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.

Excess growth hormone suppresses DNA damage repair in epithelial cells

“…ATM can be regulated via different pathways. After DNA damage, the Mre11/Rad50/Nbs1 (MRN) DNA-binding complex is required for ATM activation (87), and spectrometry-based phosphoproteomics showed that GH rapidly dephosphorylates Mre11 and Rad50 in 3T3-F442A preadipocytes, potentially affecting activity of the MRN complex (88). TRIM29, induced by DNA damage, binds and degrades the histone acetyltransferase Tip60 (46), which also induces ATM kinase activity in response to DNA damage.…”

Quantitative analysis of global phosphorylation changes with high-resolution tandem mass spectrometry and stable isotopic labeling

“…Abnormal control of phosphorylation events due to an imbalance of kinase and phosphatase activities has been linked to a large number of disease states, including cancer (3–6), diabetes (7, 8), and Alzheimer’s disease (9–11). Phosphorylation is a central process in signal transduction to activate and propagate signals through phosphorylation cascades in response to specific stimuli such as hormones, growth factors, and drugs (1, 12, 13). In addition, phosphorylation has a broad range of effects on target proteins, including control of subcellular localization, protein structure, protein interactions, and enzymatic activity.…”

“…These direct effects of phosphorylation on protein function make quantitative phosphoproteome analysis useful for identifying and characterizing changes in protein function and for mapping control pathways. The use of quantitative phosphoproteome analysis in conjunction with time course studies (1, 12, 14) and genetic interventions (1, 15), are particularly useful in deciphering signal transduction pathways and provide direct insights into many biological processes (16–26). …”

“…The efficiency and selectivity of affinity chromatography for phosphopeptide enrichment are greatly influenced by various factors, such as buffer concentration, pH, the ratio of sample to affinity support, and the levels of non-phosphorylated peptides and other ions present in the final sample. Several studies have shown that combining multiple enrichment methods into one analytical pipeline provides more comprehensive coverage of phosphopeptides in complex samples (12, 36, 41, 42). The affinity based enrichment methods have been modified and applied for enrichment of intact phosphorylated proteins.…”

“…Anti-phosphotyrosyl (pY)-antibodies are frequently used to selectively enrich Tyr-phosphorylated proteins and peptides which enhances their detection (4, 12, 35–37, 43). Immunopurification of Tyr-phosphopeptides using anti-pY-antibodies has been successfully employed for quantitative studies of Tyr-directed phosphorylations, including receptor tyrosine kinase (RTK) signaling pathway analysis (35, 44–47).…”