Phorbol 12-myristate 13-acetate induces epidermal growth factor receptor transactivation via protein kinase Cdelta/c-Src pathways in glioblastoma cells.
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.
Amos
Samson
S
Department of Pathology, University of Virginia Health System, Charlottesville 22908, USA. sa7h@virginia.edu
Martin
Patrick M
PM
Polar
Gregory A
GA
Parsons
Sarah J
SJ
Hussaini
Isa M
IM
D000098:Acetophenones / Q000494:pharmacology; D001253:Astrocytes / Q000378:metabolism; D001578:Benzopyrans / Q000494:pharmacology; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008954:Models, Biological; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D034741:RNA, Small Interfering / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013936:Thymidine / Q000737:chemistry; D013997:Time Factors; D015533:Transcriptional Activation; D014443:Tyrosine / Q000737:chemistry; D019061:src-Family Kinases / Q000378:metabolism*
D000098:Acetophenones; D001578:Benzopyrans; D002070:Butadienes; D004791:Enzyme Inhibitors; D009570:Nitriles; D020033:Protein Isoforms; D034741:RNA, Small Interfering; C113580:U 0126; D014443:Tyrosine; C085746:rottlerin; D066246:ErbB Receptors; D019061:src-Family Kinases; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate; D013936:Thymidine