. As seen in Fig. 1A, S6K1 specifically coimmunoprecipitated with p35 but not with p25, revealing the association of S6K1 with p35 and the requirement of the N terminus of p35 for the association. The reciprocal immunoprecipitation of ectopically expressed S6K1 further confirmed the p35 association (Fig. 1B). Moreover, Cdk5 coimmunoprecipitated with S6K1 only in the presence of p35 (Fig. 1B). Finally, endogenous p35 and S6K1 were coimmunoprecipitated from differentiated Neuro 2A cells (Fig. 1C). Altogether, these results indicate that S6K1 forms a ternary complex with Cdk5 and p35 via associating with p35. To demonstrate the direct binding of S6K1 to the N terminus of p35, we performed pull-down assays using recombinant proteins of S6K1 and two p35 fragments, p10 (amino acids 1–98) and p25, which were purified from bacterial expressions. In the assays, the binding of GST-p10 to S6K1 was readily detected, whereas GST and GST-p25 did not exhibit any pulldown activity with S6K1 (Fig. 1D). This established the direct interaction between S6K1 and the N terminus of p35. Cdk5-p35 Phosphorylates S6K1 at Ser-411—From scanning of the S6K1 sequence, we found that Ser-411 and Ser-424 conform to the consensus motif of Cdk5 phosphorylation. When subjected to protein phosphorylation, the recombinant protein of S6K1 was readily phosphorylated by Cdk5-p35 and about 1.1 mol/mol of phosphate was incorporated into S6K1 in the reaction for 1 h (Fig. 2, A and B). To examine whether Ser-411 and Ser-424 were targeted by Cdk5-p35 in the reactions, S6K1 mutants were generated to substitute either or both of these residues by Ala. The mutation of either Ser-411 or Ser-424 reduced S6K1 phosphorylation, while the double mutation almost completely abolished the [32P]phosphate incorporation into the protein (Fig. 2, A and B). Thus, Cdk5-p35 phosphorylated S6K1 primarily at Ser-411 and Ser-424 in the reactions. Also, Cdk5-p35 displayed a remarkably lower kinase activity toward the protein S6K1(S411A) than the mutant S6K1(S424A) (Fig. 2, A and B), suggesting that Ser-411 is a preferred site over Ser-424 for Cdk5-p35 action. Cdk5 is a ubiquitously expressed protein that depends on the neuron-specific protein p35 or p39 for kinase activity (24Dhavan R. Tsai L.H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 749-759Crossref PubMed Scopus (931) Google Scholar). To demonstrate S6K1 phosphorylation by active Cdk5 in cells, HEK293T cells transiently expressing p35 or the empty vector were serum-starved and were then subjected to S6K1 phosphorylation assessment at various sites. Expression of p35 induced Ser-411 phosphorylation of S6K1, whereas phosphorylation of Thr-421/Ser-424 and Thr-389 was unaffected in the starved cells (Fig. 2B). These results reveal specific Ser-411 phosphorylation induced by Cdk5-p35. S6K1 Activation Requires Ser-411 Phosphorylation—Ser-411 is situated in the conserved RSPRR sequence, which confers an inhibitory effect on mTOR-mediated S6K1 activation (21Schalm S.S. Tee A.R. Blenis J. J. Biol. Chem. 2005; 280: 11101-11106Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). To investigate the impact of Ser-411 phosphorylation on S6K1 activity, Ser-411 was mutated to Ala or Asp to generate a non-phosphorylatable mutant S6K1(S411A) or a phosphomimetic mutant S6K1(S411D), respectively. Upon insulin stimulation, S6K1(S411D) was phosphorylated at Thr-389 and was activated to similar extents as the wild type (Fig. 3, A and B). Thr-389 phosphorylation and activation of the kinase were sensitive to rapamycin (Fig. 3, A and B). However, insulin-induced Thr-389 phosphorylation was almost completely blocked in the mutant S411A (Fig. 3A). S6K1(S411A) also exhibited much lower basal and insulin-stimulated kinase activities than the wild type and S6K1(S411D) (Fig. 3B). We also examined the phosphorylation of the S6K1 mutants in primary cortical neurons. Similarly, the S411A mutation inhibited