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• W2004176156 abstract "Cdk5 is a proline-directed Ser/Thr protein kinase predominantly expressed in postmitotic neurons together with its activator, p35. N-terminal truncation of p35 to p25 by calpain results in deregulation of Cdk5 and contributes to neuronal cell death associated with several neurodegenerative diseases. Previously we reported that p35 occurred as a phosphoprotein, phospho-p35 levels changed with neuronal maturation, and that phosphorylation of p35 affected its vulnerability to calpain cleavage. Here, we identify the p35 residues Ser8 and Thr138 as the major sites of phosphorylation by Cdk5. Mutagenesis of these sites to unphosphorylatable Ala increased susceptibility to calpain in cultured cells and neurons while changing them to phosphomimetic glutamate-attenuated cleavage. Furthermore, phosphorylation state-specific antibodies to these sites revealed that Thr138 was dephosphorylated in adult rat, although both Ser8 and Thr138 were phosphorylated in prenatal brains. In cultured neurons, inhibition of protein phosphatases converted phosho-Ser8 p35 to dual phospho-Ser8/Thr138 p35 and conferred resistance to calpain cleavage. These results suggest phosphorylation of Thr138 predominantly defines the susceptibility of p35 to calpain-dependent cleavage and that dephosphorylation of this site is a critical determinant of Cdk5-p25-induced cell death associated with neurodegeneration. Cdk5 is a proline-directed Ser/Thr protein kinase predominantly expressed in postmitotic neurons together with its activator, p35. N-terminal truncation of p35 to p25 by calpain results in deregulation of Cdk5 and contributes to neuronal cell death associated with several neurodegenerative diseases. Previously we reported that p35 occurred as a phosphoprotein, phospho-p35 levels changed with neuronal maturation, and that phosphorylation of p35 affected its vulnerability to calpain cleavage. Here, we identify the p35 residues Ser8 and Thr138 as the major sites of phosphorylation by Cdk5. Mutagenesis of these sites to unphosphorylatable Ala increased susceptibility to calpain in cultured cells and neurons while changing them to phosphomimetic glutamate-attenuated cleavage. Furthermore, phosphorylation state-specific antibodies to these sites revealed that Thr138 was dephosphorylated in adult rat, although both Ser8 and Thr138 were phosphorylated in prenatal brains. In cultured neurons, inhibition of protein phosphatases converted phosho-Ser8 p35 to dual phospho-Ser8/Thr138 p35 and conferred resistance to calpain cleavage. These results suggest phosphorylation of Thr138 predominantly defines the susceptibility of p35 to calpain-dependent cleavage and that dephosphorylation of this site is a critical determinant of Cdk5-p25-induced cell death associated with neurodegeneration. Cyclin-dependent kinase 5 (Cdk5) 2The abbreviation used is: Cdk5, cyclin-dependent kinase 5. 2The abbreviation used is: Cdk5, cyclin-dependent kinase 5. is a unique member of the Cdk family. Its activity in postmitotic neurons is completely dependent upon association with one of two neuronal specific activators, p35 or p39. Cdk5/p35 is involved in a panoply of processes critical to central nervous system function both during development and throughout maturity including neuronal migration during corticogenesis, neurite outgrowth, regulation of the synaptic vesicle cycle, neurotransmitter release, and postsynaptic neurotransmitter receptor regulation and signaling (1Tang D. Wang J.H. Prog. Cell Cycle Res. 1996; 2: 205-216Crossref PubMed Scopus (75) Google Scholar, 2Dhavan R. Tsai L.H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 749-759Crossref PubMed Scopus (931) Google Scholar, 3Hisanaga S. Saito T. Neurosignals. 