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

Abstract

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a downstream target of PI 3-kinase, but a mutant protein in which three putative phosphorylation sites of PKB had been replaced by Ala was not recognized by PKB.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 The AFX-GFP mutant in which all three phosphorylation sites had been replaced by Ala was detected exclusively in the cell nucleus.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...

Phosphorylation and nuclear exclusion of the forkhead transcription factor FKHR after epidermal growth factor treatment in human breast cancer cells

“…This phenomenon has been observed for forkhead family members after serum treatment in other systems, however, these studies required transfection to show this e ect (Biggs et al, 1999;Brunet et al, 1999;Takaishi et al, 1999). The data shown here demonstrate endogenous FKHR translocates from the nucleus to the cytoplasm after EGF treatment.…”

“…Others have shown that transfected forkhead family members translocate from the nucleus to the cytoplasm after insulin/IGF-I or serum treatment (Biggs et al, 1999;Brunet et al, 1999;del Peso et al, 1999;Takaishi et al, 1999). To determine if EGF could promote translocation of endogenous FKHR, we stimulated cells for 0, 10 or 30 min, then puri®ed nuclear and cytoplasmic proteins.…”

B Cell Receptor Signaling Down-Regulates Forkhead Box Transcription Factor Class O 1 mRNA Expression via Phosphatidylinositol 3-Kinase and Bruton’s Tyrosine Kinase

“…It has been established that both DAF-16 and the FOXO proteins are posttranslationally controlled via Akt (also known as protein kinase B) in the PI3K signaling pathway. Phosphorylation of FOXO proteins by Akt in response to cytokines and growth factors such as insulin and insulin-like growth factor-1 results in their exclusion from the nucleus and their subsequent degradation (13)(14)(15)(16)(17)(18). Although this means of posttranslational control for the FOXO family has been well defined, other levels of regulation, such as mRNA expression, remain largely unexplored.…”

“…A major downstream target of PI3K signaling in BCR-stimulated B cells is Akt (20). Yusuf et al (21) have shown recently that BCR cross-linking leads to PI3K-dependent phosphorylation of FOXO1, a substrate of Akt (13)(14)(15)(16)(17)(18), and subsequent nuclear exclusion of the FOXO1 protein. Overexpression of either constitutively active FOXO1 or FOXO3a protein in activated primary B cells causes cell cycle arrest and apoptosis (21).…”

Deacetylation of FOXO3 by SIRT1 or SIRT2 leads to Skp2-mediated FOXO3 ubiquitination and degradation

“…FOXOs are extensively regulated by post-translational modifications, including phosphorylation, acetylation and ubiquitination (Obsil and Obsilova, 2008). For example, insulin induces the phosphorylation of FOXO1, FOXO3 and FOXO4 through Akt/protein kinase B, leading to the exclusion of FOXO from the nucleus and the inhibition of FOXO transcription activities (Biggs et al, 1999;Brunet et al, 1999;Kops et al, 1999;Rena et al, 1999;Takaishi et al, 1999). On the other hand, in response to stress, MST1 (Lehtinen et al, 2006) and JNK1 (Essers et al, 2004) phosphorylate FOXO factors at different sites, resulting in nuclear translocation and transactivation.…”