Figure 1.
Role of Gi/o proteins, Gβγ dimers and PI3K in Akt phosphorylation in response to ghrelin.
A, Time-course of the effect of ghrelin on Akt HM (S473) and A-loop (T308) phosphorylation. B, Ghrelin-induced Akt phosphorylation in the absence or presence of PTX (100 ng/mL, 12 h), PI3K inhibitor wortmannin (1 µM, 30 min) and βγ sequester β-ARK-CT. In A and B, serum-starved HEK-GHSR1a cells were treated with ghrelin (100 nM) at 37°C for the indicated times. Cells were lysed and analyzed by SDS-PAGE using specific antibodies. Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation obtained for each residue (mean±S.E. of three independent experiments). Blots are representative of three independent experiments.
Figure 2.
c-Src is required for phosphorylation of Akt in response to ghrelin.
A, Effect of siRNA depletion of c-Src on ghrelin-induced Akt phosphorylation. HEK-GHSR1a cells transfected with c-Src siRNA were serum-starved for 12 h and then stimulated with ghrelin (100 nM) at 37°C. After stimulation, equal amounts of protein in each sample were used to assess the expression of c-Src (left panel) and Akt phosphorylation (right panel) by western blotting. Expression of c-Src was quantified by densitometry and expressed as percentages of the level of c-Src in control siRNA-transfected cells (mean±S.E.). Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation at HM (S473) and A-loop (T308) after ghrelin addition to control siRNA-transfected cells (mean±S.E.). B, Effect of ghrelin on Y phosphorylation of Akt and interaction between Akt and c-Src. Cells were incubated with ghrelin (100 nM, 5 min) at 37°C, lysed and immunoprecipitated (IP) with antibodies to Akt, and then analyzed by western blotting with pAkt HM(S473), pY, pSrc (Y416) antibodies. C, Effect of ghrelin on Y phosphorylation of Akt in the presence of c-Src siRNA. HEK-GHSR1a cells transfected with c-Src siRNA were serum-starved for 12 h and then stimulated with ghrelin (100 nM, 5 min) at 37°C. Equal amounts of protein in each sample were used to assess the expression of c-Src [upper panel; values shown (mean±S.E.) are percentages of the level of c-Src in control siRNA-transfected cells]. Cells were lysed and immunoprecipitated (IP) with antibodies to Akt, and then analyzed by western blotting with with pAkt HM(S473), pY, pSrc (Y416) antibodies. Immunoblots are representative of three independent experiments.
Figure 3.
PDK1 and mTORC2 regulate Akt phosphorylation activated by ghrelin.
A, Time-course of the effect of ghrelin (100 nM) on phosphorylation of PDK1. Cell extracts were analyzed by SDS-PAGE using specific antibody against pPDK1 (S241) and Akt. PDK1 phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation (mean±S.E of three independent experiments). B, PDK1 phosphorylation induced by ghrelin (100 nM, 5 min) in the absence or presence of PTX (100 ng/mL, 12 h) and the PI3K inhibitor wortmannin (1 µM, 30 min). PDK1 phosphorylation was quantified by densitometry and expressed as the percentage of the basal phosphorylation obtained in control cells (means±S.E.). C, Effects of siRNA depletion of Rictor on ghrelin-induced Akt phosphorylation. HEK-GHSR1a cells transfected with Rictor siRNA were stimulated with ghrelin (100 nM) at 37°C. After stimulation, cell extracts were prepared as described in Experimental Procedures. Equal amounts of protein in each sample were used to assess the expression of Rictor and Akt phosphorylation by Western blotting. Expression of Rictor was quantified by densitometry. Shown values are percentages of the level of Rictor in control siRNA-transfected cells. Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation of Akt HM (S473) and A-loop (T308) after ghrelin stimulation to control siRNA-transfected cells (mean±S.E.). In A, B and C, blots are representative of three independent experiments.
Figure 4.
β-arrestins are required for the modulation of Akt phosphorylation in response to ghrelin.
A, Effect of siRNA depletion of β-arrestin 1 or β-arrestin 2 on ghrelin-induced Akt phosphorylation. HEK-GHSR1a cells transfected with β-arrestin 1 or β-arrestin 2 siRNA were serum-starved for 12 h and then stimulated with ghrelin (100 nM) at 37°C. After stimulation, equal amounts of protein in each sample were used to assess the expression of β-arrestin 1 or β-arrestin 2 (upper panel) and Akt phosphorylation (lower panel) by Western blotting. Expression of β-arrestin 1 or β-arrestin 2 was quantified by densitometry. Values shown are percentages of the level of β-arrestins in control siRNA-transfected cells. Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation at HM (S473) and A-loop (T308) after ghrelin addition to control siRNA-transfected cells (mean±S.E.). B, Effect of ghrelin on the assembly of complexes containing β-arrestins 1 and 2 and pAkt. Cells were incubated with ghrelin (100 nM, 10 min) at 37°C, lysed and immunoprecipitated (IP) with antibodies to β-arrestin 1 (left panel) or β-arrestin 2 (right panel), and then analyzed by western blotting with pAkt[HM(S473), A-loop(T308), pPDK1 (S241)], Rictor, mTOR, and β-arrestin (A1CT) antibodies. In A and B, western blots are representative of three independent experiments.
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.
Figure 6.
Model of upstream activation of Akt by ghrelin.
Binding of ghrelin to GHS-R1a activates the generation of the second messenger PtdIns(3,4,5)P3 (PIP3) by PI3K activation through Gi/o-protein dependent signaling pathway. Akt translocates to the plasma membrane by binding to PIP3 where it is phosphorylated at Y by the membrane bound c-Src. This phosphorylation (Y) is followed by phosphorylation of Akt A-loop (T308) and HM (S473) by PDK1 and mTORC2 respectively. Once the receptor is activated, a second signaling pathway is mediated by β-arrestins 1 and 2, involving the recruitment of GHS-R1a, c-Src and Akt into an β-arrestin-scaffolded complex.