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

Abstract

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. 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. Glioblastoma multiforme (GBM) 1The abbreviations used are: GBM, glioblastoma multiforme; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; BIM, bisindolylmaleimide; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase. as a biologically aggressive neoplasm has an elevated, often aberrant, proliferative capacity with a diffuse pattern of brain invasion. It is the most malignant astrocytic tumor, composed of poorly differentiated neoplastic astrocytes (1Collins V.P. Cancer Surv. 1998; 32: 37-51PubMed Google Scholar, 2Rasheed B.K. Wiltshire R.N. Bigner S.H. Bigner D.D. Curr. Opin. Oncol. 1999; 11: 162-167Crossref PubMed Scopus (195) Google Scholar, 3Kleihues P. Burger P.C. Collins V.P. Newcomb E.W. Ohgaki H. Cavene W.K. Kleihues P. Cavene W.K. Glioblastoma: Tumors of the Nervous System. IARC Press, Lyon, France2000: 29-39Google Scholar). 50–60 and 40% of GBMs have overexpression and amplification of the epidermal growth factor receptor (EGFR), respectively (4Libermann T.A. Nusbaum H.R. Razon N. Kris R. Lax I. Soreq H. Whittle N. Waterfield M.D. Ullrich A. Schlessinger J. Nature. 1985; 313: 144-147Crossref PubMed Scopus (1244) Google Scholar, 5Ekstrand A.J. Sugawa N. James C.D. Collins V.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1169-1174Crossref PubMed Scopus (508) Google Scholar, 6Wong A.J. Bigner S.H. Bigner D.D. Kinzler K.W. Hamilton S.R. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6899-6903Crossref PubMed Scopus (714) Google Scholar, 7Ohgaki H. Schauble B. Zur H. von Ammon K. Kleihues P. Virchows Arch. 1995; 427: 113-118Crossref PubMed Scopus (139) Google Scholar). The over-expression and amplification of the EGFR contribute to the malignant phenotype of human glioblastomas (6Wong A.J. 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The data that PKCδ and c-Src are involved in of the EGFR and the glioblastoma to The of between PKC and the receptor tyrosine kinase EGFR, which is in of a signaling pathway that is in astrocytic and that may a EGF, and were The against the EGFR at Tyr992, Tyr845, and Tyr1045 and were Cell against the EGFR at Tyr1068 were and Cell The Gö and bisindolylmaleimide (BIM), the MEK inhibitor U0126, rottlerin, (4-amino-5-(4-chlorophenyl)-7-(t-butyl) and the EGFR kinase inhibitor AG 1478 are of was The against was was and were PKCδ and c-Src small interfering were c-Src(S12C/S48A) was in our J. Cell human MG cell was by A. J. and the U-87 MG cell was human astrocytes were The cell lines were astrocytic that were as and have by G. S.R. J. Biol. PubMed Scopus Google Scholar). Cell lines were to of with were in with and The cells were to an of in or or and in growth at in and to that were were with to PMA In the the were to the cells for the the was and the cells were by the the cells were with phosphate-buffered and and with and in the of and were at for at The protein of the was by the protein were for in were by on and and with and were detected the as by the with EGFR phosphorylation was stimulated in cells that had in for by with PMA or EGF for The was by the of to the was and cells were with and in the of and The was at for and as with or specific for the EGFR. and were used to the protein siRNA and c-Src were and by PKCδ and c-Src were both MG and U-87 MG cells the cells were and in at a of of and PKCδ siRNA or c-Src siRNA was were the cells were in of and at K. A. J. PubMed Scopus Google Scholar). of mutant c-Src(S12C/S48A) was astrocytic cells a the cells were as The cell of were by on were and with and Tyr1068 The were detected with or and was as by the were to to for in no cells were treated with EGF or PMA for and at were treated with the PKC inhibitor BIM and the PKCδ inhibitor for the of were with and with and in the of and Cell were to at for at of and treated cells by protein of cell was with at were with protein and with and The was to and by with and the of of were by of U-87 MG cells were a for cells were to a with BIM AG 1478 or by treatment with PMA or EGF for an In of cells with siRNA against PKCδ or c-Src were a at a of the cells were treated with PMA or EGF for were with of for and with This was by with at and at were in with an of and in was in a are as between two or were of by a of with a of were PMA and EGF EGFR Phosphorylation in Glioblastoma well characterized glioblastoma cell lines (U-1242 MG and U-87 were used for phosphorylation EGF and The MG cells have mutant M. B. Oncogene. 