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Title: $Cyclin-dependent Kinase-5 Is Involved in Neuregulin-dependent Activation of Phosphatidylinositol 3-Kinase and Akt Activity Mediating Neuronal Survival
Abstract: The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays an important role in mediating survival signals in wide variety of neurons and cells. Recent studies show that Akt also regulates metabolic pathways to regulate cell survival. In this study, we reported that cyclin-dependent kinase-5 (Cdk5) regulates Akt activity and cell survival through the neuregulin-mediated PI 3-kinase signaling pathway. We found that brain extracts of Cdk5–/–mice display a lower PI 3-kinase activity and phosphorylation of Akt compared with that in wild type mice. Moreover, we demonstrated that Cdk5 phosphorylated Ser-1176 in the neuregulin receptor ErbB2 and phosphorylated Thr-871 and Ser-1120 in the ErbB3 receptor. We identified the Ser-1120 sequence RSRSPR in ErbB3 as a novel phosphorylation consensus sequence of Cdk5. Finally, we found that Cdk5 activity is involved in neuregulin-induced Akt activity and neuregulin-mediated neuronal survival. These findings suggest that Cdk5 may exert a key role in promoting neuronal survival by regulating Akt activity through the neuregulin/PI 3-kinase signaling pathway. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays an important role in mediating survival signals in wide variety of neurons and cells. Recent studies show that Akt also regulates metabolic pathways to regulate cell survival. In this study, we reported that cyclin-dependent kinase-5 (Cdk5) regulates Akt activity and cell survival through the neuregulin-mediated PI 3-kinase signaling pathway. We found that brain extracts of Cdk5–/–mice display a lower PI 3-kinase activity and phosphorylation of Akt compared with that in wild type mice. Moreover, we demonstrated that Cdk5 phosphorylated Ser-1176 in the neuregulin receptor ErbB2 and phosphorylated Thr-871 and Ser-1120 in the ErbB3 receptor. We identified the Ser-1120 sequence RSRSPR in ErbB3 as a novel phosphorylation consensus sequence of Cdk5. Finally, we found that Cdk5 activity is involved in neuregulin-induced Akt activity and neuregulin-mediated neuronal survival. These findings suggest that Cdk5 may exert a key role in promoting neuronal survival by regulating Akt activity through the neuregulin/PI 3-kinase signaling pathway. Cyclin-dependent kinase 5 (Cdk5) 1The abbreviations used are: Cdk5, cyclin-dependent kinase-5; NRG, neuregulin; PI3K, phosphatidylinositol 3-kinase; HA, hemagglutinin; PBS, phosphate-buffered saline; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; PtdIns(4,5)P2, phosphatidylinositol 4,5-diphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PDK1, phosphoinositide-dependent kinase-1; IP, immunoprecipitation; E18, embryonic day 18; TUNEL, terminal deoxynucleotidyl-transferase enzyme-mediated dUTP nick end labeling. is a serine/threonine kinase, which is predominantly expressed in postmitotic neurons (1Hellmich M.R. Pant H.C. Wada E. Battey J.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10867-10871Crossref PubMed Scopus (248) Google Scholar). Cdk5 kinase activity requires association with its neuron-specific activators, p35 and p39. Cdk5 kinase activity is essential for neuronal migration, neurite outgrowth, and laminar configuration of the cerebral cortex (2Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-426Crossref PubMed Scopus (545) Google Scholar, 3Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (818) Google Scholar, 4Humbert S. Dhavan R. Tsai L-H. J. Cell Sci. 2000; 113: 975-983Crossref PubMed Google Scholar). Recently, Cdk5 has been suggested as contributing to the control of neuronal positioning in Reelin signaling during neural development (5Ohshima T. Mikoshiba K. Mol. Neurobiol. 