Phosphorylation of the transcription factor forkhead family member FKHR byprotein kinase B

Protein kinase B lies "downstream" of phosphatidylinositide (PtdIns) 3-kina se and is thought to mediate many of the intracellular actions of insulin a nd other growth factors. Here we show that FKHR, a human homologue of the D AF16 transcription factor in Caenorhabditis elegans, is rapidly phosphoryla ted by human protein kinase B alpha (PKB alpha) 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 upst ream activator of PKB), All three residues became phosphorylated when 293 c ells were stimulated with insulin-like growth factor 1 (IGF-1), The IGF-1-i nduced phosphorylation was abolished by the PtdIns 3-kinase inhibitor wortm annin but not by PD 98059 (an inhibitor of the mitogen-activated protein ki nase cascade) or by rapamycin, These results indicate that FKHR is a physio logical 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 si gnaling pathway that has been partially dissected genetically and includes homologues of the insulin/IGF-1 receptor, PtdIns 8-kinase and PKB, The cons ervation 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.