2003; 12: 221-229Crossref PubMed Scopus (78) Google Scholar). The mechanisms by which Cdk5 activity is normally regulated remains to be fully delineated. Furthermore, because aberrant Cdk5 activity has been implicated in the etiology of neurodegenerative diseases (4Shelton S.B. Johnson G.V. J. Neurochem. 2004; 88: 1313-1326Crossref PubMed Scopus (129) Google Scholar, 5Tsai L.H. Lee M.S. Cruz J. Biochim. Biophys. Acta. 2004; 1697: 137-142Crossref PubMed Scopus (100) Google Scholar), identifying the biochemical mechanisms contributing to deregulation of Cdk5 is of substantial biomedical relevance. Deregulation of Cdk5 results from removal of the first 98 amino acids of p35 by the Ca2+-dependent cysteine protease, calpain, leaving Cdk5 associated with the N-terminal truncated form p25. Cleavage of p35 to p25 changes the subcellular distribution of active Cdk5 from membranes to the cytosolic fraction (6Patrick G.N. Zukerberg L. Nikolic M. de la Monte S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1297) Google Scholar, 7Kusakawa 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), thereby altering substrate specificity. p25 accumulates in neurons undergoing various types of cell death (6Patrick G.N. Zukerberg L. Nikolic M. de la Monte S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1297) Google Scholar, 7Kusakawa 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, 8Lee 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, 9Kerokoski P. Suuronen T. Salminen A. Soininen H. Pirttila T. Biochem. Biophys. Res. Commun. 2001; 280: 998-1002Crossref PubMed Scopus (30) Google Scholar). Expression of Cdk5/p25 in cultured cells results in increased phospho-Tau levels in comparison to cells expressing Cdk5/p35 (6Patrick G.N. Zukerberg L. Nikolic M. de la Monte S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1297) Google Scholar). Furthermore, exogenous overexpression of p25 in transgenic mice results in a neurodegenerative phenotype including the formation of paired helical filaments, Tau aggregation, and neuronal loss similar to that observed in Alzheimer disease (10Noble W. Olm V. Takata K. Casey E. Mary O. Meyerson J. Gaynor K. LaFrancois J. Wang L. Kondo T. Davies P. Burns M. Nixon R.Veeranna Dickson D. Matsuoka Y. Ahlijanian M. Lau L.F. Duff K. Neuron. 2003; 38: 555-565Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 11Cruz J.C. Tseng H.C. Goldman J.A. Shih H. Tsai L.H. Neuron. 2003; 40: 471-483Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). Cdk5/p25 has also been implicated in ischemia-induced neuronal loss in the hippocampus via increased phosphorylation of the NR2A subunit of the N-methyl-d-aspartic acid receptor (12Wang J. Liu S. Fu Y. Wang J.H. Lu Y. Nat. Neurosci. 2003; 6: 1039-1047Crossref PubMed Scopus (232) Google Scholar). In addition, several recent reports indicate that Cdk5-p25 mediates cell death via translocation to the nucleus (13O'Hare M.J. Kushwaha N. Zhang Y. Aleyasin H. Callaghan S.M. Slack R.S. Albert P.R. Vincent I. Park D.S. J. Neurosci. 2005; 25: 8954-8966Crossref PubMed Scopus (115) Google Scholar, 14Hamdane M. Bretteville A. Sambo A.-V. Schinodowski K. Begard S. Delacourte A. Bertrand P. Buee L. J. Cell Sci. 2005; 118: 1291-1298Crossref PubMed Scopus (81) Google Scholar, 15Smith P.D. Mount M.P. Shree R. Callaghan S. Slack R.S. Anisman H. Vincent I. Wang X. Mao Z. Park D.S. J. Neurosci. 