Graduation Year

2011

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Chemistry

Major Professor

Mildred Acevedo-Duncan, Ph.D.

Committee Member

Robert Potter, Ph.D.

Committee Member

Wayne Guida, Ph.D.

Committee Member

Niketa Patel, Ph.D.

Keywords

Signal Transduction, Pro-apoptotic molecule - Bad, Cyclin dependent kinase, Phosphatidylinositol (3)-kinase, Cell cycle progression

Abstract

The focus of this research was to investigate the role of protein kinase C-iota (PKC-é) in the regulation of Bad function, a pro-apoptotic member of the Bcl-2 family and Cdk7 function, a master cell cycle regulator in glioblastoma.

The results were obtained from the human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-é co-localized and directly associated with Bad as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-é directly phosphorylated Bad at phospho specific residues, S112, S136 and S155 which in turn induced inactivation of Bad and disruption of the Bad/Bcl-XL dimer. Knockdown of PKC-é by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-é may be a Bad kinase. Since, PKC-é 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-é/Bad pathway. Treatment with PI(3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-é activity and subsequent phosphorylation of Bad suggesting that PKC-é regulates the activity of Bad in a PI (3)-kinase dependent manner.

Robust expression of PKC-é is a hallmark of human glioma and benign and malignant meningiomas, however, little is understood about its role in glioma cell proliferation. The cyclin dependent kinase activating kinase complex (CAK), comprises of cyclin dependent kinase 7 (Cdk7), cyclin H and MAT1, is the master cell regulator. Cdk7 phosphorylates its downstream cyclin dependent kinases (cdks) and promotes cell proliferation. Results show that PKC-é directly associated and phosphorylated Cdk7 at T170. Furthermore, Cdk7 phosphorylated its downstream target, cyclin dependent kinase 2 (cdk2) at T160. Purified PKC-é was also observed to phosphorylate endogenous as well as exogenous Cdk7. PKC-é knockdown with siRNA, PDK1 siRNA and (PI) 3-kinase inhibitors, Wortmannin and LY294002 treatment exhibited corresponding reduction in phosphorylation of Cdk7 and subsequently cdk2. In addition, PKC-é knockdown reduced cell proliferation; led to cell cycle arrest and also induced apoptosis. Thus, these findings suggest the presence of a novel PI (3)-kinase/PKC-é/BAD mediated cell survival and PI (3)-kinase/PKC-é/Cdk7 mediated cell proliferation pathway.

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