Analysis of Phosphotyrosine Signaling in Glioblastoma Identifies STAT5 as a Novel Downstream Target of ΔEGFR

An in-frame deletion mutation in Epidermal Growth Receptor (EGFR), Delta EGFR is a common and potent oncogene in glioblastoma (GBM), promoting growth and survival of cancer cells. This mutated receptor is ligand independent and constitutively active. Its activity is low in intensity and thought to be qualitatively different from acutely ligand stimulated wild-type receptor implying that the preferred downstream targets of Delta EGFR play a significant role in malignancy. To understand the Delta EGFR signal, we compared it to that of a kinase-inactivated mutant of Delta EGFR and wild-type EGFR with shotgun phosphoproteomics using an electron-transfer dissociation (ETD) enabled ion trap mass spectrometer. We identified and quantified 354 phosphopeptides corresponding to 249 proteins. Among the Delta EGFR-associated phosphorylations were the previously described Gab 1, c-Met and Mig-6, and also novel phosphorylations including that of STAT5 on Y694/9. We have confirmed the most prominent phosphorylation events in cultured cells and in murine xenograft models of glioblastoma. Pathway analysis of these proteins suggests a preference for an alternative signal transduction pathway by Delta EGFR compared to wild-type EGFR This understanding will potentially benefit the search for new therapeutic targets for Delta EGFR expressing tumors.