2006; 26: 440-447Crossref PubMed Scopus (162) Google Scholar). p25 generation increases nuclear Cdk5 activity in cultured neurons, facilitating phosphorylation and inhibition of the pro-survival transcription factor MEF2 (13O'Hare M.J. Kushwaha N. Zhang Y. Aleyasin H. Callaghan S.M. Slack R.S. Albert P.R. Vincent I. Park D.S. J. Neurosci. 2005; 25: 8954-8966Crossref PubMed Scopus (115) Google Scholar, 15Smith P.D. Mount M.P. Shree R. Callaghan S. Slack R.S. Anisman H. Vincent I. Wang X. Mao Z. Park D.S. J. Neurosci. 2006; 26: 440-447Crossref PubMed Scopus (162) Google Scholar, 16Gong X. Tang X. Wiedmann M. Wang X. Peng J. Zheng D. Blair L.A. Marshall J. Mao Z. Neuron. 2003; 38: 33-46Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Aberrant Cdk5 activity may also contribute to neuronal cell death via phosphorylation of other survival factors such as the tumor suppressor protein p53 (17Zhang J. Krishnamurthy P.K. Johnson G.V. J. Neurochem. 2002; 81: 307-313Crossref PubMed Scopus (93) Google Scholar) and retinoblastoma protein (14Hamdane M. Bretteville A. Sambo A.-V. Schinodowski K. Begard S. Delacourte A. Bertrand P. Buee L. J. Cell Sci. 2005; 118: 1291-1298Crossref PubMed Scopus (81) Google Scholar). Ca2-dependent activation of the cytoplasmic protease calpain is involved in apoptotic and necrotic cell death (18Suzuki K. Hata S. Kawabata Y. Sorimachi H. Diabetes. 2004; 53: 12-18Crossref PubMed Google Scholar). Calpain generally recognizes motifs between conformational domains and cleaves substrate proteins in a limited manner, although the physiological function of calpain activity remains unclear. In some cases calpain cleavage is suspected to be a signaling process. Calpain-mediated cleavage of many proteins including neurofilament proteins (19Pant H.C. Biochem. J. 1988; 256: 665-668Crossref PubMed Scopus (208) Google Scholar), αII-spectrin (20Nicolas G. Fournier C.M. Galand C. Malbert-Colas L. Bournier O. Kroviarski Y. Bourgeois M. Camonis J.H. Dhermy D. Grandchamp B. Lecomte M.C. Mol. Cell. Biol. 2002; 22: 3527-3536Crossref PubMed Scopus (86) Google Scholar), NR2 subunits of N-methyl-d-aspartic acid receptors (21Bi R. Rong Y. Bernard A. Khrestchatisky M. Baudry M. J. Biol. Chem. 2000; 275: 26477-26483Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and ezrin (22Wang H. Guo Z. Wu F. Long F. Cao X. Liu B. Zhu Z. Yao X. Biochem. Biophys. Res. Commun. 2005; 333: 496-501Crossref PubMed Scopus (21) Google Scholar) is suppressed through phosphorylation. However, how such signaling works and the mechanisms of phosphorylation-dependent inhibition are unknown. Previously we demonstrated that Cdk5 phosphorylated p35, that p35 occurred as a phosphoprotein in neurons, and that the phosphorylation state of p35 affected its susceptibility to calpain cleavage (23Saito T. Onuki R. Fujita Y. Kusakawa G. Ishiguro K. Bibb J.A. Kishimoto T. Hisanaga S. J. Neurosci. 2003; 23: 1189-1197Crossref PubMed Google Scholar). Phospho-p35 predominates in fetal rat brain and is resistant to the cleavage by calpain, whereas unphosphorylated p35 present during adulthood is more vulnerable to calpain-dependent cleavage. Here we report that Ser8 and Thr138 of p35 serve as the sites of Cdk5-dependent phosphorylation. Furthermore, phosphorylation at these sites reduces the susceptibility of p35 to calpain cleavage. Moreover, specific dephosphorylation of Thr138 increases the susceptibility of p35 to the cleavage by calpain in adult rat brains, suggesting that the phosphorylation of this site is a particularly critical determinant of Cdk5-dependent neuronal cell death in neurodegenerative diseases. Chemicals and Antibodies—cDNAs encoding human Cdk5 and p35 within pCMV vectors were provided by Dr. L.