c-Src Regulates Akt Signaling in Response to Ghrelin via β-Arrestin Signaling-Independent and -Dependent Mechanisms
Figure 5
Src and β-arrestins regulate Akt phosphorylation in response to ghrelin in 3T3-L1 preadipocyte cells.
A, Effects of the Src inhibitor PP2 and its negative control, PP3, on the ghrelin-induced Akt activation in 3T3-L1 preadipocyte cells. Serum-starved cells were pretreated with PP2 (5 µM, 30 min) or PP3 (5 µM, 30 min) before ghrelin stimulation (100 nM) for the indicated time periods. Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation at HM (S473) and A-loop (T308) (mean±S.E of three independent experiments). B, Effect of ghrelin on the assembly of complexes containing β-arrestins 1 and 2 and pAkt. Preadipocyte 3T3-L1 cells were incubated with ghrelin (100 nM, 10 min) at 37°C, lysed and immunoprecipitated (IP) with antibodies to β-arrestins 1 and 2, and then analyzed by western blotting with pAkt HM (S473), pAkt A-loop (T308), pY, pSrc (Y416) antibodies. For A and B, western blots are representative of three independent experiments.