Insulin-like growth factor-1-induced phosphorylation of the forkhead family transcription factor FKHRL1 is mediated by Akt kinase in PC12 cells.
The Forkhead family transcription factor FKHRL1, a mammalian homolog of DAF16 in the nematode Caenorhabditis elegans, is an inducer of apoptosis in its unphosphorylated form and was recently reported as a substrate of Akt kinases. Insulin-like growth factor (IGF-1) is a potent stimulant of Akt kinase, leading to inhibition of the apoptotic pathway. In this study, we characterized the phosphorylation of FKHRL1 induced by IGF-1 in PC12 cells and various neuronal cell types and examined the potential role of Akt in this regard. IGF-1 rapidly induced the phosphorylation of Akt and FKHRL1 in PC12 cells. The phosphorylation of Akt and FKHRL1 induced by 10 nm IGF-1 was inhibited by the phosphatidylinositide 3-kinase (PI3K) inhibitors wortmannin (0.25-2 microm) and LY294002 (12.5-100 microm), but not by the MEK inhibitor PD98059 (50 microm) or the p70 S6 kinase pathway inhibitor rapamycin (50 nm), suggesting that the phosphorylation of FKHRL1 induced by IGF-1 is mediated by the PI3K pathway. As observed for IGF-1, an in vitro kinase assay with purified active Akt kinase demonstrated that the kinase is capable of directly phosphorylating FKHRL1 at Thr(32) and Ser(253), leading to inhibition of its pro-apoptotic properties. Moreover, transient expression of constitutively active Akt (MS-Akt, where MS is a myristylation signal) increased the phosphorylation of FKHRL1, whereas the expression of kinase-dead Akt (M179A Akt) attenuated the phosphorylation of FKHRL1 induced by 10 nm IGF-1 in PC12 cells. Interestingly, FKHRL1 co-immunoprecipitated with Akt in PC12 cells, indicating that these two proteins can associate in these cells. As IGF-1 also induced the phosphorylation of FKHRL1 in primary cortical and cerebellar neuronal cultures, these data, taken together, demonstrate that IGF-1, acting via the PI3K/Akt kinase pathway, can regulate the phosphorylation of FKHRL1, leading to inhibition of this apoptotic transcription factor in neuronal cells.
Zheng
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WH
Douglas Hospital Research Center, Departments of Psychiatry and of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H4H 1R3, Canada.
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Quirion
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D016475:3T3 Cells; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D002531:Cerebellum / Q000196:embryology; D002540:Cerebral Cortex / Q000196:embryology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D051379:Mice; D016296:Mutagenesis; D019814:Myristic Acid / Q000378:metabolism; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000378:metabolism; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000502:physiology; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D019893:Ribosomal Protein S6 Kinases / Q000037:antagonists & inhibitors; D012694:Serine / Q000737:chemistry; D020123:Sirolimus / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D007334:Insulin-Like Growth Factor I; D019814:Myristic Acid; D009419:Nerve Tissue Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; D012694:Serine; D020123:Sirolimus; D013912:Threonine; D014157:Transcription Factors; D014638:Vanadates; D000077191:Wortmannin; C085126:FOXO1 protein, human; C499517:FOXO3 protein, human; C499515:FOXO3 protein, rat; C474033:FoxO3 protein, mouse; C069276:peroxovanadate; C074639:Foxo1 protein, rat; C494918:AKT1 protein, human; C494920:Akt1 protein, rat; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one