Abstract 2162: EGFR-induced interaction of GSTP1 with the SH2-domain of Grb2 enhances Ras downstream activation in human glioma cells

Abstract

Abstract Although, Ras mutation are rare in human malignant gliomas, the Ras signaling pathway is frequently activated, particularly, in tumors with activated receptor tyrosine kinases, such as, epidermal growth factor receptor (EGFR). Glutathione S-transferase P1 (GSTP1), a major metabolizing and stress response signaling protein frequently overexpressed in human cancers, undergoes EGFR-dependent tyrosine phosphorylation, resulting in an enhancement of both its enzymatic and its cell signaling regulatory function. The EGFR-phosphorylated tyrosine 198 residue in human GSTP1 occurs within the consensus motif for high affinity binding to the SH2 domain of Grb2 (growth factor receptor-bound protein 2), an adaptor protein and critical downstream mediator of intracellular signal transduction from activated cell surface receptors via its SH2 domain. Here, we investigated, in glioma cells, the binding of GSTP1 to the Grb2 SH domain and its effect on Ras signaling and how this is impacted by EGFR. GSTP1 was co-immunoprecipitated with Grb2 in extracts of human glioblastoma (GBM) cells, following activation of EGFR with EGF, and EGFR-overexpressing GBM xenografts. Pull-down assays using biotinylated wild-type C-terminal GSTP1 peptides, containing the phospho-acceptor Y198 (Y198WT) showed it to form a complex with Grb2 and Sos1, when Y198 was phosphorylated by EGFR, and not when it was unphosphorylated. In contrast, its mutant peptide counterpart (Y198F) did not complex with Grb2 or Sos1. EGFR-induced Ras activation and ERK1/2 phosphorylation were significantly increased in wild-type GSTP1 but not GSTP1-Y198F transfectant cells. In human MGR3 GBM cells, both siRNA-mediated GSTP1 knockdown and GSTP1 inhibition with TLK199, a GSTP1 specific inhibitor, decreased ERK1/2 phosphorylation and activity markedly. The cell-permeable GSTP1-Y198 peptides also formed a complex with Grb2-Sos1 in UW228 human medulloblastoma cells with activated EGFR. Finally, following GSTP1 transfection, non-tumorigenic GSTP1-ve UW228 cells were transformed into highly tumorigenic cells that formed subcutaneous tumors in vivo. Our data show that, in human glioma cells, EGF-activated EGFR phosphorylates GSTP1 at Y198, facilitating formation (via Grb2-SH2 binding) of a GSTP1-Grb2-Sos1 complex and enhancement of Ras pathway and ERK1/2 activation. This EGFR-mediated GSTP1 phosphorylation-dependent enhancement of the Ras downstream signaling could contribute, in part, to the activated Ras signaling observed in the absence of structural Ras mutations and is a likely mechanism for the accelerated tumor growth characteristic of GBM and other tumors with elevated GSTP1 and aberrant EGFR signaling. Supported by NIH grants RO1 CA 153050, RO1CA127872, RO1 CA 112519, P50CA108786 and P30-CA14236. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2162. doi:1538-7445.AM2012-2162