Regulation of nuclear translocation of forkhead transcription factor AFX by protein kinase B.
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a downstream target of PI 3-kinase, but a mutant protein in which three putative phosphorylation sites of PKB had been replaced by Ala was not recognized by PKB.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 The AFX-GFP mutant in which all three phosphorylation sites had been replaced by Ala was detected exclusively in the cell nucleus.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 dominant-negative 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.
Takaishi
H
H
Biosignal Research Center, Faculty of Science, Kobe University, Kobe 657-8501, Japan.
Konishi
H
H
Matsuzaki
H
H
Ono
Y
Y
Shirai
Y
Y
Saito
N
N
Kitamura
T
T
Ogawa
W
W
Kasuga
M
M
Kikkawa
U
U
Nishizuka
Y
Y
D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D018797:Cell Cycle Proteins; D006224:Cricetinae; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000378:metabolism; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors; D015723:Gene Library; D006801:Humans; D015151:Immunoblotting; D008957:Models, Genetic; D016296:Mutagenesis; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D013997:Time Factors; D014157:Transcription Factors; D014158:Transcription, Genetic
D001798:Blood Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D051858:Forkhead Transcription Factors; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D014157:Transcription Factors; C108803:FOXO4 protein, human