-H. Tsai (Harvard Medical School, Boston, MA) (24Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (802) Google Scholar). Human m-calpain and the rabbit small subunit of calpain encoded within pSRD vectors using SV40 early promoters were described previously (25Sorimachi H. Toyama-Sorimachi N. Saido T.C. Kawasaki H. Sugita H. Miyasaka M. Arahata K. Ishiura S. Suzuki K. J. Biol. Chem. 1993; 268: 10593-10605Abstract Full Text PDF PubMed Google Scholar). Ionomycin, calpain inhibitor IV, and benzyloxycarbonylleucyl-leucyl-leucinal (MG132) were purchased from Calbiochem. Roscovitine, okadaic acid, and bacterial alkaline phosphatase were obtained from Wako Chemicals (Osaka, Japan). The anti-p35 antibody C19 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-Cdk5 antibody DC17 was purchased from Calbiochem. The anti-calpain-cleaved αII-spectrin antibody and the anti-active m-calpain antibody were described previously (26Takano J. Tomioka M. Tsubuki S. Higuchi M. Iwata N. Itohara S. Maki M. Saido T.C. J. Biol. Chem. 2005; 280: 16175-16184Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 27Zhang J. Miyamoto K. Hashioka S. Hao H.P. Murao K. Saido T. Nakanishi H. Dev. Brain Res. 2003; 142: 105-110Crossref PubMed Scopus (19) Google Scholar). Horseradish peroxidase-conjugated or alkaline phosphatase-conjugated secondary antibody were from DAKO (Glostrup, Denmark). The enhanced chemiluminescence (ECL) detection system was from Amersham Biosciences (Tokyo, Japan) and the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium phosphatase substrate system was from KPL (Gaithersburg, MD). Plasmid Construction of Mutant p35—The Ala mutants at Ser8, Thr138, Ser170, or Thr197 (S8A, T138A, S170A, or T197A) were generated using p35 as a template by the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Primers used were 5′-GTGCTGTCCCTGGCTCCCAGCTACC-3′ and 5′-GGTAGCTGGGAGCCAGGGACAGCAC-3′ for S8A, 5′-TCCGCAGGGGCGCCCAAACGGGTCAT-3′ and 5′-ATGACCCGTTTGGGCGCCCCTGCGGA-3′ for T138A, 5′-CTGAAGCACCTGGCCCCCACGGACC-3′ and 5′-GGTCCGTGGGGGCCAGGTGCTTCAG-3′ for S170A, and 5′-GGCTTCATCGCGCCGGCCAACGTGGT-3′ and 5′-ACCACGTTGGCCGGCGCGATGAAGCC-3′ for T197A. Adenoviral expression vectors of p35 and p354A were described previously (23Saito T. Onuki R. Fujita Y. Kusakawa G. Ishiguro K. Bibb J.A. Kishimoto T. Hisanaga S. J. Neurosci. 2003; 23: 1189-1197Crossref PubMed Google Scholar). Expression and Metabolic Phosphorylation of p35 in HEK293 Cells—HEK293 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/ml of penicillin, and 0.1 mg/ml streptomycin. Transfection of Cdk5 and p35 was performed by the Polyfect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For metabolic phosphorylation of p35, transfected cells were cultured in the presence of 3.7 MBq of [32P]orthophosphate in phosphate-free Dulbecco's modified Eagle's medium for 2 h. In some cases, roscovitine (50 μm) was added to inhibit Cdk5 activity 2 h before metabolic labeling. After washing with phosphate-buffered saline, cells were suspended in RIPA buffer (20 mm Tris-HCl, pH 7.5, 1 mm EGTA, 1 mm EDTA, 0.15 m NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.1% sodium deoxycholate) containing 10 mm β-glycerophosphate, 5 mm NaF, 0.2 mm Pefabloc SC (Merck, Darmstadt, Germany), 1 μg/ml leupeptin, and 1 mm dithiothreitol, and lysed by freezing and thawing. The extract was obtained as a supernatant of centrifugation at 17,000 × g for 15 min. p35 was isolated from the extract by immunoprecipitation with C19 anti-p35 antibody. 