Tyrosine Phosphorylation of the Human Glutathione S-Transferase P1 by Epidermal Growth Factor Receptor

Tatsunori Okamura,Simendra Singh,John Buolamwini,Timothy Haystead,Henry Friedman,Darell Bigner,Francis Ali-Osman

doi:10.1074/jbc.m808153200

Abstract

Epidermal growth factor receptor (EGFR) gene amplification, mutations, and/or aberrant activation are frequent abnormalities in malignant gliomas and other human cancers and have been associated with an aggressive clinical course and a poor therapeutic outcome. Elevated glutathione S-transferase P1 (GSTP1), a major drug-metabolizing and stress response signaling protein, is also associated with drug resistance and poor clinical outcome in gliomas and other cancers. Here, we provide evidence that GSTP1 is a downstream EGFR target and that EGFR binds to and phosphorylates tyrosine residues in the GSTP1 protein in vitro and in vivo. Mass spectrometry and mutagenesis analyses in a cell-free system and in gliomas cells identified Tyr-7 and Tyr-198 as major EGFR-specific phospho-acceptor residues in the GSTP1 protein. The phosphorylation increased GSTP1 enzymatic activity significantly, and computer-based modeling showed a corresponding increase in electronegativity of the GSTP1 active site. In human glioma and breast cancer cells, epidermal growth factor stimulation rapidly increased GSTP1 tyrosine phosphorylation and decreased cisplatin sensitivity. Lapatinib, a clinically active EGFR inhibitor, significantly reversed the epidermal growth factor-induced cisplatin resistance. These data define phosphorylation and activation of GSTP1 by EGFR as a novel, heretofore unrecognized component of the EGFR signaling network and a novel mechanism of tumor drug resistance, particularly in tumors with elevated GSTP1 and/or activated EGFR.

Highlights

Grants RO1 CA127872, RO1 CA 112519, P50CA108786, and P30-CA14236. □S The on-line version of this article contains supplemental Tables S1 and S2. 1 To whom correspondence should be addressed: 421 Medical Science phatidylinositol 3-kinase/AKT, Janus kinases/signal transducers and activators of transcription (STAT), and Ras/Raf/mitogen-activated protein kinase (MAPK) [1, 2]
In this study we provide the first evidence that the human Glutathione S-transferase P1 (GSTP1) protein is a direct downstream Epidermal growth factor receptor (EGFR) target and an important player in the EGFR signaling network
Phosphoamino acid analysis of the hydrolyzed EGFRphosphorylated GSTP1 confirmed that only tyrosine residues were phosphorylated by EGFR in the GSTP1 protein

Summary

EXPERIMENTAL PROCEDURES

Chemicals and Antibodies—Recombinant human GSTP1-1 protein was purchased from Invitrogen, and recombinant human EGFR active kinase domain and normal mouse IgG was from Upstate Biotechnology Inc., (Lake Placid, NY). [␥-32P]ATP and Protein A-Sepharose were from Amersham Biosciences. EGFR-dependent GSTP1 Phosphorylation in Glioma and Breast Cancer Cells—Tumor cells grown in serum-free medium for 24 h were treated with 100 ng/ml EGF for 10 min, rinsed with ice-cold phosphate-buffered saline, and lysed in 50 mM Tris-HCl (pH 7.5) containing 1% Triton X-100, protease and phosphatase inhibitors mixture (Pierce). For a combined immunoprecipitation (IP)-Western blotting, supernatants (1 mg total protein) were incubated (4 °C; overnight) with anti-GSTP1, phospho-EGFR antibodies, or normal mouse IgG (as a negative control). The histidine-tagged GSTP1 proteins were immunoprecipitated with TALON cobalt Dynabeads (Invitrogen) according to the manufacturer’s instructions followed by SDS-PAGE and Western blotting with anti-Tyr(P) antibody as described earlier. Extracts from cells with and without subsequent treatment with 2.5 ␮M EGFR inhibitor, lapatinib, for 30 min were assayed for specific GSTP1 activity as we described earlier [22]. Replicate cells after siRNA treatment were lysed and subjected to Western blotting for GSTP1 expression to monitor the level of GSTP1 knockdown

RESULTS

Phosphorylated residues

DISCUSSION