Cyclin-dependent kinase control of cell-division and transcription cycles

细胞分裂和转录周期的细胞周期蛋白依赖性激酶控制

• 批准号: 10378005
• 负责人: ROBERT P FISHER
• 金额: $ 46.47万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10378005
• 关键词: Antineoplastic Agents; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell division; Cells; Chemicals; Colon Carcinoma; Complex; Coupling; Cyclin-Dependent Kinases; Dependence; Disease; Drug Combinations; Drug Targeting; Elongation Factor; Ensure; Enzymes; Event; Failure; Fission Yeast; Gene Expression; Gene Expression Regulation; Genetic; Genetic Transcription; Goals; Human; Human Development; Kinetics; Lead; Link; Malignant Neoplasms; Modeling; Pathway interactions; Pharmaceutical Preparations; Phase; Phosphorylation; Polyadenylation; Polymerase; Positive Transcriptional Elongation Factor B; Protein phosphatase; Proteins; RNA Polymerase II; RNA Processing; Regulation; Signal Transduction; Specificity; Speed; TP53 gene; Testing; Therapeutic Agents; Time; Transcription Elongation; Transcriptional Regulation; Yeasts; analog; cancer cell; chemical genetics; cyclin T1; cyclin-dependent kinase-activating kinase; drug discovery; functional genomics; genetic approach; inhibitor; mutant; novel; promoter

Project Summary Cyclin-dependent kinases (CDKs) regulate cell division and transcription. To understand the mechanisms of this regulation, or to target the CDK network in cancer cells, we need to identify the functions and substrates of specific CDKs. To achieve this goal, we pioneered a chemical-genetic approach combining the specificity of genetics with the speed and reversibility of chemical inhibition, by replacement of wild-type with analog- sensitive (AS) mutant CDKs in living cells. In the next five years, we will combine chemical genetics with functional genomics in novel ways, to establish new paradigms of cell-cycle and transcriptional control, and to discover new pathways that interact with the CDK network, which might be targeted in cancer. The CAK-CDK network in cell-cycle control: Our studies revealed distinct activation pathways for CDKs that act in different phases of the cell cycle; a goal for the next five years is to understand how those pathways are regulated by upstream signaling and linked to cell cycle-regulated transcription. We will investigate how a cascade comprising the CDK-activating kinase (CAK) Cdk7 and its target Cdk4 is switched on in G1 when quiescent cells re-enter the division cycle, and how Cdk7 is specifically down-regulated as cells exit the cycle. CDK regulation of the transcription cycle: Inhibiting transcriptional CDKs perturbs RNA polymerase (Pol) II dynamics in ways reminiscent of cell-cycle arrests and checkpoint failures, but the precise mechanisms still need to be defined. Cdk7 is required to establish a promoter-proximal pause in the transition from initiation to elongation, and to promote pause release by activating positive transcription elongation factor b (P-TEFb, a Cdk9/cyclin T1 complex). We showed that normal Pol II elongation rates depend on Cdk9 activity in fission yeast, and defined sets of human and yeast Cdk9 substrates, which are enriched for proteins implicated in RNA processing. Over the next five years, we will test the idea that Cdk9 acts on different substrates to stimulate both transcription elongation and RNA processing, to ensure their kinetic coupling. Defining a transcription exit network: CDK regulation persists to the end of the transcription cycle; we validated the termination enzyme Xrn2 and protein phosphatase 1 (PP1, implicated in cleavage and polyadenylation) as bona fide Cdk9 substrates. Phosphorylation by Cdk9 activates Xrn2 but inhibits PP1, which is required to dephosphorylate the elongation factor (and Cdk9 target) Spt5. In the next five years we will test the emergent model of a bistable Cdk9-PP1 switch that controls the elongation-termination transition. Chemical-genetic discovery of synthetic-lethal interactions CDKs have emerged as targets of drugs that exploit transcriptional dependencies unique to certain cancer cells. We induced such a dependency in colon cancer cells by combining activators of the tumor suppressor p53 with inhibitors of Cdk7 to achieve synthetic lethality. In the next five years, we will uncover novel pathways that interact with the CDK network—and might lead to anti-cancer drug combinations—by synthetic-lethal screens in human cells dependent on AS CDKs.

项目概要 细胞周期蛋白依赖性激酶 (CDK) 调节细胞分裂和转录。了解其机制 这种调节，或者针对癌细胞中的 CDK 网络，我们需要确定其功能和底物 特定的 CDK。为了实现这一目标，我们开创了一种化学遗传方法，结合了 通过用类似物替换野生型，具有化学抑制的速度和可逆性的遗传学 活细胞中的敏感（AS）突变CDK。未来五年，我们将把化学遗传学与 以新的方式进行功能基因组学，建立细胞周期和转录控制的新范式，并 发现与 CDK 网络相互作用的新途径，这可能是癌症的靶点。 细胞周期控制中的 CAK-CDK 网络：我们的研究揭示了 CDK 的不同激活途径， 作用于细胞周期的不同阶段；未来五年的目标是了解这些途径是如何进行的 受上游信号传导调节并与细胞周期调节转录相关。我们将调查如何 包含 CDK 激活激酶 (CAK) Cdk7 及其靶标 Cdk4 的级联在 G1 时开启 休眠细胞重新进入分裂周期，以及当细胞退出分裂周期时 Cdk7 如何被特异性下调。 CDK 对转录周期的调节：抑制转录 CDK 扰乱 RNA 聚合酶 (Pol) II 动力学的方式让人想起细胞周期停滞和检查点故障，但精确的机制仍然存在 需要定义。需要 Cdk7 在从启动到启动的过渡中建立启动子近端暂停。 延伸，并通过激活正转录延伸因子 b（P-TEFb，a Cdk9/细胞周期蛋白 T1 复合物）。我们发现正常的 Pol II 延伸率取决于裂变中的 Cdk9 活性 酵母，以及确定的人类和酵母 Cdk9 底物组，这些底物富含与 RNA 处理。在接下来的五年里，我们将测试 Cdk9 作用于不同底物的想法 刺激转录延伸和 RNA 加工，以确保它们的动力学耦合。 定义转录出口网络：CDK 调节持续到转录周期结束；我们 验证了终止酶 Xrn2 和蛋白磷酸酶 1（PP1，涉及裂解和 聚腺苷酸化）作为真正的 Cdk9 底物。 Cdk9 的磷酸化会激活 Xrn2 但抑制 PP1， 这是使延伸因子（和 Cdk9 靶标）Spt5 去磷酸化所必需的。未来五年我们将 测试控制伸长终止转变的双稳态 Cdk9-PP1 开关的紧急模型。 合成致死相互作用的化学遗传学发现 CDK 已成为药物的靶点 利用某些癌细胞特有的转录依赖性。我们在结肠中诱导了这种依赖性 通过将肿瘤抑制因子 p53 的激活剂与 Cdk7 的抑制剂相结合来合成 杀伤力。在接下来的五年中，我们将发现与 CDK 网络相互作用的新途径，并且可能 通过依赖 AS CDK 的人类细胞中的合成致死筛选，产生抗癌药物组合。