Phorbol 12-myristate 13-acetate induces epidermal growth factor receptor transactivation via protein kinase C delta/c-Src pathways in glioblastoma cells

Authors:
Amos S, Martin PM, Polar GA, Parsons SJ and Hussaini IM
In:
Source: J Biol Chem
Publication Date: (2005)
Issue: 280(9): 7729-7738
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
U-87 MG
Species: human
Tissue Origin: brain
Platform:
Nucleofector® I/II/2b
Abstract
Both epidermal growth factor receptor (EGFR) and protein kinase C (PKC) play important roles in glioblastoma invasive growth, however, the interaction between EGFR and PKC is not well characterized in glioblastomas. Treatment with EGF stimulated global phosphorylation of tyrosine 845, 992, 1068 and 1045 residues of the EGFR 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 residues in the two glioblastoma cell lines. The phosphorylation of EGFR at Tyr 1068 was not detected in normal human astrocytes treated with the phorbol ester. The PMA-induced phosphorylation of EGFR at Tyr 1068 was blocked by BIM, a PKC inhibitor, and rottlerin, a specific PKC delta inhibitor. In contrast Gdelta 6976, an inhibitor of classical PKC isozymes had no effect on PMA-induced EGFR phosphorylation. Furthermore, gene silencing with PKC delta siRNA, siRNA against c-Src, mutant c-Src (Ser12Cys/Ser48Ala) and treatment with a c-Src inhibitor (PP2) abrogated PMA-induced EGFR phosphorylation at Y1068.PMA induced serine/threonine phosphorylation of Src which was blocked by both BIM and rottlerin. Inhibition of EGFR with AG 1478 did not alter PMA-induced EGFR (Tyr 1068) phosphorylation, but completely blocked EGF-induced phosphorylation of EGFR. The effect of PMA on mitogen activated protein kinase (MAPK) phosphorylation and glioblastoma cell proliferation were reduced by BIM, rottlerin, the MEK inhibitor UO 126, PKC delta siRNA and c-Src siRNA. Taken together, our data demonstrate that, PMA transactivates EGFR and increases cell proliferation by activating PKC delta/c-Src pathway in glioblastomas.