Abstract
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.
Highlights
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 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 insulinlike 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
Summary
Plasmids and Reagents—The DAF-16a1 HindIII/NheI insert from pGEM-FLAG-DAF-16a1 was ligated into the HindIII/XbaI site of pcDNA3 (ϩ) (Invitrogen) to generate pcDNA3-Flag DAF-16a1. The DAF-16a1 BstYI insert from pGEM-FLAG-DAF-16a1 was ligated into the BamHI site of the GAL4 DNA binding domain plasmid to generate GAL4-DAF-16 derivatives. Protein Interaction Assays—Myc epitope-tagged 14-3-3 expressed in 293 cells was absorbed to anti-Myc epitope antibodies (clone 9E10) pre-coupled to protein-A beads and incubated with 2 g of AKT- phosphorylated wild-type and mutant GST-DAF-16 for 90 min at 4 °C. The associated proteins were separated on SDS-PAGE and phosphorylated DAF-16 was detected by autoradiography Both wild-type and mutant GST-DAF-16 variants were detected by anti-GST immunoblotting. The reaction was pre-incubated with 1 g of either specific DAF-16 antibody (for detection of GST fusion proteins) or M2 antibody (against the Flag tag for detection of DAF-16 expressed in mammalian cells) for 15 min at 4 °C prior to the addition of 32P-labeled IRE probe. For the DAF-16 localization and DNA binding experiments, 293 cells were transfected with 5 g of the pcDNA3-DAF-16 variants or pcDNA3 alone
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
