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

To gain insight into the reliability of the identification of the novel GH-dependent phosphosites, we performed immunoblotting with phosphospecific antibodies. Sites with available antibodies included three sites in proteins associated with the mTOR pathway (raptor Ser863, PRAS40 Ser184, and Thr247), one site identified in a protein in the insulin and focal adhesion categories (Shc Tyr423), and one site identified in a protein involved in regulation of actin cytoskeleton (NHEI Ser707). Of these, PRAS40 Thr247 and NHE1 Ser707 fell into the category of Akt/PKA substrate sites. We also tested for phosphorylation at two additional Akt/PKA substrate consensus sites, NDRG1 Ser330 and ACLY Ser455. All of these sites were previously identified as phosphosites with regulatory function in the context of other stimuli. Within the mTOR pathway, phosphorylation of raptor on Ser863 by mTOR within mTORC1 or by Erks 1/2 promotes phosphorylation of raptor on other sites and augments mTORC1 activity (36, 57, 58) . Phosphorylation of PRAS40 Thr247 by Akt facilitates efficient phosphorylation of Ser184 in PRAS40 by mTORC1 (59) , which relieves PRAS40-dependent inhibition of mTORC1 (60) . Shc recruitment to GH receptor/JAK2 complexes and its subsequent phosphorylation by JAK2 and recruitment of grb2 is thought to initiate GH activation of a Shc/grb2/ SOS/Ras/Raf/MEK/Erk pathway. Shc Tyr423 has been Proteins identified in the phosphoproteomic screens that contain phosphosites that increased with GH treatment at 5 or 15 min (Table 1 and Supplemental Table 1 ) were subjected to KEGG pathway analysis (http://www.genome.jp/kegg/pathway.html) to categorize the proteins by function and signaling pathway.