PROTEOLYSIS AND MAMMALIAN GL/S CELL CYCLE TRANSITION

细胞周期控制是一个基本的调控过程，它确保 细胞内遗传信息的忠实复制和传递 组织。防止不可逆转的遗传损害并入 进入基因组，在G1/S和G2/M期之间的过渡 作为检查点控制的一部分，细胞周期受到高度调控 对环境提示做出反应。异常的细胞周期控制被废除 细胞周期不同阶段之间的协调，是一种 肿瘤性转化的标志。广泛的、长期的目标 这一建议的目的是了解细胞周期调控的机制 在细胞的生长和分化中，以及它的后果 肿瘤发生过程中的改变。这项建议的重点是机制 哺乳动物细胞中G1/S细胞周期转换的研究。特别是， 我们描述了研究机制的方法， 哺乳动物的G1周期蛋白和CDK抑制剂的水平受到控制 在G1/S期间通过选择性泛素依赖的降解 过渡。G1期细胞周期蛋白的积累是速率的限制 在G1/S转换过程中的步骤和CDK抑制剂通常作为 负生长反应中的G1/S检查点控制蛋白 信号。G1期细胞周期蛋白的过度表达与CDK的丢失 抑制剂通常与人类癌症有关。我们之前已经 基于其基因序列分离出一个新的p19SKP1和p45SKP2细胞周期复合体 在许多肿瘤性转化的细胞中水平高度升高。我们有 确定了这个建筑群的其他组成部分。我们的初步数据 提示这个复合体参与了对G1/S的控制 通过调节G1期细胞周期调节器的水平来实现过渡 通过泛素依赖的蛋白水解酶。进一步调查 这一重要的细胞周期调控机制，我们提出如下建议 具体目标：(1)确定和表征以下组件 参与通过p19SKP1和p19SKP1控制G/S转换 P45SKP2介导的泛素依赖的蛋白分解。(2)识别 这一规定在G1/S过渡期间的关键目标。(3)至 确定控制这一过程的细胞周期调控因素。 (4)建立泛素的体外检测方法 偶联反应及其调控机制。我们的调查 应为哺乳动物G1/S的机制提供新的见解 转变并有助于评估这些细胞周期的变化 调控过程可能有助于肿瘤的发生。这些研究 也将为设计新的策略提供分子基础 人类癌症的诊断和治疗。

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.