Inhibition of Nuclear Import by Protein Kinase B (Akt) Regulates the Subcellular Distribution and Activity of the Forkhead Transcription Factor AFX

Abstract

AFX belongs to a subfamily of Forkhead transcription factors that are phosphorylated by protein kinase B (PKB), also known as Akt. Phosphorylation inhibits the transcriptional activity of AFX and changes the steady-state localization of the protein from the nucleus to the cytoplasm. Our goal was threefold: to identify the cellular compartment in which PKB phosphorylates AFX, to determine whether the nuclear localization of AFX plays a role in regulating its transcriptional activity, and to elucidate the mechanism by which phosphorylation alters the localization of AFX. We show that phosphorylation of AFX by PKB occurs in the nucleus. In addition, nuclear export mediated by the export receptor, Crm1, is required for the inhibition of AFX transcriptional activity. Both phosphorylated and unphosphorylated AFX, however, bind Crm1 and can be exported from the nucleus. These results suggest that export is unregulated and that phosphorylation by PKB is not required for the nuclear export of AFX. We show that AFX enters the nucleus by an active, Ran-dependent mechanism. Amino acids 180 to 221 of AFX comprise a nonclassical nuclear localization signal (NLS). S193, contained within this atypical NLS, is a PKB-dependent phosphorylation site on AFX. Addition of a negative charge at S193 by mutating the residue to glutamate reduces nuclear accumulation. PKB-mediated phosphorylation of AFX, therefore, attenuates the import of the transcription factor, which shifts the localization of the protein from the nucleus to the cytoplasm and results in the inhibition of AFX transcriptional activity.

ACKNOWLEDGMENTS

We thank all members of our laboratories for their continued advice throughout the experimental and preparatory phases of the manuscript. In particular, we thank Mark Lindsay, Michael Nemergut, Kendra Plafker, Scott Plafker, Alicia Smith, Katie Welch, Nancy de Ruiter, and Hans Bos. We also thank Iain Mattaj and Dirk Görlich for their generosity in providing expression plasmids used in this study (Crm1, RanQ69L), Barbara Wolff for her gift of LMB, Anne Brunet for the anti-phospho T32 FKHRL1 antibody, Kris Reedquist for the c-cbl antibody, Marc Timmers for the RNA pol II antibody, and Bryce Paschal for the GSN2 cell line.

This work was supported by a grant awarded to I.G.M. from the National Institutes of Health, DHHS (GM-50526). A.M.B. is supported by a Postdoctoral National Service Award from the National Institutes of Health, DHHS (GM-20017). G.K. and B.B. are supported by grants from NWO and the Dutch Cancer Foundation (KWF).