PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway

Abstract

The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway.

Radiosensitization of human glioma cells by tamoxifen is associated with the inhibition of PKC-ι activity in vitro

“…In addition, TAM treatment did not induce an increase in the number of cells that underwent G2/M arrest subsequent to radiation exposure, but delayed the recovery of cell cycle progression. The activity of PKC-ι signaling, which plays an important role in the proliferation, apoptosis and cell cycle regulation of glioma cells (27,28,30), was markedly suppressed by TAM.…”

“…The present results revealed an inhibitory role of TAM in the expression levels of p-PKC-ι, which may participate in the radioresistance of glioma cells. In the human glioma T98G and U87MG cell lines, PKC-ι is able to directly phosphorylate Bad, and restrain its pro-apoptotic function (30). PKC-ι may play an equivalent role in A172 and U251 cells, since inhibition of PKC-ι by TAM was revealed to increase the expression of Bad.…”

“…A synergistic effect of TAM with radiation in C6 glioma cells has been demonstrated, and may partially be due to the inhibition of PKC activation (18). The atypical PKC family member, PKC-ι was of particular interest as it is a key regulator of cell survival, proliferation, invasion and chemoresistance in glioma (27,30,34,35). The present results revealed an inhibitory role of TAM in the expression levels of p-PKC-ι, which may participate in the radioresistance of glioma cells.…”

Growth factors and kinases in glioblastoma growth

“…There are reports indicating that PKC is important for progesterone-receptor-mediated proliferation in glioma cells, adding to the possible mechanisms of PKC function [114] . Other reports also indicated that PKC induces proliferation, invasion, and metastasis through PI3K, Bad or Cdk7/Cdk2 [116] , mTOR, and Akt activation [117] . Its interactions with other pathways include MAPK since PKC may activate the cascade through a direct interaction with EGFR and may also increase cell proliferation [118] .…”

“…It has been reported that the combined use of temozolomide and tamoxifen significantly reduces PKC phosphorylation; however, the biological effects are cell-dependent and include increased apoptosis or necrosis, and decreased proliferation or motility [115] . The importance of PKC in therapy resistance has been highlighted, particularly PKC's implication in cell cycle regulation [116] . PKCα activates MAPK and MMP, thus increasing infiltration ability [112] ; PKCδ has been linked to radiation-induced apoptosis as well as MMP activation [119,120] ; and PKCε induces proliferation and apoptosis evasion [121] .…”