Date of Award

Summer 8-2009

Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biochemistry and Molecular Biology

Department

Graduate School of Biomedical Sciences

First Advisor

Dr. Alakananda Basu

Abstract

Protein kinase C-η (PKCη) is involved in cell proliferation, differentiation and plays an anti-apoptotic role in various cancer models. The purpose of this dissertation is to understand the regulation of PKCη in cell death signaling in breast cancer cells.

Tumor promoting phorbol esters are potent activators of PKC and prolonged treatment with these activators leads to downregulation and desensitization of PKC signaling. PKCη resists phorbol ester-induced downregulation and upregulated in response to prolonged treatment. We have found that phosphorylation of PKCη at conserved serine/threonine sites is increased by phorbol ester treatment. Single mutations that prevent phosphorylation at these conserved sites resulted in downregulation of PKCη. These results suggest that phosphorylation of PKCη at conserved sites prevents its downregulation.

We have found that PKCη can be significantly downregulated by inhibition of two pathways: PKC and phosphoinositide 3-kinase (PI3K) using pharmacological inhibitors. Inhibitors of PKC and PI3K induce dephosphorylation of PKCη by a calyculin A-specific phosphatase. These inhibitors differ in their ability to degrade PKCη via the proteasome-mediated pathway. Downregulation of PKCη by PKC inhibitor is not mediated by the proteasome degradation pathway, whereas downregulation by PI3K inhibitor is dependent on the proteasome. These results demonstrate that dephosphorylation is important for PKCη downregulation and this occurs through two distinct mechanisms.

A comparison of PKC protein levels in a series of isogenic cell lines representing a breast cancer progression model revealed that PKCη protein levels increased from the normal, benign stage to the pre-malignant and metastatic stages. These results indicate that PKCη plays a potential role in breast cancer progression. Further analysis indicated that overexpression of PKCη in the MCF-7 breast adenocarcinoma cell line conferred resistance to tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL)-induced cell death. Therefore, PKCη protects breast cancer cells from TRAIL-induced apoptosis. Depletion of PKCη by small interfering RNA (siRNA) resulted in increased dimerization of pro-apoptotic Bax and decreased induction of anti-apoptotic Mcl-1 by TRAIL. These results indicate that PKCη may exert its anti-apoptotic effect by targeting Bcl-2 proteins, Bax and Mcl-1.

Share

COinS