Roles of the forkhead in rhabdomyosarcoma (FKHR) phosphorylation sites in regulating 14-3-3 binding, transactivation and nuclear targetting.
The transcription factor, forkhead in rhabdomyosarcoma (FKHR), is phosphorylated at three amino acid residues (Thr-24, Ser-256 and Ser-319) by protein kinase B (PKB)alpha. In the present study, mutagenesis has been used to study the roles of these phosphorylation events in regulating FKHR function in transfected HEK-293 cells. We find that the overexpression of FKHR[S256A] (where Ser-256-->Ala) blocks PKB activity in cells, preventing phosphorylation of the endogenous substrates FKHRL1 and glycogen synthase kinase-3. Thus some reported effects of overexpression of this and other mutants may be indirect, and result from suppression of the phosphorylation of other sites on FKHR and/or other PKB substrates. For example, we have shown that Thr-24 phosphorylation alone is critical for interaction with 14-3-3 proteins, and that the substitution of Ser-256 with an alanine residue indirectly blocks 14-3-3 protein binding by preventing the phosphorylation of Thr-24. We also found that insulin-like growth factor (IGF)-1 and serum-induced nuclear exclusion of FKHR[S256A] depends on the degree of overexpression of this mutant. Our results indicated that the interaction of FKHR with 14-3-3 proteins was not required for IGF-1-stimulated exclusion of FKHR from the nucleus. We present evidence in support of another mechanism, which depends on the phosphorylation of Ser-256 and may involve the masking of a nuclear localization signal. Finally, we have demonstrated that the failure of IGF-1 to suppress transactivation by FKHR[S256A] is not explained entirely by its failure to bind 14-3-3 proteins or to undergo nuclear exclusion. This result suggests that Ser-256 phosphorylation may also suppress transactivation by FKHR by yet another mechanism, perhaps by disrupting the interaction of FKHR with target DNA binding sites and/or the function of the transactivation domain.
MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee DD1 5EH, Scotland, UK.
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Rena
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Prescott
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Guo
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Cohen
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Unterman
D048948:14-3-3 Proteins; D021581:Active Transport, Cell Nucleus; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015533:Transcriptional Activation; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D004268:DNA-Binding Proteins; C085126:FOXO1 protein, human; C499517:FOXO3 protein, human; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D010769:Phosphothreonine; D049452:Green Fluorescent Proteins; D010768:Phosphoserine; D007334:Insulin-Like Growth Factor I; D014446:Tyrosine 3-Monooxygenase; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt