Dissertations, Theses, and Capstone Projects

Date of Degree

10-2014

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Susan A. Rotenberg

Subject Categories

Biochemistry | Oncology

Keywords

CEP4, GTPase, Mutants, Protein Kinase C, Rac, Substrate

Abstract

Aberrations in PKC signaling can lead to the development of multiple human diseases and the most prominent association of PKC with disease has been in tumor growth and metastasis. PKC and its related pathways have been recognized as promising targets for blocking the malignancy of breast cancer cells. To better understand PKC-mediated pathway in breast cancer cells, it is important to identify the cellular substrates of PKC. The main focus of this work is to identify physiologically relevant cellular substrates of PKC in human breast cells and to characterize their roles in cancer-related phenotypes. The work to be described consists of two projects: (1) identification of a new PKC substrate that contributes to cancer-related phenotypes in human breast cells; (2) characterization of the functional significance of its phosphorylation by PKC in normal and malignant human breast epithelial cells.

In the first project, the traceable kinase method was applied to identify potential PKC substrates in human breast cells. Potential substrates included those proteins that regulate or serve as effectors for the small GTPases. In light of previous studies showing that small GTPases play an important role in PKC-induced motility, the present study focused on proteins deemed to be strong candidate substrates. From ROCK1, CEP4, PAK2 and CLASP1 four candidates, it was demonstrated that CEP4 is in fact an intracellular PKC substrate, as shown by in vitro kinase assay and intracellular phosphorylation in human breast cells under DAG-lactone stimulation. CEP4 was found to serve as an intracellular substrate for PKC-&alpha, -&delta and -&zeta by testing the impact on intracellular CEP4 phosphorylation by PKC isoform-specific shRNA reagents or kinase-defective mutants.

In the second project, the functional significance of phospho-CEP4 was investigated in MCF-10A cells and MDA-MB-231 cells. Phospho-CEP4 stimulated cell motility, slowed the cell proliferation rate, and induced filopodia formation. In MCF-10A cells, the phosphorylation of CEP4 caused its disassociation from Cdc42. Rac1 was shown to participate in phospho-CEP4-induced cell motility. As shown by others, CEP4 does not interact with Rac1 directly. Phospho-CEP4 might interact with Rac1 directly or associate with another partner to engage the Rac1 pathway to induce cell migration. This study is the first to identify CEP4 as a PKC substrate in human breast cells. The demonstrated functional significance of phospho-CEP4 to malignant phenotypes of human breast cells could provide a predictive marker for human breast cancer.

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