Regulation of the Forkhead transcription factor AFX by Ral-dependent phosphorylation of threonines 447 and 451.
AFX is a Forkhead transcription factor that induces a G(1) cell cycle arrest via upregulation of the cell cycle inhibitor p27(Kip1). Previously we have shown that protein kinase B (PKB) phosphorylates AFX causing inhibition of AFX by nuclear exclusion. In addition, Ras, through the activation of the RalGEF-Ral pathway, induces phosphorylation of AFX. Here we show that the Ras-Ral pathway provokes phosphorylation of threonines 447 and 451 in the C terminus of AFX. A mutant protein in which both threonines are substituted for alanines (T447A/T451A) still responds to PKB-regulated nuclear-cytoplasmic shuttling, but transcriptional activity and consequent G(1) cell cycle arrest are greatly impaired. Furthermore, inhibition of the Ral signaling pathway abolishes both AFX-mediated transcription and regulation of p27(Kip1), while activation of Ral augments AFX activity. From these results we conclude that Ral-mediated phosphorylation of threonines 447 and 451 is required for proper activity of AFX-WT. Interestingly, the T447A/T451A mutation did not affect the induction of transcription and G(1) cell cycle arrest by the PKB-insensitive AFX-A3 mutant, suggesting that Ral-mediated phosphorylation plays a role in the regulation of AFX by PKB.
De Ruiter
N D
ND
Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands.
Burgering
B M
BM
Bos
J L
JL
D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D051858:Forkhead Transcription Factors; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D020725:ral GTP-Binding Proteins / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; C108803:FOXO4 protein, human; D051858:Forkhead Transcription Factors; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020725:ral GTP-Binding Proteins; D018631:ras Proteins