Chemical Genetic Analysis of the Human Cell Cycle

描述（由申请人提供）：正常生长、发育和基因组完整性需要细胞分裂的适当调节;在人类癌症中，这种控制的某些方面丢失或被破坏。细胞周期蛋白依赖性激酶（CDK）驱动细胞分裂周期中的所有主要转变。哺乳动物细胞有多个下游效应CDK，但如我们所示，一个单一的上游CDK激活激酶（CAK），Cdk 7。基因敲除和沉默研究表明，CDK网络冗余，通过显示Cdk 2-长期以来被认为是DNA合成（S）阶段的主要驱动程序-是可行的。然而，我们的工作-使用选择性地使靶向CDK的催化功能失活而保留非催化的“支架”功能的化学遗传方法-揭示了在转化和非转化的人细胞增殖中对Cdk 2活性的严格要求。Cdk 2的一个重要功能是防止Cdk 1的过早激活，从而确保通过S期的协调进展。与Cdk 1相比，该功能取决于Cdk 2激活的动力学更有利的途径，部分原因是Cdk 7的识别机制不同。我推测，专门的功能Cdk 2-在调节基因表达，协调S期和响应DNA损伤或复制应力出现从其独特的激活模式。我建议，此外，另一个独特的途径，Cdk 7激活Cdk 4和Cdk 6-细胞周期承诺之前的功能-直接或间接耦合到丝裂原传感途径。具体目标是：1.分析Cdk 7、Cdk 2、Cdk 4和Cdk 6组成的G1/S调控网络。探讨Cdk 7和Cdk 2在DNA损伤反应中的特异性功能3.为了用小分子抑制剂靶向Cdk 2活化途径，我们的初步研究揭示了一个单一的CAK-CDK网络，该网络通过动力学上不同的激酶活化和失活模式实现调节灵活性。我现在建议探测CAK和下游CDK在协调组成性细胞周期事件和确保对遗传毒性损伤的有效反应中的特异性功能;并测试CAK和下游CDK在细胞周期中的作用。 通过选择性靶向其活化途径抑制特异性CDK的新范例。这些研究有望推进对细胞周期调控的基本理解，并揭示抗CDK治疗人类癌症的新策略。

DESCRIPTION (provided by applicant): Proper regulation of cell division is required for normal growth, development and genomic integrity; aspects of this control are lost or disrupted in human cancers. The cyclin-dependent kinases (CDKs) drive all major transitions in the cell division cycle. Mammalian cells have multiple downstream effector CDKs but, as we show, a single upstream CDK-activating kinase (CAK), Cdk7. Gene knockout and silencing studies suggested redundancy in the CDK network, by showing that Cdk2-long thought to be the principal driver of DNA synthesis (S) phase-was dispensable for viability. However, our work-using a chemical-genetic approach that selectively inactivates catalytic function of the targeted CDK while sparing non-catalytic, "scaffold" functions-uncovered strict requirements for Cdk2 activity in proliferation of both transformed and non- transformed human cells. An important function of Cdk2 is to prevent premature activation of Cdk1, and thereby ensure coordinated progression through S phase. That function depends on a kinetically more favorable pathway for activation of Cdk2, compared to Cdk1, due in part to different mechanisms of recognition by Cdk7. I hypothesize that specialized functions of Cdk2-in regulating gene expression, coordinating S phase and responding to DNA damage or replication stress-emerge from its unique mode of activation. I propose, moreover, that another distinct pathway by which Cdk7 activates Cdk4 and Cdk6-which function prior to cell-cycle commitment-is directly or indirectly coupled to mitogen-sensing pathways. The specific aims are: 1. To dissect the G1/S regulatory network comprising Cdk7, Cdk2, Cdk4 and Cdk6 2. To probe specific functions of Cdk7 and Cdk2 in the DNA damage response 3. To target the Cdk2 activation pathway with small molecule inhibitors Our preliminary studies reveal a unitary CAK-CDK network that achieves regulatory flexibility through kinetically distinct modes of kinase activation and inactivation. I ow propose to probe specific functions of CAK and the downstream CDKs in coordinating constitutive cell-cycle events and ensuring effective responses to genotoxic insults; and to test a new paradigm for inhibiting a specific CDK by selective targeting of its activation pathway. These studies promise to advance basic understanding of cell-cycle control, and to reveal novel strategies for anti-CDK therapy of human cancer.