PROTEOLYSIS AND MAMMALIAN GL/S CELL CYCLE TRANSITION

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The cell cycle control is a fundamental regulatory process that ensures the faithful duplication and passage of genetic information during cell division. To prevent the irreversible incorporation of genetic lesions into the genome, the transitions between the G1/S and G2/M phases of cell cycle are highly regulated as part of the checkpoint control in respond to environmental cues. Aberrant cell cycle control abolishes coordination between different phases of the cell cycle and is a hallmark of neoplastic transformation. The broad, long-term objective of this proposal is to understand the mechanism of cell cycle control in cell growth and differentiation, and the consequences of its alteration during tumorigenesis. This proposal focuses on the mechanism of the G1/S cell cycle transition in mammalian cells. In particular, we describe the approaches to investigate the mechanism by which the levels of the mammalian G1 cyclins and CDK inhibitors are controlled through the selective ubiquitin-dependent degradation during the G1/S transition. The accumulation of the G1 cyclins are the rate limiting step during the G1/S transition and the CDK inhibitors usually serve as the G1/S checkpoint control proteins in response to negative growth signals. Over-expression of the G1 cyclins and the loss of CDK inhibitors are often associated with human cancer. We have previously isolated a novel p19SKP1 and p45SKP2 cell cycle complex based on its highly elevated level in many neoplastic transformed cells. We have identified additional components of this complex. Our preliminary data indicate that this complex is involved in the control of the G1/S transition by regulating the levels of the G1 cell cycle regulators through the ubiquitin-dependent proteolysis. To further investigate this important cell cycle control mechanism, we propose the following specific aims: (1) To determine and characterize the components that are involved in the control of the G/S transition through the p19SKP1 and p45SKP2-mediated ubiquitin-dependent proteolysis. (2) To identify the critical targets of this regulation during the G1/S transition. (3) To determine the cell cycle regulatory factors that control this process. (4) To establish in vitro assays to characterize the ubiquitin conjugation reaction and its regulatory mechanism. Our investigation should provide new insights into the mechanism of the mammalian G1/S transition and help to assess how the alteration of these cell cycle regulatory processes may contribute to tumorigenesis. These studies will also provide a molecular basis for designing novel strategies for the diagnostic and therapeutic treatment of human cancer.