32P incorporation into p35 was detected by a BAS 2000 image analyzer (Fuji Film, Tokyo, Japan) after SDS-PAGE. Neuronal Culture, Metabolic Phosphorylation of p35, and Adenoviral Expression of p35—Cerebral cortical neurons were prepared from rat brains at embryonic day 17 as described previously (28Tokuoka H. Saito T. Yorifuji H. Wei F. Kishimoto T. Hisanaga S. J. Cell Sci. 2000; 113: 1059-1068PubMed Google Scholar). Neuronal cultures were used 7 days after plating. In some experiments, 0.1 μm okadaic acid was added to the culture medium for the final 2 h of a 4-h labeling period. MG132 (50 μm) was also added 1 h before the addition of okadaic acid to suppress proteosome-dependent degradation of p35. After washing with phosphate-buffered saline, neurons were lysed with RIPA buffer. The supernatant of centrifugation at 17,000 × g was immunoprecipitated with C19 and phosphorylation of p35 was detected as described above. Adenoviral expression vectors encoding p35 or p354A were infected into rat primary cortical neurons cultured for 5 days in vitro. Two days after infection, neurons were treated with 5 μm Ca2+ ionophore A23187 for 1 h, and the cleavage of p35 was detected by immunoblotting with anti-p35 antibody (C19). Two-dimensional Phosphopeptide Map Analysis—Metabolically phosphorylated p35 was digested with 50 μg/ml l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma) in 10 mm (NH4)2CO3 overnight at 37 °C. The digested peptides were subjected to two-dimensional phosphopeptide map analysis using a thin layer cellulose (TLC) plate (Merck, Darmstadt, Germany) (29Sakaue F. Saito T. Sato Y. Asada A. Ishiguro K. Hasegawa M. Hisanaga S. J. Biol. Chem. 2005; 280: 31522-31529Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Preparation of Antibodies against Phosphorylated Ser8 (Anti-Ser(P)8) and Thr138 (Anti-Thr(P)138) of p35—Non-phosphorylated and phosphorylated peptides, “LSLSPSYRKRC” and “LSLpSPSYRKRC” for anti-Ser(P)8, and “ITSAGTPKRVIRC” and “ITSAGpTPKRVIRC” for anti-Thr(P)138, were chemically synthesized, and the phosphorylated peptides, LSLpSPSYRKRC and ITSAGpTPKRVIRC, were conjugated to keyhole limpet hemocyanin through the cysteine residue attached to the C terminus by AnyGen (Kwang-ju, Korea). Anti-Ser(P)8 or anti-Thr(P)138 antibody generated by immunizing rabbits was affinity purified by peptide columns, the non-phosphorylated peptide negatively and the phosphorylated peptide positively using SulfoLink (Pierce), according to the manufacturer's protocol. Expression of Cdk5-p35 in COS-7 Cells, Cleavage of p35 to p25 by Calpain, and Dephosphorylation of p35—COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 0.1 mg/ml of streptomycin. Transfection of Cdk5, p35, μ-calpain, and the small subunit of calpain was performed by the Polyfect transfection reagent according to the manufacturer's instruction. The cleavage of p35 to p25 by calpain in COS-7 cells was induced by treatment of COS-7 cells with 10 μm ionomycin at 37 °C for 30 min. To increase the phosphorylation status of p35, COS-7 cells were treated with 0.1 μm okadaic acid for 3 h before treatment with ionomycin. For inhibiting the Cdk5 activity, cells were treated with 50 μm roscovitine for 2 h before treatment with ionomycin. For inhibiting the calpain activity, cells were treated with 10 μm calpain inhibitor IV for 2 h before treatment with ionomycin. To increase the calpain activity, μ-calpain and the small subunit of calpain were coexpressed in COS-7 cells with Cdk5 and p35. Dephosphorylation of p35 expressed in HEK293 cells was performed in the cell extract (50 mm Tris-HCl, pH 8.5, 0.15 m NaCl, and 1% Nonidet P-40) by incubation with bacterial alkaline phosphatase (20 unit/ml, Wako, Osaka, Japan) at 37 °C for 1 h. Immunoprecipitation, SDS-PAGE, and Immunoblotting—Preparation of the rat brain extracts and immunoprecipitation of Cdk5-p35 were done as described previously (23Saito T. Onuki R. Fujita Y. Kusakawa G. Ishiguro K. Bibb J.A. Kishimoto T. Hisanaga S. J. Neurosci. 