1997; PubMed Scopus Google Scholar), whereas the U-87 MG cells and mutant J. M. G. P. Collins V.P. G. Cancer Res. 1998; Google Scholar). Treatment with EGF induced EGFR phosphorylation at tyrosine residues Tyr1068, Tyr992, and The phosphorylation induced by EGF was The two cell lines levels of EGFR, which was not by treatment with EGF for EGF, treatment of the cells with PMA did not global phosphorylation of the tyrosine of the autophosphorylation sites of the EGFR in MG and U-87 MG cells was phosphorylated PMA did not the of EGFR the cell were with and with Tyr1068 both PMA and EGF induced EGFR phosphorylation at Tyr1068 The specificity of the against EGFR Tyr1068 was in cells a Tyr1068 mutant in which the tyrosine has with not In contrast, treatment of normal human astrocytes with PMA for did not EGFR phosphorylation at Tyr1068, the cells the receptor phosphorylation of the EGFR at Tyr1068. MG and U-87 MG cells were treated with PMA at and astrocytic cell were and The were with against Tyr1068 and stimulates phosphorylation of the EGFR at Tyr1068. MG and U-87 MG cells were stimulated with PMA for the These cells were with Cell were with and with against Tyr1068 or EGFR. of PMA and EGF on normal human human astrocytes cells to were The cells were treated with PMA for and EGF for were and The were with to EGFR Tyr1068. The was and for of on PMA-induced EGFR PMA with and both classical and and and PKC isozymes, to the PKC PMA-induced phosphorylation of the EGFR at Tyr1068. our and have that BIM of both and classical whereas Gö the of classical classical or PKC are involved in PMA-induced Tyr1068 phosphorylation, the cells with BIM or Gö for to PMA to the BIM PMA-induced EGFR phosphorylation in both U-87 MG and whereas Gö had no effect on PMA-induced phosphorylation of Tyr1068 BIM but not Gö PMA-induced EGFR phosphorylation, the PKC to the of the PMA PKCδ is used the PKCδ-specific inhibitor In the of rottlerin, PMA-induced phosphorylation of the EGFR was that PKCδ the to of both MG and U-87 MG cells with a did not EGFR at Tyr1068 with cells not may not for S. J. Biol. 2001; PubMed Google Scholar). an to PKCδ was PKCδ siRNA was astrocytic cells for by the of PMA for however, PKCδ gene silencing PKCδ by in MG and by in U-87 MG cells and PKCδ gene silencing PMA-induced EGFR Tyr1068 phosphorylation. for the of to demonstrate the specificity of PKCδ The of or as was not had no effect on signaling of in PMA of the The data that PMA-induced EGFR tyrosine phosphorylation is by PKCδ is a serine/threonine kinase and not a tyrosine kinase. is the kinase between PKCδ and the The tyrosine kinase c-Src has to the EGFR at in the cell and the cell Cancer Res. 1999; PubMed Google and at Tyr1068 in human cells W. PubMed Scopus Google Scholar). the effect of the c-Src inhibitor and the on EGFR phosphorylation in glioblastoma cell lines to PMA the of PMA phosphorylation of the EGFR at Tyr1068. of glioblastoma cell lines for with blocked PMA-induced phosphorylation of the EGFR at Tyr1068 by but the did not effect our data on the of c-Src in the of EGFR Tyr1068 phosphorylation by PMA, used siRNA against c-Src siRNA was astrocytic cells as The that the siRNA the of c-Src and phosphorylation of the EGFR at Tyr1068 by PMA and These data that PMA-induced tyrosine phosphorylation of the EGFR is or by kinase. PMA the Phosphorylation of c-Src at is a tyrosine kinase that as a of transmembrane Cancer Res. 1999; PubMed Google Scholar). c-Src has a poorly conserved domain that is in and and that activated by PKC PubMed Scopus Google Scholar). the of c-Src by PMA, cell were with and the were with The a of c-Src serine/threonine residues PMA which was by BIM and the of the domain of c-Src, of c-Src by and which are the phosphorylation for with and and of were by The c-Src mutant was our glioblastomas cell and effect on PMA-induced EGFR phosphorylation at Tyr1068. The c-Src(S12C/S48A) mutant abrogated phosphorylation of the EGFR at Tyr1068 by the that PKCδ may with c-Src in our cell were MG and U-87 MG cell were with and with PKCδ was detected in the c-Src treatment with PMA did not the of PKCδ with not These data demonstrate that PKCδ with c-Src in glioblastoma cell that PKCδ may in c-Src and phosphorylation of the EGFR at Tyr1068. The that PMA with PKCδ to or c-Src, which as a of EGFR Tyr1068 phosphorylation. Inhibition of EGFR PMA-induced Phosphorylation of the EGFR at PMA EGFR kinase activity phosphorylation of the EGFR at Tyr1068, the effect of AG a inhibitor of EGFR kinase MG and U-87 MG cells were with AG 1478 for and stimulated with PMA or EGF for and with the of EGFR kinase AG 1478 the EGFR phosphorylation at Tyr1068 EGF In contrast, the of EGFR kinase activity by AG 1478 had no effect on the phosphorylation of Tyr1068 in cells activated with PMA and These that PMA induced phosphorylation of the EGFR at Tyr1068 which to an in EGFR kinase activity and of the pathway as Inhibition of PMA-induced of the pathway is a in the regulation of important such as cell In cell the MAPK pathway has in both and PMA-induced growth had that PMA and EGF of in glioblastoma cell lines and that of MEK with PMA-induced cell proliferation G. S.R. J. Biol. PubMed Scopus Google Scholar). 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