Small molecules targeting aberrant RAF activity, like Vemurafenib (PLX4032), are highly effective against cancers harboring the V600E BRAF mutation, and are now approved for clinical use against metastatic melanoma. However, in tissues showing elevated RAS activity and in RAS-mutant tumors, these inhibitors stimulate RAF dimerization, resulting in inhibitor resistance and downstream paradoxical ERK activation. To understand the global signaling response of cancer cells to RAF inhibitors, we profiled the temporal changes of the phosphoproteome of two colon cancer cell lines (Colo205 and HCT116) that respond differently to Vemurafenib. Comprehensive data mining and filtering identified a total of 37910 phosphorylation sites, 660 of which were dynamically modulated upon treatment with Vemurafenib. We established that 83% of these dynamic phosphorylation sites were modulated in accordance with the phospho-ERK profile of the two cell lines. Accordingly, kinase-substrate prediction algorithms linked most of these dynamic sites to direct ERK1/2-mediated phosphorylation, supporting a low off-target rate for Vemurafenib. Functional classification of target proteins indicated the enrichment of known (nuclear pore, transcription factors, RAS-RTK signaling) and novel (Rho GTPases signaling, actin cytoskeleton) ERK-controlled functions. Our phosphoproteomic data combined with experimental validation established novel dynamic connections between ERK signaling and the transcriptional regulators TEAD3 (Hippo pathway), MKL1 and MKL2 (Rho-SRF pathway). We also confirm that an ERK docking site found in MKL1 is directly antagonized by overlapping actin binding, defining a novel mechanism of actin-modulated phosphorylation. Altogether, time-resolved phosphoproteomics further documented Vemurafenib selectivity and identified novel ERK downstream substrates.
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