2003; 23: 1189-1197Crossref PubMed Google Scholar, 28Tokuoka H. Saito T. Yorifuji H. Wei F. Kishimoto T. Hisanaga S. J. Cell Sci. 2000; 113: 1059-1068PubMed Google Scholar). SDS-PAGE was performed using a 12.5% polyacrylamide gel. Proteins were transferred to polyvinylidene difluoride membrane (Millipore, Bedgord, MA). The membranes were blocked with 5% skim milk in TBST (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.05% Tween 20). The membranes were incubated with primary antibodies followed by horseradish peroxidase- or alkaline phosphatase-conjugated secondary antibody (DAKO). The reactions were detected using an ECL system for horseradish peroxidase or the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium phosphatase substrate system for alkaline phosphatase and quantified by densitometry using NIH Image software. All experiments in this study were performed more than three times with similar results and representative results are shown in the figures. p35 Is Phosphorylated at Ser8 and Thr138 by Cdk5—Substantial evidence has been presented to suggest that Cdk5 autophosphorylates its activator p35 in vitro and in cultured cells (24Tsai L.H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (802) Google Scholar, 30Lew J. Huang Q.Q. Qi Z. Winkfein R.J. Aebersold R. Hunt T. Wang J.H. Nature. 1994; 371: 423-4363Crossref PubMed Scopus (536) Google Scholar, 31Patrick G.N. Zhou P. Kwon Y.T. Howley P.M. Tsai L.H. J. Biol. Chem. 1998; 273: 24057-24064Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 32Saito T. Ishiguro K. Onuki R. Nagai Y. Kishimoto T. Hisanaga S. Biochem. Biophys. Res. Commun. 1998; 252: 775-778Crossref PubMed Scopus (38) Google Scholar). To further investigate this autophosphorylation, we expressed both Cdk5 and p35 in HEK293 cells and cultured them with [32P]orthophosphate for 2 h in the absence or presence of the Cdk5 inhibitor, roscovitine. 32P incorporation was readily detected in p35 immunoprecipitated from the cell extracts by autoradiography (Fig. 1A). The highest levels of radiolabeled p35 were immunoprecipitated from cells cotransfected with Cdk5 (Fig. 1A, lane 4), but not with kinase-dead Cdk5 (Fig. 1A, lane 6). Furthermore, a large portion of the radioactivity associated with p35 could be attenuated by treating the cells with roscovitine (Fig. 1A, lane 5). Interestingly radiolabeled p35 was detected in cells transfected with p35 alone (Fig. 1A, lane 2). Moreover, treatment of cells transfected with p35 alone with roscovitine reduced radiolabeled p35 to almost undetectable levels (Fig. 1A, lane 3), suggesting that the endogenous Cdk5 was responsible for phosphorylation of p35 in the absence of exogenous Cdk5. Cdk5 is a proline-directed protein kinase and exhibits the requirement of Pro following the substrate Ser/Thr sites, with a preference for the consensus motif Ser/Thr-Pro-X-His/Lys/Arg. There are only four Ser/Thr-Pro sites, Ser8, Thr138, Ser170, and Thr197, within the primary sequence of p35 (Fig. 1B). Only Thr138 is encompassed within a consensus motif sequence (138TPKR), although Ser8 is also followed by two positively charged amino acid residues (8SPSYRK). Based on these observations, site-directed mutagenesis was used to replace Ser8 or Thr138 with Ala residues and the resulting plasmid was used together with one encoding Cdk5 to cotransfect HEK293 cell cultures. Mutation of Ser8 or Thr138 resulted in a large loss in radiolabel associated with immunoprecipitated p35 (data not shown). To further characterize