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

In summary, these studies are the first studies to use mass-spectrometry and SILAC-based phosphoproteomics to identify novel proteins involved in GH signaling. Using this approach, we have identified 132 GH-up-regulated phosphosites in 95 proteins and 96 GH-downregulated phosphosites in 46 proteins. Of the seven sites subjected to further testing by immunoblotting, all were confirmed to be phosphorylated in response to GH, which suggests that the phosphoproteomic screen identified GH-dependent phosphorylation sites with high accuracy. Motif analysis suggested that Akt, PKA, CamK2, GSK3, CK1, and CK2 (or kinases with similar substrate specificity) are important kinases in GH signaling. Manual annotation (Supplemental Table 1 ) and KEGG pathway analysis revealed that the identified proteins included multiple adaptor proteins, focal adhesion proteins, proteins involved in the cytoskeleton, and proteins involved in transcription. Overall, these results provide multiple new candidates for study, some of which will extend our understanding of known GH-signaling pathways and functions, and others that have the potential to break new ground in our quest to understand the full range of GH responses.