Regulation of the Forkhead Transcription Factor AFX by Ral-Dependent Phosphorylation of Threonines 447 and 451

Abstract

AFX is a Forkhead transcription factor that induces a G₁ 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 G₁ 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 G₁ 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.

ACKNOWLEDGMENTS

We thank Geert Kops for continuous discussions and help during this project, Lydia de Vries and René Medema for help with certain experiments, Kris Reedquist for critically reading of the manuscript, and other members of our laboratory for support.

This work was supported by the Dutch Cancer Society.