2002; 26: 153-166Crossref PubMed Scopus (41) Google Scholar) and to mediate neuronal guidance by regulating semaphorin-3A with Fyn kinase (6Sasaki Y. Cheng C. Uchida Y. Nakajima O. Ohshima T. Yagi T. Taniguchi M. Nakayama T. Kishida R. Kudo Y. Ohno S. Nakamura F. Goshima Y. Neuron. 2002; 35: 907-920Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Cdk5 and p35 have recently also been shown to regulate presynaptic and postsynaptic activity by phosphorylating Munc-18, amphiphysin, and the NR2A subunit of the N-methyl-d-aspartate receptor (7Shuang R. Zhang L. Fletcher A. Groblewski G.E. Pevsner J. Stuenkel E.L. J. Biol. Chem. 1998; 273: 4957-4966Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 8Fletcher A.I. Shuang R. Giovannucci D.R. Zhang L. Bittner M.A. Stuenkel E.L. J. Biol. Chem. 1999; 274: 4027-4035Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 9Rosales J.L. Nodwell M.J. Johnston R.N. Lee K.Y. J. Cell. Biochem. 2000; 78: 151-159Crossref PubMed Scopus (57) Google Scholar, 10Floyd S.R. Porro E.B. Slepnev I, V. Ochoa G.C. Tsai L.H. De Camilli P. J. Biol. Chem. 2001; 276: 8104-8110Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 11Li B.S. Sun M.K. Zhang L. Takahashi S. Ma W. Kulkarni A.B. Pant H.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12742-12747Crossref PubMed Scopus (228) Google Scholar). Cdk5 activity also regulates dopamine signaling by phosphorylating DARPP-32 protein (12Bibb J.A. Snyder G.L. Nishi A. Yan Z. Meijer L. Fienberg A.A. Tsai L.H. Kwon Y.T. Girault J.A. Czernik A.J. Huganir R.L. Hemmings Jr., H.C. Nairn A.C. Greengard P. Nature. 1999; 402: 669-671Crossref PubMed Scopus (495) Google Scholar) and has been shown to up-regulate the expression of acetylcholine receptor at the neuromuscular junction (13Fu A.K. Fu W.Y. Cheung J. Tsim K.W. Ip F.C. Wang J.H. Ip N.Y. Nat. Neurosci. 2001; 4: 374-381Crossref PubMed Scopus (153) Google Scholar). These studies indicate that Cdk5 is a multifunctional protein kinase in the central nervous system. Cdk5 has been implicated in both cell survival and programmed cell death in neuronal and nonneuronal systems (14Ahuja H.S. Zhu Y. Zakeri Z. Dev. Genet. 1997; 21: 258-267Crossref PubMed Scopus (53) Google Scholar, 15Zhang D. Sliwkowski M.X. Mark M. Frantz G. Akita R. Sun Y. Hillan K. Crowley C. Brush J. Godowski P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9562-9567Crossref PubMed Scopus (334) Google Scholar). Cells induced to apoptosis by exogenous signals such as staurosporin display increased Cdk5 activity (16Gao C. Negash S. Wang H.S. Ledee D. Guo H. Russell P. Zelenka P. J. Cell Sci. 2001; 114: 1145-1153PubMed Google Scholar, 17Zhu X. Lai C. Thomas S. Burden S.J. EMBO J. 1995; 14: 5842-5848Crossref PubMed Scopus (159) Google Scholar). Recently, Cdk5 in association with p25, a truncated form of p35, has been thought to be deregulated to produce hyperphosphorylated tau protein and to disrupt the neuronal cytoskeleton, and it may be involved in neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral secrosis, Parkinson's disease, and Niemann-Pick disease (18Patrick G.N. Zukerberg L. Nikolic M. de la Monte S. Dikkes P. Tsai L.H. Nature. 2001; 411: 764-765Crossref PubMed Scopus (17) Google Scholar, 19Lee M.S. Kwon Y.T. Li M. Peng J. Friedlander R.M. Tsai L.H. Nature. 2000; 405: 360-364Crossref PubMed Scopus (922) Google Scholar, 20Kusakawa 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 (340) Google Scholar, 21Nguyen M.D. Lariviere R.C. Julien J.P. Neuron. 