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

Our motif analysis of the newly identified phosphosites that were up-regulated by GH determined that the dominant motifs phosphorylated were Akt/PKA, PKA, CK1, CamK2, GSK3, and CK2 consensus phosphorylation motifs. This analysis suggests that pathways that activate these kinases or kinases with similar substrate specificity may play an important role in GH signaling. Of these kinases, Akt is the only one known to be stimulated by GH (13) . Our present findings and previous work of others (14) provide evidence that a number of the identified Akt phosphorylation consensus sites are phosphorylated by Akt in response to GH (e.g. TSC2 Ser939, Tensin-3 Ser769 2.0/Ϫ/Ϫ PRAS40 Thr247, ACLY Ser455). However, because of overlap within kinase substrate consensus sequences and because the MaxQuant analysis of consensus phosphorylation motifs does not use a comprehensive list of kinases, some of the identified "consensus" phosphosites are likely to be phosphorylated primarily by a related kinase. For instance, GH signaling reportedly inhibits GSK3 (45) . Thus, our finding that GH signaling increases phosphorylation of GSK3 consensus sites suggests that GSK3 does not represent the physiological kinase for these sites. Instead, another, perhaps related, kinase that responds positively to GH, seems more likely to be the responsible kinase. Akt is a member of the AGC family of kinases. Family members Akt and SGK1 are both reported to phosphorylate Ser and Thr that lie in RXRXXS/T motifs (83, 84) . Rather than Akt/PKA, which was identified as the most likely kinase(s) by MaxQuant, the AGC kinase member SGK1 is thought to phosphorylate NDRG1 Thr328 and Ser330 (65) . The AGC family member p90 RSK (RXXS motif) is thought to be the kinase that phosphorylates NHE1 Ser707 (85) . At least one of the kinases identified in our motif analysis, CK2, is thought to be constitutively active (86) . CK2 was recently reported to both associate with JAK2 and, in an in vitro assay, phosphorylate JAK2 (86) . Additionally, CK2 inhibitors were found to depress GH-induced activation of STAT5, consistent with CK2 playing a role in GH signaling. With kinases like CK2 that are constitutively active, the regulation of ligand-dependent signaling likely requires a change in the conformation of protein substrates or altered subcellular localization of substrates or kinases to permit phosphorylation. Therefore, for potential substrates of CK2, it may be informative to determine whether any GH-dependent changes in subcellular localization of the substrates can be detected.