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: Bridgette N Ray, Hye Kyong Kweon, Lawrence S Argetsinger, Diane C Fingar, Philip C Andrews, Christin Carter-Su
• Journal: Molecular endocrinology (Baltimore, Md.) (Mol Endocrinol) Vol. 26 Issue 6 Pg. 1056-73 (Jun 2012) ISSN: 1944-9917 [Electronic] United States
• PMID: 22570334 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Peptide Fragments
• Phosphoproteins
• Proteome
• Receptors, Somatotropin
• Growth Hormone
• Phosphatidylinositol 3-Kinases
• Proto-Oncogene Proteins c-akt

Topics

• 3T3 Cells
• Amino Acid Motifs
• Animals
• Chromatography, Ion Exchange
• Consensus Sequence
• Growth Hormone (physiology)
• Mice
• Peptide Fragments (chemistry, isolation & purification)
• Phosphatidylinositol 3-Kinases (metabolism)
• Phosphoproteins (chemistry, isolation & purification, metabolism)
• Protein Processing, Post-Translational
• Proteome (chemistry, isolation & purification, metabolism)
• Proteomics
• Proto-Oncogene Proteins c-akt (metabolism)
• Receptors, Somatotropin (metabolism)
• Signal Transduction
• Tandem Mass Spectrometry