2001; 30: 135-147Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 22Nakamura S. Kawamoto Y. Nakano S. Akiguchi I. Kimura J. Acta Neuropathol. (Berl.). 1997; 94: 153-157Crossref PubMed Scopus (80) Google Scholar, 23Bu B. Klunemann H. Suzuki K. Li J. Bird T. Jin L.W. Vincent I. Neurobiol. Dis. 2002; 11: 285-297Crossref PubMed Scopus (55) Google Scholar). On the other hand, Cdk5 may also be involved in neuronal survival. Cdk5 knockout mice exhibit a unique phenotype with perinatal mortality, associated with extensively disrupted cerebral cortical layering due to abnormal neuronal migration, absence of cerebella foliation, and degeneration of neurons in the brain stem and the spinal cord (24Ohshima T. Ward M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (815) Google Scholar, 25Ohshima T. Gilmore E.C. Longenecker G. Jacobowitz D.M. Brady R.O. Herrup K. Kulkarni A.B. J. Neurosci. 1999; 19: 6017-6026Crossref PubMed Google Scholar). The p35 knockout mice are viable and fertile (26Chae T. Kwon Y.T. Bronson R. Dikkes P. Tsai L.H. Neuron. 1997; 18: 29-42Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 27Kwon Y.T. Tsai L-H. Crandall J.E. J. Comp. Neurol. 1999; 415: 218-229Crossref PubMed Scopus (69) Google Scholar), with fewer severe abnormalities of laminar structures in the cerebral cortex, compared with Cdk5 null mice (28Ohshima T. Ogawa M. Veeranna Hirasawa M. Longenecker G. Ishiguro K. Pant H.C. Brady R.O. Kulkarni A.B. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2764-2769Crossref PubMed Scopus (115) Google Scholar). Recent studies on Cdk5 null mice expressing transgenic Cdk5, regulated by a p35 promoter to ensure specific expression in brain neurons (TgKO), were rescued and exhibited the wild type phenotype (29Tanaka T. Veeranna Ohshima T. Rajan P. Amin N.D. Cho A. Sreenath T. Pant H.C. Brady R.O. Kulkarn A.B. J. Neurosci. 2001; 21: 550-558Crossref PubMed Google Scholar). This suggests that neuronal survival (and host survival) is dependent upon Cdk5 activity. In addition, our recent studies demonstrated that Cdk5 prevents neuronal apoptosis by negative regulation of c-Jun N-terminal kinase 3 in cortical neurons (30Li B.S. Zhang L. Takahashi S. Ma W. Jaffe H. Kulkarni A.B. Pant H.C. EMBO J. 2002; 21: 324-333Crossref PubMed Scopus (122) Google Scholar). These studies indicate that the role of Cdk5 activity in neuronal survival may depend on the specific cell type as well as its site of action in survival and/or apoptotic signal pathways. A clue as to other possible sites of Cdk5 activity in regulating neuronal survival is suggested by the observation that Cdk5 activity, mediated by the neuregulin/ErbB pathway, up-regulates acetylcholine receptor expression at the neuromuscular junction in embryonic myotubes and adult muscle (13Fu A.K. Fu W.Y. Cheung J. Tsim K.W. Ip F.C. Wang J.H. Ip N.Y. Nat. Neurosci. 2001; 4: 374-381Crossref PubMed Scopus (153) Google Scholar). The neuregulins (NRGs) are a class of epidermal growth factor-like molecules; a multigene family encodes them. All NRG proteins contain an extracellular epidermal growth factor-like domain, which is essential for their function (31Adlkofer K. Lai C. Glia. 2000; 29: 104-111Crossref PubMed Scopus (158) Google Scholar, 32Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N Raab H. Lewis G.D. Shepard H.M. Kuang W.J. Wood W.I. Goeddel D.V. Vandlen R.L. Science. 1992; 256: 1205-1210Crossref PubMed Scopus (937) Google Scholar, 33Peles E. Lamprecht R. Ben-Levy R. Tzahar E.Y. Arden Y. J. Biol. Chem. 1992; 267: 12266-12274Abstract Full Text PDF PubMed Google Scholar, 34Falls D.L. Rosen K.M. Corfas G. Lane W.S. Fischbach G.D. Cell. 1993; 72: 801-815Abstract Full Text PDF PubMed Scopus (562) Google Scholar, 35Chang H. Riese D.J. Gilbert W. Stern D.F. McMahan U.J. Nature. 1997; 387: 509-512Crossref PubMed Scopus (259) Google Scholar,15Zhang D. Sliwkowski M.X. Mark M. Frantz G. Akita R. Sun Y. Hillan K. Crowley C. Brush J. Godowski P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9562-9567Crossref PubMed Scopus (334) Google Scholar). The NRG-1/neuregulin family includes heregulin, acetylcholine receptor-inducing activity, neu differentiation factor, and glial growth factor (36Burden S. Yarden Y. Neuron. 1997; 18: 847-855Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 37Meyer D. Birchmeier C. Nature. 1995; 378: 386-390Crossref PubMed Scopus (1065) Google Scholar). Neuregulin proteins mediate their action through the ErbB family of receptor tyrosine kinases, including ErbB2, ErbB3, and ErbB4 (36Burden S. Yarden Y. Neuron. 1997; 18: 847-855Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 38Gassmann M. Lemke G. Curr. Opin. Neurobiol. 1997; 7: 87-92Crossref PubMed Scopus (108) Google Scholar). NRG receptors differ in kinase activity and substrate selectivity. Each ErbB protein has an extracellular ligand binding domain, a single transmembrane domain, a short intracellular juxtamembrane region, a tyrosine kinase domain, and a protein-rich carboxyl-terminal tail (36Burden S. Yarden Y. Neuron. 1997; 18: 847-855Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). NRG-1 binds to ErbB3 and induces the formation of heterodimers between ErbB2 and ErbB3 and thus activates the receptor (36Burden S. Yarden Y. Neuron. 1997; 18: 847-855Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Targeted disruptions of the NRG-1 gene as well as the neuregulin receptors demonstrate that neuregulins are essential for the formation of the heart and nervous system (39Lin W. Sanchez H.B. Deerinck T. Morris J.K. Ellisman M. Lee K.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1299-1304Crossref PubMed Scopus (157) Google Scholar, 40Gassmann M. Casagranda F. Orioli D. Simon H. Lai C. Klein R. Lemke G. Nature. 1995; 378: 390-394Crossref PubMed Scopus (957) Google Scholar). Neuregulins have been implicated in a number of events in cell survival, mitosis, migration, and differentiation (41Dong Z. Brennan A. Liu N. Yarden Y. Lefkowitz G. Mirsky R. Jessen K.R. Neuron. 1995; 15: 585-596Abstract Full Text PDF PubMed Scopus (403) Google Scholar, 42Anton E.S. Marchionni M.A. Lee K.F. Rakic P. Development. 1997; 124: 3501-3510Crossref PubMed Google Scholar, 43Rio C. Rieff H.I. Qi P. Khurana T.S. Corfas G. Neuron. 1997; 19: 39-50Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 44Shah N.M. Marchionni M.A. Isaacs I. Stroobant P. Anderson D.J. Cell. 1994; 77: 349-360Abstract Full Text PDF PubMed Scopus (461) Google Scholar). The survival of Schwann cells, for example, is mediated by neuregulin signaling through the phosphoinositide 3-kinase (PI3K)/Akt pathway, a critical survival pathway in neurons and in most cell types (45Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4994) Google Scholar). Accordingly, it seemed possible that an alternative site for Cdk5 regulation of neuronal cell survival might be the neuregulin-mediated PI3K/Akt signaling pathway. To explore this possibility, we took advantage of the Cdk5 null mouse, which exhibited no Cdk5 activity (24Ohshima T. Ward M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (815) Google Scholar), and examined the activities of PI3K and Akt. In this study, we report that Cdk5 regulates Akt activity through phosphorylation of the neuregulin receptors (ErbB2/ErbB3) and regulation of PI3K/Akt kinase signaling pathways. We found that brain extracts and cortical neurons from Cdk5 knockout mice exhibited reduced PI3K/Akt activities and increased apoptosis. The Ser-1120 residue in the RSRSPR sequence in the ErbB3 receptor is a novel Cdk5 consensus sequence. Here we see that Cdk5 activity sustains neuronal survival by activating a survival-signaling pathway, upstream at the receptor itself. Cell Culture—Cortical neurons from embryonic day 16.5 and 18 Cdk5 wild type and knockout mice (Cdk5–/–) were prepared as described by Li et al. (30Li B.S. Zhang L. Takahashi S. Ma W. Jaffe H. Kulkarni A.B. Pant H.C. EMBO J. 2002; 21: 324-333Crossref PubMed Scopus (122) Google Scholar). In brief, embryos were dissected and minced well with scissors. The dissociated cells were collected by centrifugation and resuspended in a serum-free neurobasal medium supplemented with B27 and 0.5 mm l-glutamine. Cells (25 × 104) were plated in 35-mm plastic dishes precoated with polylysine (10 μg/ml) (Gibco) for 7 days. HEK-293T and COS-7 cells were cultured in Dulbecco's modified Eagle's medium with 5% fetal calf serum. Preparation of Brain Extracts—Brain of E18.5 from Cdk5–/– and wild type mice were rinsed with cold phosphate-buffered saline (PBS) and homogenized in a buffer containing 0.32 m sucrose, 5 mm HEPES, pH 7.4, and protease inhibitor mixture using poltroon. The homogenate was centrifuged at 10,000 × g for 60 min. The supernatant was analyzed for in vitro phosphorylation and PI3K activity. Phospholipid Extraction—Equal aliquots of 50 μl (1 μg/μl) of supernatant from Cdk5–/– and wild type mice brain (E18) were phosphorylated as described by Li et al. (30Li B.S. Zhang L. Takahashi S. Ma W. Jaffe H. Kulkarni A.B. Pant H.C. EMBO J. 2002; 21: 324-333Crossref PubMed Scopus (122) Google Scholar). In brief, the samples were incubated in 15 μCi of [32P]ATP in a buffer containing 50 mm Tris (pH 7.5), 50 mm NaCl, 5 mm MgCl2, 2 mm dithiothreitol for 2 h at 30 °C with constant mixing. The reactions were stopped by the addition of chloroform/methanol (2:1, v/v). Following vigorous overtaxing, tubes were spun at 3000 rpm for 10 min. the organic phase containing phospholipids was collected, and a 0.8-ml mixture of chloroform/methanol/H2O (3:48:47) was added to the organic phase vortexes and spun for 10 min at 3000 rpm. The aqueous phase was discarded completely, and the organic phase was used to analyze the 32P incorporation into the lipids. The extracted phospholipids were quantified by liquid scintillation. PI3K Assay—Brain tissue from Cdk5–/– and wild type mice (E18) was homogenized in a buffer containing Nonidet P-40 (1%), 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm sodium orthovanadate, 2 mm sodium pyrophosphate, 50 mm NaF, 2 mm EDTA, 3 mm EGTA, and protease and phosphatase inhibitors. Cell lysates (200 μg of total protein) were immunoprecipitated with anti-p85 subunit antibody. The precipitates were resuspended in 50 μl of kinase assay buffer (10 mm Tris-HCl, pH 7.6, 100 mm NaCl, 1 mm EDTA, and 100 μm Na3VO4). The activity of PI3K was assayed essentially as described by Hii et al. (73Hii C.S. Moghadammi N. Dunbar A. Ferrante A. J. Biol. Chem. 2001; 276: 27246-27255Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Briefly, 10 μl of MgCl2 (stock concentration of 100 mm), 10 μl of PtdIns (stock concentration of 1 μg), and 5 μl of 400 mm Tris-HCl, pH 7.6, were added to the 50-μl precipitates, and the reaction was started by the addition of 25 μCi of [γ-32P]ATP. The reaction was incubated at room temperature for 10 min with constant mixing. The reaction was terminated by the addition of 40 μl of 8 n HCl and 320 μl of chloroform/methanol (1:1). After vigorous mixing and centrifugation, the lipid phase was collected and washed with 500 ml of CH3OH/H2O (1:1). The inositol 3,4,5-trisphosphate and phosphatidylinositols were separated by TLC using CHCl3/CH3OH/H2O/30% NH4OH (90:70:14.6:5.4, v/v/v/v) developing mixture. Phosphorylated phospholipids were detected by autoradiography. Phosphorylation Studies—P7 rat brain extracts were prepared, and Cdk5 was immunoprecipitated using anti-Cdk5 antibody as described by Li et al. (30Li B.S. Zhang L. Takahashi S. Ma W. Jaffe H. Kulkarni A.B. Pant H.C. EMBO J. 2002; 21: 324-333Crossref PubMed Scopus (122) Google Scholar). The peptides were synthesized commercially (Peptide Technologies Inc.). For in vitro phosphorylation studies, we incubated peptides or histone H1 with the Cdk5 immunoprecipitate (IP). An Akt IP was used to phosphorylate H2B to determine Akt activity. Substrates (peptides or H2B) were incubated with [γ-32P]ATP (0.1 mm) in a buffer containing 50 mm Tris-HCl (pH 7.4) with 1 mm EGTA, 1 mm dithiothreitol, 5 mm MgCl2, 0.5 μm microcystin LR, and immunoprecipitated Cdk5 or Akt for 30 min at room temperature as described by Li et al. (30Li B.S. Zhang L. Takahashi S. Ma W. Jaffe H. Kulkarni A.B. Pant H.C. EMBO J. 2002; 21: 324-333Crossref PubMed Scopus (122) Google Scholar). To study the phosphorylation of ErbB-specific sites by Cdk5, the mutant and wild type C-terminal domain (residues 663–1339) of rat ErbB3 plasmids were transfected into COS-7 cells using LipofectAMINE. HA-tagged ErbB3 wild type and mutant (T871A and S1120A) C-terminal domains (residues 663–1339) of the receptor were generated by standard cloning methods. The putative phosphorylation sites in ErbB3 were mutated using the QuikChange™ site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The mutations were verified by DNA sequencing. Western Blot Analysis—Brain tissues or cells were homogenized in a buffer (5 mm HEPES, pH 7.4, 150 mm NaCl, 1% Triton X-100, 10 mm glycerol, 1 mm EDTA, 2 mm Na3VO4, 5 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, leupeptin, and pepstatin). Proteins were resolved by 10–20% SDS-PAGE, blotted onto a polyvinylidene difluoride membrane (Roche Applied Science); blocked in 5% skim milk, 1× PBS, 0.05% Tween 20; and probed with primary antibodies. Anti-Cdk5 polyclonal antibody, anti-ErbB3 polyclonal antibody (C-17) or anti-ErbB3 monoclonal antibody (G-4) (C-8; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-phospho-Ser-473 and Thr-308 Akt, or anti-total Akt was used for Western blot analysis using the ECL kit (Amersham Biosciences) following the manufacturer's procedures. Apoptosis Assays—DNA fragmentation associated with apoptosis was detected by TUNEL histochemistry staining. Cortical neurons with or without inhibitor treatment were directly mounted on cover slides and fixed with 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 (20 min at room temperature and then incubated for nick end-labeling for 2 h at 37 °C with TdT according to standard procedures (Roche Applied Science). Immunofluorescence—Cortical neurons or brain sections were fixed in 4% paraformaldehyde in PBS for 30 min, washed in several changes of PBS for 30 min, and permeabilized in 0.2% Triton X-100 in PBS for 15 min. Monoclonal or polyclonal anti-Cdk5 antibody (1:200; Santa Cruz Biotechnology), polyclonal antibody ErbB3 (C-17; Santa Cruz), and phospho-Akt (Thr308) (1:200; New England Biolabs) was incubated