c-Src Regulates Akt Signaling in Response to Ghrelin via β-Arrestin Signaling-Independent and -Dependent Mechanisms

The aim of the present study was to identify the signaling mechanisms to ghrelin-stimulated activation of the serine/threonine kinase Akt. In human embryonic kidney 293 (HEK293) cells transfected with GHS-R1a, ghrelin leads to the activation of Akt through the interplay of distinct signaling mechanisms: an early Gi/o protein-dependent pathway and a late pathway mediated by β-arrestins. The starting point is the Gi/o-protein dependent PI3K activation that leads to the membrane recruitment of Akt, which is phosphorylated at Y by c-Src with the subsequent phosphorylation at 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 at least β-arrestins, c-Src and Akt. This β-arrestin-scaffolded complex leads to full activation of Akt through PDK1 and mTORC2, which are not associated to the complex. In agreement with these results, assays performed in 3T3-L1 preadipocyte cells indicate that β-arrestins and c-Src are implicated in the activation of Akt in response to ghrelin through the GHS-R1a. In summary this work reveals that c-Src is crucially involved in the ghrelin-mediated Akt activation. Furthermore, the results support the view that β-arrestins act as both scaffolding proteins and signal transducers on Akt activation

Role of G_i/o proteins, Gβγ dimers and PI3K in Akt phosphorylation in response to ghrelin.

<p>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.</p

c-Src is required for phosphorylation of Akt in response to ghrelin.

<p>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.</p

Obestatin controls skeletal muscle fiber-type determination

Abstract Obestatin/GPR39 signaling stimulates skeletal muscle growth and repair by inducing both G-protein-dependent and -independent mechanisms linking the activated GPR39 receptor with distinct sets of accessory and effector proteins. In this work, we describe a new level of activity where obestatin signaling plays a role in the formation, contractile properties and metabolic profile of skeletal muscle through determination of oxidative fiber type. Our data indicate that obestatin regulates Mef2 activity and PGC-1α expression. Both mechanisms result in a shift in muscle metabolism and function. The increase in Mef2 and PGC-1α signaling activates oxidative capacity, whereas Akt/mTOR signaling positively regulates myofiber growth. Taken together, these data indicate that the obestatin signaling acts on muscle fiber-type program in skeletal muscle

The SHP-1 protein tyrosine phosphatase negatively modulates Akt signaling in the ghrelin/GHSR1a system

The SHP-1 tyrosine phosphatase is a negative regulator of ghrelin activity, being a critical signaling component for proper regulation of Akt-dependent processes. Based on the SHP-1 expression pattern in white adipose tissue (WAT) and its regulation in a positive energy balance situation, it is possible to speculate about its role in the enlargement of WAT in obesity