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

We also tested whether the GH-dependent phosphorylation detected at PRAS40 Thr247, ACLY Ser455, and raptor Ser863 is downstream of Akt as predicted by the motif analysis (Supplemental Tables 3-5) . We treated 3T3-F442A preadipocytes for 30 min with wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor that blocks GH activation of Akt (68) and then with GH for 15 or 30 min. The efficacy of wortmannin at inhibiting Akt activity was determined by blotting with antibody to Akt pSer473 or with a phosphospecific antibody to the S6 kinase 1 substrate Ser235/Ser236 in ribosomal S6. We observed robust GH-dependent phosphorylation of PRAS40 Thr247, ACLY Ser455 (Fig. 7A) , and raptor Ser863 (Fig. 7B) . Phosphorylation at all of these sites was suppressed by wortmannin treatment, indicating that GH-induced phosphorylation of PRAS40 Thr247, ACLY Ser455, and raptor Ser863 lies downstream of PI3K/Akt. Phosphorylation of raptor Ser863 is catalyzed by several kinases (Fig. 3) , including Erks1/2 (36, 57, 58) . Because GH is known to stimulate Erk activation (9), we also investigated whether the MEK inhibitor CL-1040 inhibits GH-dependent phosphorylation of raptor Ser863. The efficacy of CL-1040 to inhibit MEK substrates Erk1 and Erk2 was assessed by blotting with antibody against the doubly phosphorylated activated form of Erk1/2. GHdependent stimulation of raptor phosphorylation at 6 . ACLY, NDRG1 and NHE1 are phosphorylated in response to GH. 3T3-F442A preadipocytes were treated with GH (100 ng/ml) (panel A), or with GH (500 ng/ml) (panels B and C) for the times indicated. A and B, Whole-cell lysates were immunoblotted with ␣pS455-ACLY, ␣ACLY, ␣pS330-NDRG, or ␣NDRG as indicated. C, NHE1 was immunoprecipitated from clarified cell lysates using ␣NHE1 and then immunoblotted with ␣pS707-NHE1 and ␣NHE1. ␣pS707-NHE1 band intensity was quantified and normalized to ␣NHE-1 band intensity. The average intensity of three experiments Ϯ SEM was graphed, *, P Ͻ 0.05. IP, Immunoprecipitation; ns, nonspecific.