overnight at 4 °C. After a wash in PBS (three times for 15 min each), Cells or sections were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG and rhodamine-labeled goat anti-rabbit IgG or rhodamine-labeled goat anti-mouse IgG secondary antibody for1hat room temperature. Fluorescent images were obtained using a Zeiss LSM-410 laser-scanning confocal microscope. Images were processed and merged using Adobe PhotoShop software. Mass Spectrometry—Peptides were phosphorylated using nonradioactive ATP as described above. Phosphorylation of the peptides was determined by MALDI-TOF mass spectra on a Voyager-DE STR Biospectrometry Work station (Applied Biosystems) operating in the negative linear mode. α-Cyano-4-hydroxycinnamic acid matrix was utilized. Reduced Brain Lipid Phosphorylation and PI3K Activity in Cdk5–/– Mouse Brain—Previous studies have shown that neuronal Cdk5 activity is necessary for survival and nervous system development (29Tanaka T. Veeranna Ohshima T. Rajan P. Amin N.D. Cho A. Sreenath T. Pant H.C. Brady R.O. Kulkarn A.B. J. Neurosci. 2001; 21: 550-558Crossref PubMed Google Scholar, 30Li B.S. Zhang L. Takahashi S. Ma W. Jaffe H. Kulkarni A.B. Pant H.C. EMBO J. 2002; 21: 324-333Crossref PubMed Scopus (122) Google Scholar, 46Ko j. humbert S. Bronson R.T. Takahashi S. Kulkarni A.B. Li E. Tsai L.H. J. Neurosci. 2001; 21: 6758-6771Crossref PubMed Google Scholar). Phospholipid kinases, the phosphoinositide kinases, are responsible for the phospholipid phosphorylation. Among three general families, PI3K selectively phosphorylates phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P2) and produces phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) upon stimulation by a variety of ligands in vivo as well as in vitro (47Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (139) Google Scholar). PtdIns(3,4,5)P3 is a key molecule involved in cell growth and survival signaling (48Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2145) Google Scholar). To determine the identity of this fraction, phospholipids were extracted from wild type and Cdk5–/– mouse brain extracts after in vitro phosphorylation in the presence of [γ-32P]ATP as described under "Experimental Procedures." The data presented in Fig. 1A show a 6-fold decrease in γ-32P incorporation into the phospholipids in brain extracts of Cdk5–/– as compared with wild type mice. In the absence of Cdk5, the synthesis of phospholipids is compromised. In order to explore the possibility of Cdk5-mediated interaction with the phospholipid-dependent PI3K signaling pathway, we compared PI3K activity in the cortical neurons derived from wild type and Cdk5–/– mice. The cortical neurons of Cdk5–/– mice showed a significantly decreased PI3K activity (Fig. 1B). Akt Phosphorylation and Activity Is Decreased in Cdk5–/– Mouse Brain—Since Akt phosphorylation on both Thr-308 and Ser-473 by 3′-phosphoinositide-dependent kinase-1 (PDK1) is dependent on the levels of PtdIns(3,4,5)P3, the key product of PI3K activity and PtdIns(4,5)P2 levels (49Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar), we compared the Akt phosphorylation and activity levels in cortical extracts of E18-wild type and Cdk5–/– mice (Fig. 2, A and B). The total Akt and phosphorylated Akt expression levels were determined by Western blot analysis with specific anti-phospho-Akt (Thr-308 or Ser-473) and anti-total Akt antibodies. Akt kinase activity was assessed using IPs from brain extracts obtained with total Akt antibody and histone H2B as a substrate. We found that expression levels of Akt protein of wild type and Cdk5–/– mice were similar (Fig. 