Phosphatidylinositol 3-kinase signaling inhibits DAF-16 DNA binding and function via 14-3-3-dependent and 14-3-3-independent pathways.
In Caenorhabditis elegans, an insulin-like signaling pathway to phosphatidylinositol 3-kinase (PI 3-kinase) and AKT negatively regulates the activity of DAF-16, a Forkhead transcription factor. We show that in mammalian cells, C. elegans DAF-16 is a direct target of AKT and that AKT phosphorylation generates 14-3-3 binding sites and regulates the nuclear/cytoplasmic distribution of DAF-16 as previously shown for its mammalian homologs FKHR and FKHRL1. In vitro, interaction of AKT- phosphorylated DAF-16 with 14-3-3 prevents DAF-16 binding to its target site in the insulin-like growth factor binding protein-1 gene, the insulin response element. In HepG2 cells, insulin signaling to PI 3-kinase/AKT inhibits the ability of a GAL4 DNA binding domain/DAF-16 fusion protein to activate transcription via the insulin-like growth factor binding protein-1-insulin response element, but not the GAL4 DNA binding site, which suggests that insulin inhibits the interaction of DAF-16 with its cognate DNA site. Elimination of the DAF-16/1433 association by mutation of the AKT/14-3-3 sites in DAF-16, prevents 14-3-3 inhibition of DAF-16 DNA binding and insulin inhibition of DAF-16 function. Similarly, inhibition of the DAF-16/14-3-3 association by exposure of cells to the PI 3-kinase inhibitor LY294002, enhances DAF-16 DNA binding and transcription activity. Surprisingly constitutively nuclear DAF-16 mutants that lack AKT/14-3-3 binding sites also show enhanced DNA binding and transcription activity in response to LY294002, pointing to a 14-3-3-independent mode of regulation. Thus, our results demonstrate at least two mechanisms, one 14-3-3-dependent and the other 14-3-3-independent, whereby PI 3-kinase signaling regulates DAF-16 DNA binding and transcription function.
Cahill
C M
CM
Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114, USA.
Tzivion
G
G
Nasrin
N
N
Ogg
S
S
Dore
J
J
Ruvkun
G
G
Alexander-Bridges
M
M
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051858:Forkhead Transcription Factors; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008322:Mammals; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D029742:Caenorhabditis elegans Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D051858:Forkhead Transcription Factors; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D014446:Tyrosine 3-Monooxygenase; C108831:daf-16 protein, C elegans; C106207:par-5 protein, C elegans; C494918:AKT1 protein, human; C494915:akt-1 protein, C elegans