Abstract 4002: C-terminal phosphorylation of human GSTP1 by EGFR and site-directed mutation allows JNK reactivation in glioma cells

Abstract

Abstract In malignant gliomas, the c-jun N-terminal kinase (JNK) pathway regulates tumor growth and resistance to apoptosis, and correlates with histologic grade and epidermal growth factor receptor (EGFR) tyrosine kinase expression. We recently reported that glutathione S-transferase P1 (GSTP1), a major metabolizing and stress response signaling protein that is frequently overexpressed in brain tumors, is a downstream target of EGFR and undergoes EGFR-dependent tyrosine phosphorylation, in vitro and in vivo, resulting in an enhancement of its enzymatic function and increased drug resistance. Although the dimeric GSTP1 exists in a reversible equilibrium with its monomeric form, the monomeric GSTP1 binds to JNK and acts as an endogeneous JNK inhibitor. In our previous study, the EGFR-phosphorylation of GSTP1 shifted the GSTP1 dimer-monomer equilibrium toward the monomeric state and the c-Jun phosphorylation by JNK was enhanced by phosphorylated-GSTP1 and suppressed by unphosphorylated-GSTP1. EGFR activation resulted in dissociation of GSTP1 from the GSTP1-JNK1 complex in a cell-free system and in glioma cells. The C-terminal domain of GSTP1 is reported to be involved in the interaction with JNK. Accordingly, we synthesized the C-terminal domain peptides of human GSTP1 containing the phospho-acceptor Tyr198, unique to hominidae among species, by EGFR (Y198WT) and its phenylalanine-replaced mutant peptide (Y198F). Both the C-terminal domain peptides significantly suppressed the c-Jun phosphorylation by JNK1 in a cell-free system. The binding assay using the synthetic peptides showed that the C-terminal domain peptides of human GSTP1 bound to JNK1 with a lower affinity when Tyr 198 was phosphorylated by EGFR than when it was unphosphorylated. Mutagenesis in human glioma cells revealed that the EGF-induced c-Jun phosphorylation by JNK decreased in the GSTP1-Y198F mutant, whereas increased in the phospho-mimic GSTP1-Y198D mutant, compared with vector control. Together these results indicate that in malignant glioma cells the C-terminal tyrosine phosphorylation of human GSTP1 by EGFR leads to dissociation of GSTP1 from the GSTP1-JNK1 complex. Thus, the GSTP1-JNK1 direct interaction rather than JNK upstream signaling elicits reactivation of JNK. This regulation of JNK signaling function by the EGFR-GSTP1 crosstalk, defines a novel signaling network with a potential to regulate the biology and therapeutic outcome in malignant gliomas. Supported by NIH grants RO1 CA127872, RO1 CA 112519, P50CA108786 and P30-CA14236. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4002.