2C). However, the phosphorylation of Akt and its activity were significantly decreased in the Cdk5–/– mice compared with wild type mouse brain extracts (Fig. 2, C and D). Although the Akt activity was reduced to low levels in Cdk5–/– mice brain extracts, the corresponding Akt phosphorylation was not reduced proportionally (Fig. 2, C and D). This may be due to some cross-reactively of phospho-Akt antibodies used in these experiments. In the absence of any Cdk5 activity in Cdk5–/– mice, the PI3K/Akt kinases are down-regulated, suggesting that their activities are dependent upon Cdk5 phosphorylation. Neuregulin Receptor ErbB3 and ErbB2 Are Phosphorylated by Cdk5—The above observations suggest that Cdk5 activity is involved in the PI3K signaling pathway. To determine the target substrate for Cdk5, we based our analysis on the observation that the Cdk5-p35 complex is implicated in neuregulin-induced acetylcholine receptor expression at the neuromuscular junction (13Fu A.K. Fu W.Y. Cheung J. Tsim K.W. Ip F.C. Wang J.H. Ip N.Y. Nat. Neurosci. 2001; 4: 374-381Crossref PubMed Scopus (153) Google Scholar). This suggested that ErbB receptors might be a substrate for Cdk5 phosphorylation. Indeed, we found that the putative Cdk5 phosphorylation consensus sequence motifs are present in the ErbB2 and ErbB3 receptor molecules. Ser-1176 in the sequence SPGK of ErbB2, Thr-871 in the TPIK sequence, and Ser-1204 in the SPPR sequence of ErbB3 are putative phosphorylation sites in Cdk5 consensus motifs. To test whether these and other proline-directed serine residues in the peptides derived from ErbB2 and ErbB3 (see Fig. 3A) are the sites phosphorylated by Cdk5, we incubated these peptides and their mutant forms with Cdk5 IP from brain extracts and determined their phosphorylation by in vitro kinase assays. We found that Cdk5 significantly phosphorylated the Ser-1176 in the ErbB2 peptide RPKTLSPGKN and Thr-871 in the ErbB3 peptide AKTPIKWAL but weakly phosphorylated Ser-1204 in the ErbB3 proline-rich peptide RRGSPPRPPR (Fig. 3C). Interestingly, we found that the Ser-1120 in the basic amino acid-rich sequence RSRSRSPRPR of ErbB3 was the most favorable substrate for Cdk5 (Fig. 3C). The peptide RSRSRSPRPR does not have the conventional consensus sequence ((K/R)(S/T)PX(K/R), where X represents a basic residue) of cyclin-dependent kinase including Cdk5, but it is rich in basic amino acids and appears to be a novel Cdk5 phosphorylation consensus sequence. To confirm whether Cdk5 phosphorylates the Ser-1120 residue, we conducted in vitro kinase assays using various mutant peptides derived from RSRSRSPRPR in which alanines were substituted for serines (Fig. 3, B and D). Ser-1120 appeared to be the site for Cdk5 phosphorylation. The phosphorylation of Thr-871 of RPKTLSPGKN in ErbB2 and Ser-1120 of RSRSRSPRPR in ErbB3 were further investigated and confirmed by mass spectrometry (Fig. 3, E and F). To investigate the Cdk5 phosphorylation of ErbB3 in vivo, the Thr-871 and Ser-1120 in the C-terminal domain of rat ErbB3 were mutated to Ala (T871A and S1120A). COS-7 cells were cotransfected with wild type HA-ErbB3 or mutant HA-ErbB3 (T871A and S1120A) C-terminal domains with Cdk5-p35. The levels of phosphorylated wild type HA-ErbB3 C terminus and HA-ErbB3 (T871A and S1120A) were measured by immunoprecipitation using anti-HA-tagged antibody, and their phosphorylation state was detected by Western blot using anti-phosphoserine and anti-phosphothreonine antibody. We found that mutation of Thr-871 or Ser-1120 to Ala in ErbB3 showed no detectable Cdk5-dependent serine and threonine phosphorylation in HA-ErbB3 (T871A and S