Two novel phosphorylation sites on FKHR that are critical for its nuclear exclusion.
FKHR is phosphorylated by protein kinase B (PKB) at Thr24, Ser256 and Ser319 in response to growth factors, stimulating the nuclear exit and inactivation of this transcription factor. Here we identify two further residues, Ser322 and Ser325, that become phosphorylated in insulin-like growth factor-1 (IGF-1)-stimulated cells and which are mediated by the phosphatidylinositol 3-kinase-dependent PKB-catalysed phosphorylation of Ser319. Phosphorylation of Ser319 forms a consensus sequence for phosphorylation by CK1, allowing it to phosphorylate Ser322, which in turn primes the CK1-catalysed phosphorylation of Ser325. IGF-1 stimulates the phosphorylation of Thr24, Ser256, Ser319, Ser322 and Ser325 in embryonic stem (ES) cells, but not in PDK1-/- ES cells, providing genetic evidence that PDK1 (the upstream activator of PKB) is required for the phosphorylation of FKHR in mammalian cells. In contrast, the phosphorylation of Ser329 is unaffected by IGF-1 and the phosphorylation of this site is not decreased in PDK1-/- ES cells. The cluster of phosphorylation sites at Ser319, Ser322, Ser325 and Ser329 appears to accelerate nuclear export by controlling the interaction of FKHR with the Ran-containing protein complex that mediates this process.
Rena
Graham
G
MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK. grena1@biochem.dundee.ac.uk
Woods
Yvonne L
YL
Prescott
Alan R
AR
Peggie
Mark
M
Unterman
Terry G
TG
Williams
Michayla R
MR
Cohen
Philip
P
D000595:Amino Acid Sequence / Q000502:physiology; D000730:Androstadienes / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology; D017124:Conserved Sequence / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D038362:Glycogen Synthase Kinase 3; D006801:Humans; D010766:Phosphorylation; D020123:Sirolimus / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D000077191:Wortmannin; D020931:ran GTP-Binding Protein / Q000502:physiology
D000730:Androstadienes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C085126:FOXO1 protein, human; D005419:Flavonoids; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D014157:Transcription Factors; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D020931:ran GTP-Binding Protein; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D020123:Sirolimus; D000077191:Wortmannin