Malignant gliomas are the most common adult brain tumors, are refractory to classical chemotherapy and radiotherapy and have poor prognosis (45
). The EGFR is overexpressed in 50- 60% of GBM and amplified in 40% of the tumors (7
), which contribute to the malignant phenotype of human glioblastomas (4
). In the last ten years, the molecular mechanisms underlying astrocytic neoplastic transformation have been widely studied and a number of signaling pathways, including that of PKC, are altered in GBM (46
). The expression and activity of PKC isozymes are highly elevated in gliomas and glioma cell lines compared with normal astrocytes (12
) and PKC inhibitors markedly reduced glioma cell proliferation (38
Both EGFR expression and PKC activity play a significant role in astrocytic tumor biology. Our data provide evidence for the first time that treatment of glioblastoma cell lines with PMA resulted in EGFR phosphorylation at Tyr 1068 but not at other tyrosine residues and that this phosphorylation was mediated by a PKC δ/c-Src-dependent pathway. PMA-induced phosphorylation of Tyr 1068 was blocked by BIM, an inhibitor of classical as well as novel PKC isozymes but not by Go6976, an inhibitor of classical PKC isozymes. The Tyr 1068 is a major Grb2 binding and autophosphorylation site of the EGFR (49
). In contrast, EGF induced phosphorylation of the EGFR at multiple sites, including Tyr 992, 845, 1045 and 1068.
The mechanism of EGFR transactivation is not well characterized. Some studies have shown that EGFR is involved in signaling networks activated by a number of stimuli that do not interact directly with this receptor (50
). These stimuli include G protein-coupled receptor agonists thrombin and lysophosphatidic acid (51
), calcium (52
) and UV irradiation (53
). In our study prior incubation with the pharmacologic inhibitor of PKCδ, rottlerin, attenuated PMA-induced phosphorylation of EGFR (), which suggests a putative role for PKC δ in this process. To further confirm the role of PKC δ in mediating PMA-induced EGFR (Tyr 1068) phosphorylation, we used siRNA duplexes for PKC δ to knockdown the expression of PKC δ and to immunoblot for EGFR (Tyr 1068). Our results convincingly showed that gene silencing of PKC δ attenuated PMA-induced EGFR (Tyr 1068) phosphorylation (). Our immunoprecipitation data reveal an association between PKC δ and c-Src in astrocytic tumor cells, as reported in other cell types. PKC δ is a widely expressed member of the novel PKCs, and this isoform has been associated with cell proliferation in a number of cell types, including NIH 3T3 fibroblast, smooth muscle cells, and human keratinocytes (54
) and also shown to phosphorylate c-Src and to interact with c-Src (55
Since PKC isozymes do not directly phosphorylate proteins at tyrosine residues, we examined the role of c-Src as an intermediate kinase between PKC and EGFR. Cellular Src and EGFR interact in the progression of certain human malignancies and act synergistically to induce enhanced signaling in cells that express these kinases (43
). Elevated levels of Src family kinases, and mutational activation have been observed in colon carcinoma, and similar findings also have been reported in lung, breast and brain tumors (43
). Evidence derived from experimental models indicates that Src activation potentiates EGF-induced mitogenesis and transformation (43
). Our study shows that the c-Src inhibitor (PP2) inhibited PMA-induced EGFR phosphorylation () and mitogenic response (), while the inactive form, PP3, had no effect on the phosphorylation of the receptor. To further confirm the role of c-Src in mediating PMA-induced EGFR transactivation, we used siRNA directed against c-Src to knock down the expression of c-Src. Gene silencing using the c-Src siRNA abrogated PMA-induced EGFR (Y1068) phosphorylation (). These data imply that PMA interacts with PKC-δ, which in turn phosphorylates Src to activate EGFR. Src structurally has a poorly conserved unique domain that contains serine/threonine residues (44
). The unique domain of c-Src, which is the least conserved region among Src family members, mediates protein interaction and is phosphorylated by protein kinase A, Protein kinase C, and Cdc2-cyclin complex (59
). To test the hypothesis that PMA may be indirectly activating Src through PKCδ, we used mutant c-Src (ser12cys/ser48ala). Transient transfection of this mutant into astrocytic tumor cells abrogated the phosphorylation of EGFR (Y1068) induced by PMA ().
The EGFR kinase inhibitor AG 1478 did not affect PMA-induced EGFR phosphorylation at Tyr 1068 but completely abrogated EGF-induced phosphorylation of EGFR. This suggests that PMA-induced EGFR (Tyr 1068) phosphorylation through PKC δ and c-Src is upstream of EGFR kinase activity. The result agrees with similar studies that used H2O2 to transactivate EGFR (62
The activation of the ERK/MAPK pathway is a key step in the regulation of important cellular responses such as cell proliferation (63
). Extracellular regulated kinases (ERK) 1 and 2 are 44- and 42-kDa members of the MAP kinase family and are involved in the regulation of gene expression, protein synthesis, cell growth and proliferation, and in some cases cell differentiation and secretion (64
). ERK phosphorylation was initially observed after ligand activation of such receptor tyrosine kinases as the EGF receptor, but many Gq-, Gi-, and Gs- coupled receptors also initiate the ERK cascade through transactivation of the EGFR (65
). The ERK activation often involves sequential steps that include transactivation of the receptor tyrosine kinases (EGFR) in a c-Src kinase-dependent manner (51
). In our study, we found that BIM (PKC inhibitor), Rottlerin (PKC δ specific inhibitor), PP2 (Src inhibitor) significantly inhibited ERK/MAPK activation by PMA (). Similarly, AG 1478, which did not block EGFR Tyr 1068 phosphorylation and the MEK inhibitor (UO 126) inhibited PMA-evoked ERK phosphorylation (). These data further suggest that the PMA-induced Tyr 1068 phosphorylation could lead to increase in EGFR kinase activity (blocked by AG 1478) to activate the Ras/Raf/MEK/MAPK pathway in glioblastoma cells.
Activation of PKC leads to the phosphorylation of several proteins that are involved in the regulation of cell growth, differentiation and apoptosis (28
). Pretreament of GBM cells with BIM or rottlerin blocked PMA-induced as well as EGF-induced increase in [3H] thymidine uptake, suggesting that PKC isoforms play a critical role in glioblastoma proliferation (38
). Gene silencing of PKC δ and c-Src with siRNA and pharmacological inhibition with PP2 and rottlerin also attenuated PMA-induced cell proliferation in U-87 MG cells. It is well established that following binding to EGFR, EGF increases PKC activity in a variety of cells (67
). In addition, PKC δ has been shown to increase transformation and metastatic progression of a number of tumors (69
) and activation of PKC δ has been shown to be involved in proliferation of epithelial breast cells (70
). In conclusion, our data provide evidence for the first time to demonstrate that PMA phosphorylates EGFR at Tyr 1068 through a PKC δ/Src-dependent pathway to activate MAPK and to increase cell proliferation in glioblastoma cells (). Furthermore, our study reveals a novel pathway that may contribute to glioblastoma invasive growth.
Schematic representation of the signaling pathways involved in PMA transactivation of EGFR (Tyr 1068) in glioblastoma cell lines.