The regulation of intracellular localization of AFX, a human Forkhead transcription factor, was studied. AFX was recovered as a phosphoprotein from transfected COS-7 cells growing in the presence of FBS, and the phosphorylation 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a downstream target of PI 3-kinase, but a mutant protein in which three putative phosphorylation sites of PKB had been replaced by Ala was not recognized by PKB. In Chinese hamster ovary cells (CHO-K1) cultured with serum, the AFX protein fused with green fluorescence protein (AFX-GFP) is localized mainly in the cytoplasm, and wortmannin induced transient nuclear translocation of the fusion protein. The AFX-GFP mutant in which all three phosphorylation sites had been replaced by Ala was detected exclusively in the cell nucleus.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 adenovirus vector encoding a dominantnegative form of either PI 3-kinase or PKB, whereas the fusion protein stayed in the cytoplasm when the cells expressed constitutively active PKB. In CHO-K1 cells expressing AFX-GFP, DNA fragmentation was induced by the stable PI 3-kinase inhibitor LY294002, and the expression of the active form of PKB suppressed this DNA fragmentation. The phosphorylation site mutant of AFX-GFP enhanced DNA fragmentation irrespective of the presence and absence of PI 3-kinase inhibitor. These results indicate that the nuclear translocation of AFX is negatively regulated through its phosphorylation by PKB. P hosphatidylinositol (PI) 3-kinase mediates the signal from various growth factors to regulate cell proliferation and survival (1, 2). A Ser/Thr protein kinase, termed protein kinase B (PKB) or Akt, is identified as a downstream target of PI 3-kinase. This protein kinase is activated by interaction of its pleckstrin homology domain with PI 3-kinase products and/or by phosphorylation of its catalytic domain by some upstream protein kinases (3, 4). The potential role of PKB in insulin action has been explored extensively (2-4). In Caenorhabditis elegans, DAF-2, AGE-1, and Ce-Akt, which are homologues of the mammalian insulin receptor, p110 catalytic subunit of PI 3-kinase, and PKB, respectively, have been isolated (5-7). In this organism, DAF-16, a transcription factor containing the Forkhead motif, is a major downstream target of the AGE-1/ Ce-AKT signaling cascade (7-9). This protein is shown to mediate insulin-like metabolic and longevity signals, and genetic analysis reveals that the AGE-1/Ce-AKT pathway suppresses the activity of DAF-16 for gene transcription. DAF-16 contains three repeats of the consensus sequence for phosphorylation by PKB (10), ArgXaa-Arg-Xaa-Xaa-Ser/Thr, where Xaa is any amino acid, and thus this protein is thought to be a direct target of Ce-Akt (7).Some members of the Forkhead family of human transcription factors, FKHR (11), its related gene products (FKHRL1 and FKHR1) (12, 13), and AFX (14, 15), are structur...