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. 2008 Jul 25;283(30):20978–20988. doi: 10.1074/jbc.M802588200

FIGURE 7.

FIGURE 7.

GSK-3β phosphorylation inhibits p190A GAP activity. A, in vitro assay of p190A GAP activity toward the Rho GTPase, demonstrating that MAPK priming-dependent phosphorylation by GSK-3β reduced GAP activity as reflected in an increase in the percentage of [32P]GTP-loaded GTPase retained on filters. Error bars reflect S.D. from four independent assays. B, in vitro phosphorylation reactions using [32P]ATP, demonstrating the kinetics of priming and subsequent phosphorylation of p190A by GSK-3β following SDS-PAGE and autoradiography (lower panel). The upper panel demonstrates equivalent levels of p190A as shown by Coomassie Blue staining. Similar conditions (leading to maximal phosphorylation; 60 min priming and 60 min GSK-3β phosphorylation) were used to prepare the material for use in the assays presented in A. C, two distinct types of morphologies are seen in COS-7 cells transiently transfected with pEGFP-p190A-WT or the various indicated mutants. The left panel exemplifies a cell demonstrating strong RhoGAP activity, as evidenced by the rounded morphology and thin cellular processes. The fluorescent signal is derived from the GFP portion of the p190A fusion protein. D, the indicated p190A constructs were transiently transfected into COS-7 cells. Immunoblots demonstrate the protein levels of p190A in transfected cells. GSK-3β was immunoblotted as a loading control. E, quantification of the percentage of transfected (GFP-positive) cells exhibiting the morphology characteristic of strong RhoGAP activity for each of the various p190A constructs. Error bars indicate S.D. (n = 200 for each construct). F, COS-7 cells were transiently transfected with either GFP or pEGFP-p190A-WT. At 40 h post-transfection, the cells were treated with DMSO, GSK-3β inhibitor XII, SB203580, U0126, or roscovitine for 4 h as indicated. The cells were then fixed and counted. The graph illustrates the average percentage of transfected cells exhibiting the strong RhoGAP phenotype (“GAP cells”) from three independent experiments, with the error bars representing S.D.