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.
Published by Elsevier B.V.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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14-3-3 Proteins / chemistry
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14-3-3 Proteins / metabolism
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Androstadienes / pharmacology
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Apoptosis
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Blotting, Western
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Cell Line, Tumor
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Cell Proliferation
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Cell Survival
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Chromones / pharmacology
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Enzyme Inhibitors / pharmacology
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Glioma / genetics
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Glioma / metabolism
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Glioma / pathology
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Humans
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Immunoprecipitation
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Isoenzymes / antagonists & inhibitors
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Isoenzymes / genetics
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Isoenzymes / metabolism*
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Models, Biological
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Morpholines / pharmacology
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Phosphatidylinositol 3-Kinases / metabolism*
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Phosphoinositide-3 Kinase Inhibitors
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Phosphorylation / drug effects
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Protein Binding
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Protein Kinase C / antagonists & inhibitors
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Protein Kinase C / genetics
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Protein Kinase C / metabolism*
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Protein Multimerization
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism
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Pyruvate Dehydrogenase Acetyl-Transferring Kinase
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RNA Interference
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Serine / metabolism
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Signal Transduction*
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Wortmannin
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bcl-Associated Death Protein / chemistry
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bcl-Associated Death Protein / metabolism*
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bcl-X Protein / metabolism
Substances
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14-3-3 Proteins
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Androstadienes
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BAD protein, human
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Chromones
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Enzyme Inhibitors
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Isoenzymes
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Morpholines
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PDK1 protein, human
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Phosphoinositide-3 Kinase Inhibitors
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Pyruvate Dehydrogenase Acetyl-Transferring Kinase
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bcl-Associated Death Protein
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bcl-X Protein
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2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
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Serine
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Protein Serine-Threonine Kinases
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Protein Kinase C
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protein kinase C lambda
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Wortmannin