title: Research resource: identification of novel growth hormone-regulated phosphorylation sites by quantitative phosphoproteomics.
abstract: GH and GH receptors are expressed throughout life, and GH elicits a diverse range of responses, including growth and altered metabolism. It is therefore important to understand the full spectrum of GH signaling pathways and cellular responses. We applied mass spectrometry-based phosphoproteomics combined with stable isotope labeling with amino acids in cell culture to identify proteins rapidly phosphorylated in response to GH in 3T3-F442A preadipocytes. We identified 132 phosphosites in 95 proteins that exhibited rapid (5 or 15 min) GH-dependent statistically significant increases in phosphorylation by more than or equal to 50% and 96 phosphosites in 46 proteins that were down-regulated by GH by more than or equal to 30%. Several of the GH-stimulated phosphorylation sites were known (e.g. regulatory Thr/Tyr in Erks 1 and 2, Tyr in signal transducers and activators of transcription (Stat) 5a and 5b, Ser939 in tuberous sclerosis protein (TSC) 2 or tuberin). The remaining 126 GH-stimulated sites were not previously associated with GH. Kyoto Encyclopedia of Genes and Genomes pathway analysis of GH-stimulated sites indicated enrichment in proteins associated with the insulin and mammalian target of rapamycin (mTOR) pathways, regulation of the actin cytoskeleton, and focal adhesions. Akt/protein kinase A consensus sites (RXRXXS/T) were the most commonly phosphorylated consensus sites. Immunoblotting confirmed GH-stimulated phosphorylation of all seven novel GH-dependent sites tested [regulatory sites in proline-rich Akt substrate, 40 kDA (PRAS40), regulatory associated protein of mTOR, ATP-citrate lyase, Na(+)/H(+) exchanger-1, N-myc downstream regulated gene 1, and Shc]). The immunoblot results suggest that many, if not most, of the GH-stimulated phosphosites identified in this large-scale quantitative phosphoproteomics analysis, including sites in multiple proteins in the Akt/ mTOR complex 1 pathway, are phosphorylated in response to GH. Their identification significantly broadens our thinking of GH-regulated cell functions.
authors.affiliation: Departments of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Bridgette N
authors.initials: BN
authors.lastname: Ray
authors.forename: Hye Kyong
authors.initials: HK
authors.lastname: Kweon
authors.forename: Lawrence S
authors.initials: LS
authors.lastname: Argetsinger
authors.forename: Diane C
authors.initials: DC
authors.lastname: Fingar
authors.forename: Philip C
authors.initials: PC
authors.lastname: Andrews
authors.forename: Christin
authors.initials: C
authors.lastname: Carter-Su
mesh_terms: D016475:3T3 Cells; D020816:Amino Acid Motifs; D000818:Animals; D002852:Chromatography, Ion Exchange; D016384:Consensus Sequence; D013006:Growth Hormone / Q000502:physiology*; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011986:Receptors, Somatotropin / Q000378:metabolism; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry
chemical_list: D010446:Peptide Fragments; D010750:Phosphoproteins; D020543:Proteome; D011986:Receptors, Somatotropin; D013006:Growth Hormone; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt