Fig. 2.

RhoGAP22 binds 14-3-3 at both Ser¹⁶ and Ser³⁹⁵. (A) The phosphorylation of FLAG-RhoGAP22 under basal and insulin-stimulated conditions was analyzed by semiquantitative mass spectrometry. Results are shown as the mean increase in phosphopeptide abundance, expressed as the fold change over the basal level. Values are means from three experiments; error bars indicate standard deviations. Residues on RhoGAP22 were mutated either singly (B) or in combination (C) to determine the insulin-responsive 14-3-3 binding site. FLAG-tagged proteins were overexpressed in CHO IR/IRS-1 cells. Cells were serum starved and treated with no additions (B), with 100 nM insulin for 30 min (I), or with 100 nM wortmannin for 30 min and then 100 nM insulin for 30 min (W). Cell lysates were subjected to FLAG immunoprecipitation and immunoblotted with the indicated antibodies or used in a far-Western blot with GST–14-3-3 as a probe. (D) Conservation of 14-3-3 binding sites of RhoGAP22 across various species. Red text indicates a putative 14-3-3 binding site. Sequence numbering refers to human RhoGAP22 isoform 1.