Gαi3 is necessary for phosphorylation of GIV by Akt. (A) Phosphorylation of GIV is decreased upon depletion of Gαi3 (Gαi3 siRNA) and restored upon transfecting rGαi3 (Gαi3 siRNA and rGαi3). GIV was immunoprecipitated from HeLa cells treated as indicated and phosphorylation determined by immunoblotting with anti-pSer/Thr IgG. (B, top) Gαi3 binding to GIV promotes phosphorylation of GIV at S1416 by Akt. Immunoprecipitated GIV was preincubated in the presence or absence of ∼5 μg GST (lane 3), GST-Gαi3wt (lane 4), Gαi3 Q204L (lane 5), or Gαi3 G203A (lane 6) and subsequently phosphorylated in vitro with recombinant Akt1, followed by SDS-PAGE. Phosphorylation was determined by immunoblotting with anti-pSer IgG. (B, bottom) Equal loading of preimmune or anti-GIV IgG and GST proteins was confirmed by Ponceau S staining. (C) Gαi3 binding to GIV increases its susceptibility to trypsin-mediated proteolysis. Immunoprecipitated GIV was incubated in the presence or absence of GST-Gαi3 proteins as in B and digested with increasing concentrations of trypsin at 37°C for 8 min. The amount of uncleaved (trypsin resistant) GIV was determined by immunoblotting (inset) and displayed as percentage of starting amount (y axis) plotted against trypsin concentration (x axis). (D) GST-Gαi3 interacts similarly with in vitro–translated GIV wild-type and phosphorylation mimic (S1416D) or nonphosphorylatable (S1416A) GIV mutants. Semiquantitative densitometry revealed that 14 ± 2, 13 ± 1, and 14 ± 3% of wild-type, S1416A, and S1416D, respectively, bound to GST-Gαi3 (n = 5). (E) Schematic representation of how binding of Gαi3 to GIV may mediate phosphorylation of GIV by bringing the C-terminal Akt binding site closer to the Akt phosphorylation site.