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.