Research Resource: Identification of Novel Growth Hormone-Regulated Phosphorylation Sites by Quantitative Phosphoproteomics

Ray, Bridgette N.; Kweon, Hye Kyong; Argetsinger, Lawrence S.; Fingar, Diane C.; Andrews, Philip C.; Carter-Su, Christin

Additionally, among the sites in our phosphoproteomics analysis that we have verified by immunoblotting, several potential novel functions of GH have been revealed. Because phosphorylation of Ser455 in ACLY has been shown to increase ACLY-dependent synthesis of acetyl-CoA (67), our finding that GH stimulates phosphorylation of ACLY Ser455 suggests that GH may increase acetyl-CoA synthesis. Acetyl-CoA is carboxylated to malonyl-CoA in fatty acid synthesis. Thus, activation of ACLY would be consistent with the known rapid and transitory insulin-like stimulatory effects of GH on lipogenesis (78) . Similarly, because phosphorylation of NHE1 at Ser707 has been shown to increase NHE1 activity (35) , our finding that GH stimulates the phosphorylation of NHE1 Ser707 suggests that GH may activate the Na Ï© /H Ï© exchange function of NHE1. Consistent with a multipronged regulation of proteins and pathways, GH may also increase NHE1 activity in a calmodulindependent manner. GH binds its receptor and activates the tyrosine kinase, JAK2. JAK2 activation has previously been reported to phosphorylate calmodulin, resulting in increased interaction of calmodulin with NHE1 and increased NHE1 activity (79) . Although these proteins clearly merit and will receive more study, our SILAC and MS-based phosphoproteomic study reveals an additional 214 GH-dependent phosphosites (118 which undergo increased phosphorylation and 96 with decreased phosphorylation) in 133 proteins that have yet to be studied.