Phosphorylation of the Transcription Factor Forkhead Family Member FKHR by Protein Kinase B

• Published in: The Journal of biological chemistry Vol. 274; no. 24; pp. 17179 - 17183
• Main Authors: Rena, G, Guo, S, Cichy, S C, Unterman, T G, Cohen, P
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 11.06.1999
• Subjects: 3-Phosphoinositide-Dependent Protein Kinases; Amino Acid Sequence; Androstadienes - pharmacology; Antibody Specificity; bcl-Associated Death Protein; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Carrier Proteins - metabolism; Consensus Sequence; DNA-Binding Proteins - genetics; DNA-Binding Proteins - immunology; DNA-Binding Proteins - metabolism; Enzyme Activation; Forkhead Box Protein O1; Forkhead Transcription Factors; Humans; Insulin-Like Growth Factor I - pharmacology; Molecular Sequence Data; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Phosphoserine - immunology; Phosphothreonine - immunology; Protein Processing, Post-Translational; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Recombinant Proteins - metabolism; Serine - metabolism; Signal Transduction; Sirolimus - pharmacology; Threonine - metabolism; Transcription Factors - genetics; Transcription Factors - immunology; Transcription Factors - metabolism; Wortmannin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.24.17179

Protein kinase B lies “downstream” of phosphatidylinositide (PtdIns) 3-kinase and is thought to mediate many of the intracellular actions of insulin and other growth factors. Here we show that FKHR, a human homologue of the DAF16 transcription factor in Caenorhabditis elegans , is rapidly phosphorylated by human protein kinase Bα (PKBα) at Thr-24, Ser-256, and Ser-319 in vitro and at a much faster rate than BAD, which is thought to be a physiological substrate for PKB. The same three sites, which all lie in the canonical PKB consensus sequences (Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr)), became phosphorylated when FKHR was cotransfected with either PKB or PDK1 (an upstream activator of PKB). All three residues became phosphorylated when 293 cells were stimulated with insulin-like growth factor 1 (IGF-1). The IGF-1-induced phosphorylation was abolished by the PtdIns 3-kinase inhibitor wortmannin but not by PD 98059 (an inhibitor of the mitogen-activated protein kinase cascade) or by rapamycin. These results indicate that FKHR is a physiological substrate of PKB and that it may mediate some of the physiological effects of PKB on gene expression. DAF16 is known to be a component of a signaling pathway that has been partially dissected genetically and includes homologues of the insulin/IGF-1 receptor, PtdIns 3-kinase and PKB. The conservation of Thr-24, Ser-256, and Ser-319 and the sequences surrounding them in DAF16 therefore suggests that DAF16 is also a direct substrate for PKB in C. elegans .