Fig. 3.

Akt phosphorylates RhoGAP22 at the 14-3-3 binding site and is required for insulin-stimulated 14-3-3 binding. (A) Insulin-stimulated phosphorylation of Ser¹⁶ and Ser³⁹⁵ is inhibited by wortmannin and Akti. CHO IR/IRS-1 cells expressing FLAG-RhoGAP22 were serum-starved and either left untreated (B), treated with 100 nM insulin for 30 min (I), or treated with 100 nM insulin for 30 min after one of the following: 100 nM wortmannin for 30 min (Wm), 5 μM Akti for 30 min (Akti), 20 nM rapamycin for 30 min (RAP), 10 μM Y27632 for 30 min (Y27632), 50 μM PD98059 for 30 min (PD98059), or 100 nm Gö6983 for 30 min (Gö6983). Treatment was followed by lysis and Western blotting with the indicated antibodies. (B) Insulin-stimulated 14-3-3 binding to endogenous RhoGAP22 is inhibited by wortmannin and Akti. L6 myotubes were serum starved and treated (I) or not treated (B) with 100 nM insulin for 30 min or with 100 nM insulin for 30 min after treatment with the indicated kinase inhibitors. (C) Recombinant Akt2 can phosphorylate Ser¹⁶ of RhoGAP22 in vitro. FLAG-GFP-tagged RhoGAP22 constructs were expressed in HEK cells by transfection with Lipofectamine 2000. Cells were treated with 100 nM wortmannin for 30 min, lysed, and subjected to FLAG immunoprecipitation. Eluted proteins were diluted in reaction buffer and incubated with 74 kBq [γ-³²P]ATP with or without 200 ng of recombinant Akt2 as indicated and incubated for various times. Reactions were run on a 10% SDS-PAGE gel, and products were dried on filter paper. Incorporation of ³²P was measured using a phospho-imager (Auto-Rad). The gel was stained with Coomassie blue to show total protein loading (Coomassie). Quantitation of these data are shown on the right as percentages of the phosphorylation of the 1-147 N-terminal fragment at the 60-min data point, normalized for protein loading. Significance was determined by one-way ANOVA followed by Tukey's posttest. *, P < 0.05; ***, P < 0.001; ns, not significant. (D) Wortmannin and Akti inhibit insulin-stimulated 14-3-3 binding to a RhoGAP22 construct that lacks the Ser¹⁶ site. CHO IR/IRS-1 cells were transfected with Myc-tagged p68RacGAP, serum starved, and treated (I) or not treated (B) with 100 nM for 30 min insulin or with 100 nM insulin for 30 min after treatment with the indicated kinase inhibitors. (E) Akt is required for insulin-stimulated 14-3-3 binding to RhoGAP22. Embryonic fibroblasts from either wild-type mice (WT MEF) or Akt 1/2 knockout mice (Akt DKO MEF) were serum starved and treated (Ins) or not treated (B) with 100 nm insulin for 30 min or with 100 nM wortmannin for 30 min and then 100 nM insulin (Wm). Cells were lysed, and lysates were used in a 14-3-3 pulldown before being immunoblotted with the indicated antibodies.