the phosphorylation of p35, radiolabeled protein immunoprecipitated from cotransfected cells was next analyzed by two-dimensional phosphopeptide mapping after trypsin digestion (Fig. 1C). Two radioactive spots representing two tryptic phosphopeptides were derived from wild-type p35. The S8A mutation resulted in disappearance of one of these phosphopeptides (spot 1), whereas the T138A mutation caused lose of the other phosphopeptide (spot 2). Taken together, these data indicate that p35 is phosphorylated by Cdk5 in transfected HEK293 cell cultures at two proline-directed sites, Ser8 and Thr138. Phosphorylation at Ser8 or Thr138 in p35 Suppresses the Calpain-dependent Cleavage of p35 to p25—Previously, we presented data suggesting that Cdk5-dependent autophosphorylation of p35 affected its susceptibility to cleavage by calpain (23Saito T. Onuki R. Fujita Y. Kusakawa G. Ishiguro K. Bibb J.A. Kishimoto T. Hisanaga S. J. Neurosci. 2003; 23: 1189-1197Crossref PubMed Google Scholar). To further investigate the potential role of p35 phosphorylation in controlling calpain cleavage, p35 and Cdk5 were next exogenously expressed in COS-7 cells. In preliminary analyses, cotransfected COS-7 cells were treated with various concentrations of the Ca2+ ionophores, A23187 or ionomycin, or an inhibitor of intracellular Ca2+ pumps, thapsigargin. For each of these treatments, a small but distinct amount of p25 was found to be produced by immunoblotting the cell lysates (data not shown). However, the largest amount of p35 cleavage was consistently derived using ionomycin. Therefore, 10 μm ionomycin was selected as the method of choice for calpain activation in this paradigm. A time course for p25 generation in COS-7 cells treated with 10 μm ionomycin is shown in Fig. 2A. After 30 min of ionomycin treatment, p25 can be clearly observed, with levels increasing thereafter until 24 h with mild attenuation of p25 levels at 3 h. Based on these results, the 30-min time point with 10 μm ionomycin was chosen for all subsequent experiments as the minimal treatment necessary to induce the cleavage of p35 to p25. To demonstrate that p25 production in response to ionomycin was indeed calpain-dependent, cells were either left untreated or treated with ionomycin in the absence or presence of 10 μm calpain inhibitor IV (Fig. 2B, lanes 3 and 4). Treatment of cells with the calpain inhibitor resulted in a reduction in p25 levels both in the absence and presence of ionomycin. Furthermore, p35 cleavage was increased substantially by coexpression of μ-calpain (Fig. 2B, lanes 5 and 6). This effect corresponded to increased active μ-calpain levels as assessed in immunoblots using an antibody to this cleaved form of the protease (27Zhang J. Miyamoto K. Hashioka S. Hao H.P. Murao K. Saido T. Nakanishi H. Dev. Brain Res. 2003; 142: 105-110Crossref PubMed Scopus (19) Google Scholar). Because the predominant form of endogenous calpain in COS-7 cells is m-calpain, active μ-calpain was detected only in cultures transfected with the corresponding plasmid encoding μ-calpain (Fig. 2B, second panel). Calpain activity was also assessed by immunoblot analysis of calpain-dependent cleavage of spectrin, a major component of the neuronal cytoskeleton (26Takano J. Tomioka M. Tsubuki S. Higuchi M. Iwata N. Itohara S. Maki M. Saido T.C. J. Biol. Chem. 2005; 280: 16175-16184Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Spectrin cleavage closely mirrored p25 production in response to ionomycin and μ-calpain expression and was similarly blocked by calpain inhibition (Fig. 2B, third panel). These results demonstrate that p25 generation is tightly coupled to