| beta2-chimaerin is a diacylglycerol-regulated Rac GTPase activating protein implicated in development, neuritogenesis and cancer. The mechanisms of regulation and the physiological functions of this protein are currently only partially understood. This thesis work has attempted to elucidate how beta2-chimaerin is regulated and how it functions through two different means. Firstly, a novel mechanism of serine phosphorylation that regulates localization and activation was identified. Cellular stimulation by EGF or the phorbol ester PMA causes rapid phosphorylation of beta2-chimaerin through cross-talk with another DAG-regulated protein, PKCdelta. Phosphorylated beta2-chimaerin is primarily localized to the cytosol and is unable to translocate to membranes. Further, a non-phosphorylatable beta2-chimaerin mutant has enhanced Rac-GAP activity in cells and increased association with activated Rac in cells due to increased membrane association. Secondly, to investigate the function of beta2-chimaerin, a knock in mouse model was generated in which a hyperactivated I130A-beta2-chimaerin mutant was introduced. The initial generation and characterization of this mouse is described, and further phenotypical evaluation of this animal is ongoing. As decreased expression of beta2-chimaerin has been identified in human breast cancer, the I130A-beta2-chimaerin knock in mouse model and a beta2-chimaerin knock out mouse model are both being utilized to analyze whether beta2-chimaerin can modulate tumor formation in a mouse mammary model of tumorigenesis in which the ErbB2 growth factor receptor is overexpressed. Tumor incidence, age of onset, growth and rate of metastasis are being assessed to determine whether modulation of beta2-chimaerin activity or expression can affect mammary tumorigenesis. All in all, this thesis identifies a novel mechanism of regulation for beta2-chimaerin through serine phosphorylation, provides the first evidence that beta2-chimaerin is regulated by cross-talk with an alternative DAG receptor, PKCdelta, and begins to assess the physiological and pathological function of beta2-chimaerin through analysis of a knock in mouse model and through mammary tumorigenesis studies. This work not only contributes to our general understanding of the regulation and function of beta2-chimaerin, but will also begin to help us understand how modulation of beta2-chimaerin expression may contribute to cancer progression or other diseases. |
