Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B.
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 Balpha (PKBalpha) 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.
Rena
G
G
Department of Biochemistry, Medical Research Council Protein Phosphorylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
Guo
S
S
Cichy
S C
SC
Unterman
T G
TG
Cohen
P
P
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000918:Antibody Specificity; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins / Q000378:metabolism; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004789:Enzyme Activation; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008969:Molecular Sequence Data; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D010768:Phosphoserine / Q000276:immunology; D010769:Phosphothreonine / Q000276:immunology; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000077191:Wortmannin; D051025:bcl-Associated Death Protein
D000730:Androstadienes; C494819:BAD protein, human; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D051025:bcl-Associated Death Protein; D010769:Phosphothreonine; D010768:Phosphoserine; D013912:Threonine; D012694:Serine; D007334:Insulin-Like Growth Factor I; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C494915:akt-1 protein, C elegans; D020123:Sirolimus; D000077191:Wortmannin