Abstract
Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs.
Highlights
glutathione Stransferase P1 (GSTP1) is a downstream epidermal growth factor receptor (EGFR) phosphorylation target
We report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with Jun N-terminal kinases (JNKs), on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival
These results demonstrate that GSTP1 suppresses activation of JNK and its downstream target, cJun, and as shown with the UW228 and MGR3 cell lines, EGFR
Summary
GSTP1 is a downstream EGFR phosphorylation target. Results: EGFR-dependent C-terminal Tyr-198 phosphorylation shifts GSTP1 to the monomeric state, facilitates JNK binding and inhibition, and suppresses apoptosis in brain tumor cells. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. Phosphotyrosine GSTP1 Suppresses JNK Signaling and Apoptosis leukemia [11], non-small cell lung cancer [12], and malignant glioma [13,14,15] In several of these tumors, the level of GSTP1 increases with their increasing malignant progression, and this could function in part to down-regulate the activity of the high tumor JNK to growth supportive levels and to drive tumor resistance to therapy (16 –19). Using human brain tumor cell lines and xenografts, we examined in vitro and in vivo the functional effects of the EGFR-dependent GSTP1 tyrosine phosphorylation on GSTP1-JNK physical interaction and on JNK downstream signaling and apoptotic response
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