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

Using immunoblot analysis, we confirmed the phosphoproteomics results for all seven of the GH-stimulated sites tested, which included nearly all of the phosphosites for which phosphospecific antibodies were available. The change in phosphorylation observed by immunoblotting was consistent with our MS data, even when the MS data revealed only a 50% increase in signal with GH, providing strong validation of our phosphoproteomics approach. We identified phosphorylation sites in proteins previously reported to be phosphorylated in response to GH (Erk1, Erk2, Stat5a, Stat5b, and Shc) but the vast majority of the identified phosphosites (125 of the 132 GH-up-regulated sites) were in proteins not previously known to be regulated by GH. Based on the high degree of reproducibility (Ͼ90%) for GH regulation when a phosphosite was detected in more than one screen, and the high degree of validation (100%) of sites by Western blotting (including sites identified in only one screen), SILACbased phosphoproteomics analysis is a very powerful method to detect novel GH-regulated proteins. In a single experiment, we detected 1 order of magnitude more candidate GH-regulated proteins than we have detected using other techniques (yeast two-hybrid, GST pull-down, immobilized phosphopeptide pull-down, Tap-tagged and Flag-tagged coimmunoprecipitation, and cloning of ligand targets assays involving JAK2 and/or GH receptor) and observed a much higher rate of verification of candi-date proteins (i.e. the proportion of false positives was substantially lower). This is probably at least in part due to: 1) our SILAC-based phosphoproteomics analysis method not requiring overexpression of any proteins or artificial treatment or manipulation of any protein, other than growing some of the cells in heavy-labeled amino acids, and 2) the fact that the GH-treated and untreated cell lysates were processed together in a combined sample.