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

Recently, phosphoproteomic analysis has enabled large-scale, unbiased profiling of the sites of phosphorylation present in a population of cells and the ability to monitor dynamic changes in the phosphorylation at these sites after cellular stimulation (19 -24) . However, due to the low stoichiometry of phosphorylation and high complexity of samples isolated from whole cells, global analysis of protein phosphorylation in cells by liquid chromatography (LC)-mass spectrometry (MS) remains challenging. Enrichment of phosphopeptides before LC-MS using metal oxides (TiO 2 , ZrO 2 ) (25, 26) , immobilized metal affinity chromatography (27, 28) , and phosphopeptide-specific antibodies (29) have been used to selectively enrich phosphopeptides from complex peptide mixtures and dramatically improve the detection of phosphopeptides. We employed a MS-based quantitative phosphoproteomic approach that combined immunoaffinity purification, ZrO 2 , and LC-tandem MS (MSMS) with SILAC (stable isotope labeling with amino acids in cell culture) (30) to identify and quantify rapid (5 and 15 min) GH-dependent changes in protein phosphorylation in the highly GH-sensitive 3T3-F442A preadipocyte cell line. SILAC-based protein quantification has been widely used to study dynamic changes in protein expression levels (31) (32) (33) (34) because it enables samples to be combined and processed simultaneously, eliminating the differences within individual runs that are due to variation in sample handling during the extensive sample processing. This proteomic analysis enabled us to identify close to 1800 unique phosphorylation sites, of which 132 were found to be up-regulated by GH and 96 down-regulated by GH. The many new GH-regulated sites of phosphorylation identified by in this study suggest a broad array of proteins that are impacted by GH and are thus likely to contribute to the multiple and diverse physiological responses to GH. Western blotting confirmed GH stimulation of multiple phosphosites that lie downstream of Akt, underscoring the importance of the PI3K/Akt pathway in cellular responses to GH.