Regulation of nuclear translocation of Forkhead transcription factor AFX by protein kinase B

The regulation of intracellular localization of AFX, a human Forkhead trans cription factor, was studied. AFX was recovered as a phosphoprotein from tr ansfected COS-7 cells growing in the presence of FBS, and the phosphorylati on was eliminated by wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase. AFX was phosphorylated in vitro by protein kinase B (PKB), a downstream target of PI 3-kinase, hut a mutant protein in which three put ative phosphorylation sites of PKB had been replaced by Ala was not recogni zed by PKB. In Chinese hamster ovary cells (CHO-K1) cultured with serum, th e AFX protein fused with green fluorescence protein (AFX-GFP) is localized mainly in the cytoplasm, and wortmannin induced transient nuclear transloca tion of the fusion protein. The AFX-GFP mutant in which all three phosphory lation sites had been replaced by Ala was detected exclusively in the cell nucleus. AFX-GFP was in the nucleus when the cells were infected with an ad enovirus vector encoding a dominant-negative form of either PI 3-kinase or PKB, whereas the fusion protein stayed in the cytoplasm when the cells expr essed constitutively active PKB. In CHO-K1 cells expressing AFX-GFP, DNA fr agmentation was induced by the stable PI 3-kinase inhibitor LY294002, and t he expression of the active form of PKB suppressed this DNA fragmentation. The phosphorylation site mutant of AFX-GFP enhanced DNA fragmentation irres pective of the presence and absence of PI 3-kinase inhibitor. These results indicate that the nuclear translocation of AFX is negatively regulated thr ough its phosphorylation by PKB.