LSF AND MAMMALIAN CELL CYCLE PROGRESSION

LSF 和哺乳动物细胞周期进展

• 批准号: 2830596
• 负责人: ULLA M HANSEN
• 金额: $ 24.91万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-15 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2830596
• 关键词: 3T3 cells; DNA binding protein; DNA replication; autoradiography; biological signal transduction; cell cycle; cell growth regulation; cyclin dependent kinase; enzyme substrate; gel mobility shift assay; gene induction /repression; immunoprecipitation; phosphorylation; polymerase chain reaction; site directed mutagenesis; thymidylate synthase; transcription factor; transfection; western blottings

The growth of mammalian cells is driven by signal transduction pathways, whose targets include transcription factors that regulate expression of genes critical to cell cycle progression. At one pivotal point in late G1 phase, the restriction point, cellular signals are integrated to determine whether to replicate cellular DNA (progress to S phase) or revert to a resting state. The signalling molecules required for transit from G1 to S include both D and E cyclins complexed with cyclin-dependent kinases (CDKs). Cyclin D/CDKs phosphorylate Rb and activate members of the E2F family of transcription factors, resulting in activation of genes important for cellular DNA synthesis. Little is known, however, about the regulation of other transcription factors that activate additional components of the DNA synthesis machinery, including potential transcription factors that are targeted by cyclin E/CDK at this key point in the cell cycle. Our recent studies have shown that the mammalian transcription factor LSF plays a key role in cell cycle progression by regulating thymidylate synthase (TS) expression at the G1/S transition. Because TS is essential for cell growth, it has been a cancer chemotherapeutic target for over two decades. Likewise, as an essential regulator of TS expression, LSF may ultimately prove a valuable target for cancer therapies. The DNA-binding activity of LSF is enhanced in late G1, via the modification of pre-existing pools of nuclear LSF. In vitro, LSF is an effective substrate for cyclin E/cdk2. An understanding of the signaling pathway that regulates LSF, and the molecular mechanism by which LSF regulates TS expression may therefore illuminate novel signaling events in cell cycle progression in mammalian cells. Specifically, this proposal will address the following: 1. Is LSF phosphorylated by cyclin E/cdk2 during the late G1 phase? 2. Does phosphorylation of LSF by cyclin E/cdk2 (or other relevant modifications) alter the DNA-binding and transcriptional activities of LSF? 3. Is LSF a major target of cdk2 (or another pathway) in progression of cells into S? 4. Does LSF critically regulate genes other than TS during cell cycle progression?

哺乳动物细胞的生长是由信号转导途径驱动的，其目标包括调节对细胞周期进程至关重要的基因表达的转录因子。在 G1 期晚期的一个关键点（限制点），细胞信号被整合以确定是复制细胞 DNA（进展到 S 期）还是恢复到静息状态。从 G1 过渡到 S 所需的信号分子包括与细胞周期蛋白依赖性激酶 (CDK) 复合的 D 和 E 细胞周期蛋白。 细胞周期蛋白 D/CDK 磷酸化 Rb 并激活转录因子 E2F 家族的成员，从而激活对细胞 DNA 合成重要的基因。 然而，人们对激活 DNA 合成机制其他组件的其他转录因子的调节知之甚少，包括细胞周期中这一关键点上细胞周期蛋白 E/CDK 靶向的潜在转录因子。我们最近的研究表明，哺乳动物转录因子 LSF 通过调节 G1/S 转变时的胸苷酸合酶 (TS) 表达，在细胞周期进程中发挥关键作用。 由于 TS 对于细胞生长至关重要，因此二十多年来它一直是癌症化疗靶点。 同样，作为 TS 表达的重要调节因子，LSF 最终可能被证明是癌症治疗的一个有价值的靶点。 通过修改预先存在的核 LSF 库，LSF 的 DNA 结合活性在 G1 晚期得到增强。 在体外，LSF 是细胞周期蛋白 E/cdk2 的有效底物。 因此，了解调节 LSF 的信号传导途径以及 LSF 调节 TS 表达的分子机制可能会阐明哺乳动物细胞细胞周期进程中的新信号传导事件。具体来说，该提案将解决以下问题： 1. LSF 在 G1 后期是否被细胞周期蛋白 E/cdk2 磷酸化？ 2. 细胞周期蛋白 E/cdk2（或其他相关修饰）对 LSF 的磷酸化是否会改变 LSF 的 DNA 结合和转录活性？ 3. LSF 是细胞进展为 S 过程中 cdk2（或其他途径）的主要靶标吗？ 4. 在细胞周期进展过程中，LSF 是否对 TS 以外的基因有重要调控作用？