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

The phosphoproteins that exhibited increased phosphorylation in response to GH were assigned to functional groups based on manual annotation performed by the authors using information about protein function tabulated in PhosphositePlus (http://www.phosphosite.org) (Supplemental Table 1 ). During the early period (5-15 1 Because 3T3-F442A cells are of mouse origin, we have used the amino acid numbering for the mouse sequence used in PhosphositePlus (http://www.phosphosite.org) throughout the paper, unless specifically noted. Ϫ/1.8/Ϫ Unknown min) after GH treatment, the largest protein functional groups undergoing GH-dependent increases in phosphorylation were proteins that served as adaptors or were involved in regulating transcription, the cytoskeleton, or mRNA processing (Fig. 4, A and B) . The broad range of functions for these 132 GH-responsive proteins, both metabolic and structural, suggests that further study of these proteins has the potential to provide a molecular basis for the many GH responses (51) that are only beginning to be understood. To gain insight into pathways activated by GH, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (http://www.genome.jp/kegg/pathway.html) to categorize the proteins that contain phosphosites that increased with GH treatment at 5 or 15 min (Table 2) . Four pathways contained five or more proteins that exhibited GH-dependent phosphorylation. The four pathways identified by KEGG have obvious functional overlaps and not surprisingly, several proteins are present in two or more pathways. The identification of previously unknown, GHregulated proteins in two of the categories ("regulation of the actin cytoskeleton," and "focal adhesions") highlights proteins that have the potential to broaden our understanding of the mechanistic basis underlying the ability of GH to promote changes in the actin cytoskeleton and cell motility (52) (53) (54) (55) (56) . In the category "regulation of the actin cytoskeleton," the phosphosites in protein phosphatase 1 regulatory subunit 12a (Ppp1r12a)/Mypt1/myosin-binding subunit of myosin phosphatase, NHE1, and myosin heavy chain 9 were not previously known to be GH regulated. In the category "focal adhesion," the phosphosites in filamin, zyxin, parvin, and Ppp1r12a were not previously known to be GH regulated. The identification of proteins in the categories "insulin signaling pathway" and "mTOR signaling pathway" that exhibit increased levels of phosphorylation in GH-treated cells are consistent with the known role of GH in the regulation of metabolism and cell growth (1, 2). Erk1, Erk2, and TSC2 were known previously to be phosphorylated in response to GH on the sites we identified. The phosphosites in raptor and eukaryotic initiation factor 4B (eIF4B) were not previously known to be GH-regulated sites. Manual examination of the functions reported for proteins in our proteomic analysis revealed two additional proteins in the mTOR signaling pathway, each with two GH-up-regulated sites: PRAS40, which, like raptor, is part of the mTORC1 complex, and programmed cell death protein 4 (Pdcd4), which, like eIF4B, is a target of S6 kinase (Fig. 3) . Thus, a total of 10 GH-dependent phosphosites in seven proteins lie within the mTOR signaling pathway. These Table 1 was used to construct the pie charts. C and D, The GH-dependent phosphopeptides identified during the SILAC-based phosphoproteomics screens after 5-min (C) or 15-min (D) GH treatment were an