Thr-389 phosphorylation, whereas the mutation S411D did not show any significant effect (Fig. 3C). These results reveal that Ser-411 phosphorylation is required for Thr-389 phosphorylation and the full activation of the kinase. It was also noted that neither of these mutations affected the Thr-421/Ser-424 phosphorylation, which remained unchanged upon insulin stimulation (Fig. 3, A and C). In short, Ser-411 phosphorylation is a prerequisite for mTOR-mediated Thr-389 phosphorylation, which leads to S6K1 activation. It is noteworthy that the phosphomimetic mutation S411D did not enhance basal Thr-389 phosphorylation and S6K1 activity (Fig. 3), indicating that Ser-411 phosphorylation is insufficient for S6K1 activation. To further explore the role of Ser-411 phosphorylation in S6K1 activation, we substituted Ala for Ser-411 in the context of T389E. As shown previously, the substitution of Glu for Thr-389 to mimic the phosphorylation conferred a high basal activity on S6K1 in serum-starved cells (Fig. 4; Ref. 13Pearson R.B. Dennis P.B. Han J.W. Williamson N.A. Kozma S.C. Wettenhall R.E. Thomas G. EMBO J. 1995; 14: 5279-5287Crossref PubMed Scopus (387) Google Scholar). The T389E mutation significantly enhanced the basal and insulin-stimulated activities of S6K1(S411A), and the activities of S6K1(T389E/S411A) were similar to those of S6K1(T389E) (Fig. 4). Therefore, phosphomimetic mutation of Thr-389 renders the kinase resistant to the inhibitory effect of the S411A mutation. CDK Activities Are Required for Ser-411 Phosphorylation and S6K1 Activation—We explored whether CDKs are involved in Ser-411 phosphorylation in proliferating cells by using roscovitine to selectively inhibit CDK activities. The use of roscovitine on asynchronous cells almost completely eliminated Ser-411 phosphorylation, whereas Thr-421/Ser-424 phosphorylation was not changed (Fig. 5A), suggesting that Ser-411 is specifically targeted by roscovitine-sensitive CDKs. Also, the treatment strongly suppressed Thr-389 phosphorylation (Fig. 5A), which is consistent with the idea that Ser-411 phosphorylation is required prior to Thr-389 phosphorylation. Consequently, the kinase activity of S6K1 was inhibited and the phosphorylation of the S6K1 substrate rpS6 dramatically decreased (Fig. 5, A and B). Therefore, the CDK inhibition blocked Ser-411 and Thr-389 phosphorylation and abolished S6K1 activation. Given the observation that Cdk5-p35 binds to S6K1 and catalyzes Ser-411 phosphorylation, we examined the effect of Cdk5 inhibition and depletion on S6K1 phosphorylation in primary neuronal cultures. GW8510 is a recently developed indolone derivative that specifically inhibits Cdk5 with low cytotoxicity toward cultured neurons (33Johnson K. Liu L. Majdzadeh N. Chavez C. Chin P.C. Morrison B. Wang L. Park J. Chugh P. Chen H.M. D'Mello S.R. J. Neurochem. 2005; 93: 538-548Crossref PubMed Scopus (46) Google Scholar). The GW8510 treatment of cortical neurons led to a dramatic decrease of Ser-411 and Thr-389 phosphorylation, while Thr-421/Ser-424 phosphorylation remained unchanged (Fig. 6A), indicating the requirement of Cdk5 activity for Ser-411 and Thr-389 phosphorylation in vivo. Next, we reduced levels of the Cdk5 protein by RNA interference. Two non-overlapping siRNA sequences of cdk5 were employed to ensure the specificity of the siRNA effects. The introduction of either cdk5 siRNAs effectively and specifically reduced Cdk5 protein levels, while levels of S6K1 and rpS6 were unaffected (Fig. 6B). Depletion of Cdk5 strongly inhibited S6K1 phosphorylation at Ser-411 and Thr-389, whereas Thr-421/Ser-424 phosphorylation remained unaffected (Fig. 6B). These data are in complete agreement with those from Cdk5 inhibition by GW8510. As a result of the inhibition of Thr-389 phosphorylation, S6K1 was inactivated, as shown by the dramatically reduced phosphorylation of rpS6 (Fig. 6B). Therefore, the kinase activity of Cdk5 is required