In virtually all epithelial tumors, growth factor receptor activity is deregulated by activating mutations, genomic amplification, and autocrine loops (1
). Accumulating evidence from mouse models and human drug response suggests that signals emanating from the activated tyrosine kinase domain of growth factor receptors are required for tumor initiation and maintenance (2
). 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
). 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 (ribosomal S6 kinase) pathway involved in cell proliferation (6
), the mTOR (mammalian target of rapamycin)-p70 S6 kinase pathway involved in nutrient sensing and cell growth (8
), and the PI3K (phosphatidylinositol 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 (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 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 RNA (siRNA) screens, we also identified a subset of molecules that participate in a regulatory loop to stabilize RTKs.