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

Although GH signaling has been shown to phosphorylate and activate Akt (13, 45) , the prominent representation of Akt/mTOR pathway components in our phosphoproteomics analysis (Table 3 ) was unexpected. The consensus substrate recognition sequence for Akt was one of the top motifs in our analysis of consensus phosphorylation motifs, and the Akt-regulated mTOR signaling pathway was one of the top four pathways in KEGG pathway analysis of our data. Given the shared roles of GH and mTORC1 in regulating protein synthesis as well as organ and body growth, it makes sense that GH activates Akt-regulated mTORC1 signaling. Before this work, only one GH-initiated pathway leading from Akt to the mTORC1 complex had been identified: phosphorylation of TSC2 Ser939 (14) . Our phosphoproteomics analysis coupled with studies using wortmannin to inhibit the PI3K/Akt pathway revealed that GH stimulates the phosphorylation of numerous proteins in the Akt-mTORC1 pathway, including: 1) Akt-mediated TSC2 Ser939 phosphorylation, which suppresses TSC2 function and thus inhibits the activity of mTORC1 (49, 50) ; 2) Akt-mediated PRAS40 Thr247 phosphorylation, which is thought to contribute to relief of PRAS40-dependent inhibition of mTORC1 (59, 60) ; and 3) mTOR as well as Erk1/2-mediated raptor Ser863 phosphorylation, which has been shown to promote multisite raptor phosphorylation and augment mTORC1 activity (36, 57, 58) .