Receptor tyrosine kinases (RTKs) activate pathways mediated by serine/threonine (Ser/Thr) kinases

Receptor tyrosine kinases (RTKs) activate pathways mediated by serine/threonine (Ser/Thr) kinases like the PI3K (phosphatidylinositol 3-kinase)-Akt pathway the Ras-MAPK (mitogen-activated protein kinase)-RSK pathway and the mTOR (mammalian target of rapamycin)-p70 S6 pathway that control important aspects of cell growth proliferation and survival. (EGFR) or platelet-derived growth element receptor a (PDGFRα) RTKs. We recognized a subset of proteins with RxRxxS/T sites for which phosphorylation was decreased by RTKIs as well as by inhibitors of the PI3K mTOR and MAPK pathways and identified the effects of siRNA directed against these substrates on cell viability. We found that phosphorylation of the protein chaperone SGTA (small glutamine-rich tetratricopeptide repeat-containing protein alpha) at Ser305 is essential for PDGFRα stabilization and cell survival in PDGFRα-dependent tumor cells. Our approach provides a fresh look at of RTK and Akt-RSK-S6 kinase signaling exposing many previously unidentified Akt-RSK-S6 kinase substrates that merit further thought as focuses on for combination therapy with RTKIs. Intro In virtually all epithelial tumors growth element receptor activity is definitely deregulated by activating mutations genomic amplification and autocrine loops (1). Accumulating evidence from mouse models and human drug response suggests that signals emanating from your triggered tyrosine kinase website of growth factor receptors are required for tumor initiation and maintenance (2-4). This dependence of tumor cell survival upon the driving oncogene has been called “oncogene addiction” and demonstrates the acute sensitivity of cancer cells to inhibition of the pathways driving their proliferation growth and survival (4 5 However the complexity of the pathways and multiplicity of kinases activated downstream of RTKs has made it difficult to identify the key substrates that mediate oncogene dependence. Three core signaling pathways activated downstream of oncogenic RTKs are the Ras-Raf-MAPK (mitogen-activated protein kinase)-RSK Serpinb1a (ribosomal S6 kinase) pathway involved in cell proliferation (6 7 the mTOR (mammalian target of rapamycin)-p70 S6 kinase pathway involved in nutrient sensing and cell growth (8 9 and the Pitolisant hydrochloride PI3K (phosphatidylinositol Pitolisant hydrochloride 3-kinase)-Akt pathway involved in metabolic and cell survival signaling (10). Each of these pathways activates members of the AGC (cAMP-dependent cGMP-dependent and protein kinase C) family of serine/threonine Pitolisant hydrochloride (Ser/Thr) kinases including Akt RSK and p70 S6 kinase that phosphorylate substrates at the basophilic motif RxRxxS/T (R= arginine S = serine T = threonine and x = any amino acid) (11). Although inhibition of these three pathways often correlates with the beneficial effects of tyrosine kinase inhibitors (for instance the induction of cell death) the downstream targets of these inhibitors remain largely unidentified. To characterize the cell circuitry activated downstream of Akt RSK and p70 S6 kinase we first developed antibodies that recognized and selectively immunoprecipitated phosphorylated substrates of Akt RSK and p70 S6 kinase. Analysis of arginine-rich phosphopeptides by tandem mass spectrometry (MS/MS) is complicated by “neutral loss” in which arginine residues destabilize nearby phosphorylated amino acids resulting in the preferential loss of phosphate during the conventional peptide backbone fragmentation necessary for MS/MS based identification. Here we used two different approaches to overcome neutral loss electron transfer dissociation (ETD) (12) and two-step protease-based collision-induced dissociation (CID) analysis (13). Developing appropriate antibodies and overcoming neutral loss enabled us to use a large-scale phosphoproteomic approach to investigate Akt-RSK-S6 kinase signaling downstream of oncogenic EGFR c-Met and PDGFRα. Using selective RTK inhibitors (RTKIs) as well as inhibitors specific for the PI3K mTOR or MAPK Pitolisant hydrochloride signaling pathways (“pathway inhibitors”) we identified over 200 substrates and identified new circuitry not previously implicated in RTK signaling including connections to metabolic activity cell cycle control transforming growth factor (TGF)-Smad signaling and regulation of protein stability. Using short interfering Pitolisant hydrochloride RNA (siRNA) screens we also identified a subset of molecules that participate in a regulatory loop to stabilize RTKs. Results Monoclonal Antibodies Directed against Phosphorylated Akt RSK and p70 S6 Kinase Substrates We used an approach described previously (14) to.