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Molecular and Cellular Biology, December 2001, p. 8225-8235, Vol. 21, No. 23
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.23.8225-8235.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Nancy D. De Ruiter, Boudewijn M. T. Burgering, and Johannes L. Bos*

Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands

Received 7 May 2001/Returned for modification 12 June 2001/Accepted 20 August 2001

AFX is a Forkhead transcription factor that induces a G1 cell cycle arrest via upregulation of the cell cycle inhibitor p27Kip1. 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 p27Kip1, 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.

* Corresponding author. Mailing address: Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. Phone: 31-30-2538977. Fax: 31-30-2539035. E-mail: j.l.bos{at}med.uu.nl.

Molecular and Cellular Biology, December 2001, p. 8225-8235, Vol. 21, No. 23
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.23.8225-8235.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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