calpain activity. Having established optimal conditions for p25 generation and demonstrated calpain dependence, the effect of phosphorylation on the cleavage of p35 was next assessed (Fig. 3A). Treatment of COS-7 cells expressing p35 and Cdk5 with ionomycin resulted in increased p25 levels, as observed earlier. Pretreatment with roscovitine resulted in increased basal levels of p25, as well as the levels achieved in response to ionomycin (Fig. 3A, compare lane 3 with lane 1 and lane 4 with lane 2). Furthermore, increased basal and ionomycin-induced levels were observed in cells transfected with p35 alone in comparison to those expressing both exogenous Cdk5 and p35. These results further confirm that calpain-dependent cleavage of p35 is inhibited via autophosphorylation by Cdk5. To more specifically investigate the regulation of calpain cleavage of p35 by Cdk5-dependent phosphorylation of Ser8 and Thr138, a site-directed mutagenesis approach was again taken (Fig. 3B). The ability of ionomycin to induce p25 generation was substantially enhanced by the 2A (Ser8 and Thr138 to Ala) double mutation. On the other hand, the 2E (Ser8 and Thr138 to Glu) phosphomimetic double mutation substantially attenuated this effect. These observations indicate that the ability of Cdk5-dependent autophosphorylation to protect p35 from calpain cleavage occurs through phosphorylation of Ser8 and/or Thr138. Phosphorylation-dependent cleavage was also examined in neurons. p35 or p354A was expressed in primary cultured neurons and the cleavage was compared by immunoblotting with C19 after Ca2+ ionophore A23187 treatment for 1 h. Upper bands of p35 and p25 exogenously expressed p35 species with FLAG tag at the C-terminal. p354A was cleaved more than the wild type of p35 (compare p25-F in lanes 4 and 6 of Fig. 3C). Exogenously expressed p35 appeared to be more resistant to calpain cleavage than endogenous p35 by unknown reasons. The individual contribution of each of these two sites to this regulatory mechanism was further evaluated by site-directed mutagenesis (Fig. 3D). Interestingly, mutation of either Ser8 or Thr138 to Ala enhanced p25 generation in response to ionomycin treatment of COS7 cells cotransfected with Cdk5 and each of the respective forms of p35, suggesting phosphorylation of both of these sites contributes to protection of p35 from calpain cleavage. Phosphorylation at Ser8 and Thr138 of p35 in Cultured Cortical Neurons and Rat Brains—To further assess the physiological role of Cdk5-depedent phosphorylation of these two sites in regulating the cleavage of p35 to p25, the phosphorylation state of Ser8 and Thr138 was evaluated in cultured cortical neurons and intact brain tissue. Phosphorylation of p35 was first examined in cultured cortical neurons metabolically labeled with [32P]orthophosphate by two-dimensional phosphopeptide map analyses (Fig. 4A). Interestingly, maps of radiolabeled p35 immunoprecipitated from cultured neurons produced a single major spot that corresponded to the phosphopeptide (spot 1 of Fig. 1C) associated with phospho-Ser8. Treatment of the neuronal cultures with the Ser/Thr protein phosphatase 1 and 2A inhibitor okadaic acid (0.1 μm) together with the proteosome inhibitor MG132 (50 μm, to prevent phospho-p35 from being degraded, Refs. 31Patrick G.N. Zhou P. Kwon Y.T. Howley P.M. Tsai L.H. J. Biol. Chem. 1998; 273: 24057-24064Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar and 32Saito T. Ishiguro K. Onuki R. Nagai Y. Kishimoto T. Hisanaga S. Biochem. Biophys. Res. Commun. 1998; 252: 775-778Crossref PubMed Scopus (38)" @default.
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