in nervous system neurons for the phosphorylation and activation of S6K1. By controlling protein translation, mTOR and its effector S6K1 mediate cell growth in response to stimulating signals and modulate neuronal plasticity, learning, and memory (34Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3377) Google Scholar). In the present study, we have demonstrated that the neuronal kinase Cdk5-p35 physically associates with S6K1 and catalyzes S6K1 phosphorylation at Ser-411. Similarly, in asynchronous populations of proliferating cells, Ser-411 phosphorylation is sensitive to roscovitine, which selectively inhibits Cdk1, Cdk2, Cdk5, Cdk7, and Cdk9 (35Bach S. Knockaert M. Reinhardt J. Lozach O. Schmitt S. Baratte B. Koken M. Coburn S.P. Tang L. Jiang T. Liang D.C. Galons H. Dierick J.F. Pinna L.A. Meggio F. Totzke F. Schachtele C. Lerman A.S. Carnero A. Wan Y. Gray N. Meijer L. J. Biol. Chem. 2005; 280: 31208-31219Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Prior to this study, the biological role of Ser-411 phosphorylation is obscure, but now we have shown that Ser-411 is a critical phosphorylation site for regulating S6K1 catalytic activity through the control of Thr-389 phosphorylation. Indeed, elimination of Ser-411 phosporylation by either a mutational approach or the suppression of the CDK activities abrogates S6K1 activation. Among the Ser/Thr-Pro sites at the C terminus of S6K1, only the RSPRR sequence encompassing Ser-411 is conserved in S6K2 and across several biological species. Moreover, the RSPRR sequence imparts inhibition to S6K1 by suppressing Thr-389 and Thr-229 phosphorylation (21Schalm S.S. Tee A.R. Blenis J. J. Biol. Chem. 2005; 280: 11101-11106Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Our results in this study suggest that Ser-411 phosphorylation is crucial for relieving the negative modulation. It has been proposed that phosphorylation of the Ser/Thr-Pro cluster including Ser-411 in the autoinhibitory domain disrupts the interaction between the N and C termini of S6K1, rendering the kinase to adopt a partially open conformation (11Dennis P.B. Pullen N. Pearson R.B. Kozma S.C. Thomas G. J. Biol. Chem. 1998; 273: 14845-14852Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Therefore, Ser-411 phosphorylation is required for the unmasking of the Thr-389 site for action of the Thr-389 kinase (e.g. mTOR/raptor). In addition, nonphosphorylatable mutation of Ser-411 did not affect the kinase activity of the Thr-389 phosphomimetic mutant. Therefore, Ser-411 phosphorylation mediates S6K1 activation via the control of Thr-389 phosphorylation but does not directly participate in the kinase activation. S6K1 contains the conserved TOS motif at the N terminus, which suppresses the inhibitory activity associated with the RSPRR segment (20Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 21Schalm S.S. Tee A.R. Blenis J. J. Biol. Chem. 2005; 280: 11101-11106Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The F5A mutation within the TOS motif inhibits the phosphorylation of Thr-389 and Thr-229 and hence S6K1 activation (20Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Phosphomimetic mutation of the four phosphorylation sites (Ser-411, Ser-418, Thr-421, and Ser-424) at the C terminus along with the T389E mutation partially rescues the activity of the mutant S6K1(F5A), while the S6K1(F5A/T389E) variants lacking the RSPRR sequence either by C-terminal truncations or by multiple Ala substitutions within RSPRR exhibit the full activity of S6K1 (20Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 21Schalm S.S. Tee A.R. Blenis J. J. Biol. Chem. 2005; 280: 11101-11106Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Collectively, these observations suggest that the inhibition conferred by the RSPRR segment cannot be completely overcome by the C-terminal phosphorylation. Therefore, the RSPRR-conferred inhibition may involve multiple