Phorbol 12-Myristate 13-Acetate Induces Epidermal Growth Factor Receptor Transactivation via Protein Kinase Cδ/c-Src Pathways in Glioblastoma Cells

Samson Amos,Gregory A. Polar,Patrick M. Martin,Isa M. Hussaini,Sarah J. Parsons

doi:10.1074/jbc.m409056200

Samson Amos, Gregory A. Polar + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m409056200

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Abstract

Both the epidermal growth factor receptor (EGFR) and protein kinase C (PKC) play important roles in glioblastoma invasive growth; however, the interaction between the EGFR and PKC is not well characterized in glioblastomas. Treatment with EGF stimulated global phosphorylation of the EGFR at Tyr(845), Tyr(992), Tyr(1068), and Tyr(1045) in glioblastoma cell lines (U-1242 MG and U-87 MG). Interestingly, phorbol 12-myristate 13-acetate (PMA) stimulated phosphorylation of the EGFR only at Tyr(1068) in the two glioblastoma cell lines. Phosphorylation of the EGFR at Tyr(1068) was not detected in normal human astrocytes treated with the phorbol ester. PMA-induced phosphorylation of the EGFR at Tyr(1068) was blocked by bisindolylmaleimide (BIM), a PKC inhibitor, and rottlerin, a PKCdelta-specific inhibitor. In contrast, Go 6976, an inhibitor of classical PKC isozymes, had no effect on PMA-induced EGFR phosphorylation. Furthermore, gene silencing with PKCdelta small interfering RNA (siRNA), siRNA against c-Src, and mutant c-Src(S12C/S48A) and treatment with a c-Src inhibitor (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine) abrogated PMA-induced EGFR phosphorylation at Tyr(1068). PMA induced serine/threonine phosphorylation of Src, which was blocked by both BIM and rottlerin. Inhibition of the EGFR with AG 1478 did not significantly alter PMA-induced EGFR Tyr(1068) phosphorylation, but completely blocked EGF-induced phosphorylation of the EGFR. The effects of PMA on MAPK phosphorylation and glioblastoma cell proliferation were reduced by BIM, rottlerin, the MEK inhibitor U0126, and PKCdelta and c-Src siRNAs. Taken together, our data demonstrate that PMA transactivates the EGFR and increases cell proliferation by activating the PKCdelta/c-Src pathway in glioblastomas.

Highlights

Glioblastoma multiforme (GBM)1 as a biologically aggressive neoplasm has an elevated, often aberrant, proliferative capacity with a diffuse pattern of brain invasion
We examined the relationship between protein kinase C (PKC) and epidermal growth factor receptor (EGFR) expression in glioblastoma cell lines to identify the PKC isoform and the intermediate downstream target involved in activation of the EGFR
The existence of cross-talk between PKC activation and the receptor tyrosine kinase EGFR, which is overexpressed in Ͼ50% of primary GBMs, provides a novel signaling pathway that is altered in astrocytic tumors and that may provide a useful therapeutic target

Summary

Introduction

Glioblastoma multiforme (GBM)1 as a biologically aggressive neoplasm has an elevated, often aberrant, proliferative capacity with a diffuse pattern of brain invasion. Treatment of normal human astrocytes with PMA (100 nM) for 30 min did not induce EGFR phosphorylation at Tyr1068, even though the cells express the receptor (Fig. 4). To determine whether classical or novel PKC isozymes are involved in PMA-induced Tyr1068 phosphorylation, we pretreated the cells with either BIM (1 ␮M) or Go 6976 (10 ␮M) for 60 min prior to PMA addition to the cultures.

Results

Conclusion