Pharmaceutical Sciences Faculty Publications

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

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


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 Tyr845, Tyr992, Tyr1068, and Tyr1045 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 Tyr1068 in the two glioblastoma cell lines. Phosphorylation of the EGFR at Tyr1068 was not detected in normal human astrocytes treated with the phorbol ester. PMA-induced phosphorylation of the EGFR at Tyr1068 was blocked by bisindolylmaleimide (BIM), a PKC inhibitor, and rottlerin, a PKCδ-specific inhibitor. In contrast, Gö 6976, an inhibitor of classical PKC isozymes, had no effect on PMA-induced EGFR phosphorylation. Furthermore, gene silencing with PKCδ 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 Tyr1068. 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 Tyr1068 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 PKCδ and c-Src siRNAs. Taken together, our data demonstrate that PMA transactivates the EGFR and increases cell proliferation by activating the PKCδ/c-Src pathway in glioblastomas.