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

• Published in: The Journal of biological chemistry 2005-03, Vol.280 (9), p.7729-7738
• Main Authors: Amos, Samson, Martin, Patrick M., Polar, Gregory A., Parsons, Sarah J., Hussaini, Isa M.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M409056200

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 super(845), Tyr super(992), Tyr super(1068), and Tyr super(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 super(1068) in the two glioblastoma cell lines. Phosphorylation of the EGFR at Tyr super(1068) was not detected in normal human astrocytes treated with the phorbol ester. PMA-induced phosphorylation of the EGFR at Tyr super(1068) was blocked by bisindolylmaleimide (BIM), a PKC inhibitor, and rottlerin, a PKC delta -specific inhibitor. In contrast, Goe 6976, an inhibitor of classical PKC isozymes, had no effect on PMA-induced EGFR phosphorylation. Furthermore, gene silencing with PKC delta 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 super(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 super(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 PKC delta and c-Src siRNAs. Taken together, our data demonstrate that PMA transactivates the EGFR and increases cell proliferation by activating the PKC delta /c-Src pathway in glioblastomas.