Quantitative phosphoproteomic analysis of EphA2 signaling in PC-3 prostate cancer cells

The receptor tyrosine kinase EphA2 is frequently upregulated in cancer, where it plays an intriguing dichotomous role that may lend itself to therapeutic intervention. However, much remains to be understood about how ephrin-mediated activation of EphA2 signaling can reverse its tumour-promoting ligand-independent effects and instead inhibit migration and invasion. We used quantitative phosphoproteomics to analyze EphA2 signaling in PC-3 prostate cancer cells with the aim to further elucidate its downstream signaling and pinpoint phosphorylation-dependent cellular processes that may explain its dichotomous role. Stable isotope labeling by amino acids in cell culture (SILAC) combined with liquid chromatography-coupled mass spectrometry (LC-MS/MS) was used to quantify changes in relative phosphosite abundance in response to EphA2 activation. We identified 113 significantly regulated phosphosites comprising 73 phosphoproteins, including many novel and several previously described downstream effector proteins of EphA2 signaling. More than two thirds of the regulated phosphosites showed reduced phosphorylation in response to EphA2 activation. This is consistent with EphA2 activation inhibiting oncogenic signaling pathways, including Akt and ERK-MAPK signaling. Protein association analysis using the STRING database showed that a large proportion of these differentially regulated proteins are downstream targets of Akt and ERK-MAPK, respectively. Gene ontology enrichment analysis found our dataset to be strongly enriched in proteins associated with the cytoskeleton and its regulation. Overall, our data suggest that diminished phosphorylation of cytoskeletal regulators downstream of Akt and ERK-MAPK signaling may be a major mechanism by which stimulation of EphA2 can alter cell morphology and inhibit cell motility