mechanisms, some of which cannot be relieved by the C-terminal phosphorylation including the phosphorylation of Ser-411. We show that CDKs are the primary regulators of S6K1 phosphorylation at Ser-411. Ser-411 phosphorylation is insensitive to rapamycin and remains unaffected by the TOS mutation F5A (Fig. 3A; Refs. 20Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar and 36Ferrari S. Pearson R.B. Siegmann M. Kozma S.C. Thomas G. J. Biol. Chem. 1993; 268: 16091-16094Abstract Full Text PDF PubMed Google Scholar), which inhibits Thr-389 phosphorylation and S6K1 activation. Therefore, Ser-411 phosphorylation is independent from the TOR signaling, which is consistent with the notion that S6K1 activation requires inputs from multiple signaling pathways. S6K1 plays a crucial role in cell cycle progression through G1 into S phase. S6K1 inhibition by using rapamycin or by microinjecting S6K1-neutralizing antibodies blocks cell entry into S phase (37Lane H.A. Fernandez A. Lamb N.J. Thomas G. Nature. 1993; 363: 170-172Crossref PubMed Scopus (317) Google Scholar, 38Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1012) Google Scholar, 39Kuo C.J. Chung J. Fiorentino D.F. Flanagan W.M. Blenis J. Crabtree G.R. Nature. 1992; 358: 70-73Crossref PubMed Scopus (559) Google Scholar). Our findings in the present report support a direct role of CDKs in initiating protein translation, which is crucial for G1 progression. It has been reported that Cdk1 catalyzes the mitotic phosphorylation of S6K1 at multiple Ser/Thr-Pro sites including Ser-411 (22Papst P.J. Sugiyama H. Nagasawa M. Lucas J.J. Maller J.L. Terada N. J. Biol. Chem. 1998; 273: 15077-15084Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 23Shah O.J. Ghosh S. Hunter T. J. Biol. Chem. 2003; 278: 16433-16442Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This phosphorylation is correlated with reduced Thr-389 phosphorylation and S6K1 inactivation. It appears that S6K1 adopts a mitotic regulatory mechanism that is distinct from the regulation in G1 phase. At present, it is unclear how Cdk1 mediates S6K1 inactivation at mitosis. In nervous system neurons, the mTOR-S6K1 pathway controls the synaptic synthesis of proteins that are required for long term synaptic plasticity (34Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3377) Google Scholar). Cdk5 has been implicated to regulate synaptic plasticity by a number of mechanisms, including the regulation of vesicle cycling, ion channel conductance, protein clustering, and signal transduction (24Dhavan R. Tsai L.H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 749-759Crossref PubMed Scopus (931) Google Scholar, 40Angelo M. Plattner F. Giese K.P. J. Neurochem. 2006; 99: 353-370Crossref PubMed Scopus (110) Google Scholar). We show that the Cdk5 activity is required for S6K1 activation and thus the rpS6 phosphorylation. This process induces translation initiation of 5′-oligopyrimidine tract-containing mRNAs, which encode components of the protein translation apparatus. Therefore, our findings suggest that Cdk5 may participate in the control of protein synthesis, which plays an important role in response to synaptic demands. In summary, we have revealed the function of S6K1 phosphorylation at Ser-411 in the control of S6K1 activation. Phosphorylation of Ser-411 is mediated by the multifunctional kinase Cdk5-p35 in post-mitotic neurons and by roscovitine-sensitive CDKs in proliferating cells. These findings suggest a link between the CDKs and protein translational control. We thank Dr. J. Blenis (Harvard Medical School) for providing S6K1 constructs and for productive discussions and critical review of the manuscript. We are also grateful to Dr. N. Y. Ip and Dr. Zhenguo Wu (Hong Kong University of Science and Technology) for providing plasmids and reagents and to Dr. Amy K. Y. Fu for her assistance on nucleofection." @default.
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