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 arres t via upregulation of the cell cycle inhibitor p27(Kip1). Previously we hav e shown that protein kinase B (PKB) phosphorylates AFX causing inhibition o f 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/T4151A) still responds to PKB-regulated nuclear-cytopl asmic shuttling, but transcriptional activity and consequent G(1) cell cycl e arrest are greatly impaired. Furthermore, inhibition of the Rai signaling pathway abolishes both AFX-mediated transcription and regulation of p27(Ki p1), while activation of Rai augments AFX activity. From these results we c onclude that Rai-mediated phosphorylation of threonines 447 and 451 is requ ired for proper activity of AFX-WT. Interestingly, the T447A/T451A mutation did not affect the induction of transcription and G(1) cell cycle arrest b y the PKB-insensitive AFX-A3 mutant, suggesting that Ral-mediated phosphory lation plays a role in the regulation of AFX by PKB.