PROTEOLYSIS AND MAMMALIAN GL/S CELL CYCLE TRANSITION

蛋白质水解和哺乳动物 GL/S 细胞周期转变

• 批准号: 6150049
• 负责人: HUI ZHANG
• 金额: $ 28.07万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6150049
• 关键词: HeLa cells; SDS polyacrylamide gel electrophoresis; autoradiography; carcinogenesis; cell cycle; cell differentiation; cell growth regulation; cyclin dependent kinase; cyclins; enzyme inhibitors; immunoprecipitation; laboratory rabbit; phosphorylation; polymerase chain reaction; protein purification; protein structure function; proteolysis; synchronous cell division; ubiquitin; western blottings

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.

细胞周期控制是一个基本的调节过程， 细胞内遗传信息的忠实复制和传递 师. 为了防止不可逆转的遗传损伤 G1/S和G2/M期之间的转换， 细胞周期作为检查点控制的一部分受到高度调节， 对环境线索作出反应。 异常细胞周期控制消除 细胞周期的不同阶段之间的协调， 肿瘤转化的标志。 广泛的长期目标 这项建议的一个重要目的是了解细胞周期控制的机制， 在细胞生长和分化中， 肿瘤发生过程中的改变。 这一建议的重点是机制 哺乳动物细胞G1/S细胞周期转换。 特别是， 我们描述的方法来调查的机制， 控制哺乳动物G1细胞周期蛋白和CDK抑制剂的水平 通过G1/S期间的选择性泛素依赖性降解， 过渡 G1细胞周期蛋白的积累是速率限制 在G1/S转换过程中的步骤和CDK抑制剂通常作为 G1/S检查点控制蛋白响应负生长 信号. G1期细胞周期蛋白的过度表达和CDK的丢失 抑制剂通常与人类癌症有关。 我们先前已经 分离了一种新的p19 SKP 1和p45 SKP 2细胞周期复合物， 在许多肿瘤转化细胞中水平高度升高。 我们有 确定了这个复合体的其他组成部分。 我们的初步数据 表明该复合物参与了G1/S的控制， 通过调节G1期细胞周期调节因子的水平 通过泛素依赖的蛋白水解作用。 为了进一步研究 这一重要的细胞周期控制机制，我们提出以下建议 具体目标：（1）确定和表征 通过p19 SKP 1参与G/S转换的控制， p45 SKP 2介导的泛素依赖性蛋白水解。(2)识别 在G1/S过渡期间，这一规定的关键目标。 (3)到 确定控制这一过程的细胞周期调节因子。 (4)建立体外测定方法，以表征泛素 结合反应及其调控机制。 我们的调查 这将为哺乳动物G1/S的机制提供新的见解。 并帮助评估这些细胞周期的改变 调节过程可能有助于肿瘤发生。 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。