Chemical Genetic Analysis of the Human Cell Cycle

描述(申请人提供)：细胞分裂的适当调节是正常生长、发育和基因组完整性所必需的；在人类癌症中，这种调控的某些方面丢失或中断。细胞周期蛋白依赖性蛋白激酶(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物CDK，但如我们所示，只有一个上游CDK激活激酶(CAK)，即CDK7。基因敲除和沉默研究表明，CDK网络中存在冗余，表明CDK2--长期以来一直被认为是DNA合成(S)阶段的主要驱动力--对于生存是必不可少的。然而，我们的工作-使用一种化学遗传方法，选择性地灭活靶向CDK的催化功能，同时保留非催化的“支架”功能-揭示了在转化和未转化的人类细胞的增殖中对CDK2活性的严格要求。CDK2的一个重要功能是防止CDK1的过早激活，从而确保通过S阶段的协调进展。这一功能依赖于CDK2比CDK1在动力学上更有利的激活途径，部分原因是CDK7识别的机制不同。我推测，CDK2的特殊功能--调节基因表达、协调S阶段以及应对DNA损伤或复制压力--来自于其独特的激活模式。此外，我还提出，CDK7激活CDK4和CDK6的另一个不同的途径--在细胞周期承诺之前发挥作用--直接或间接耦合到有丝分裂原感应途径。具体目标是：1.剖析由CDK7、CDK2、CDK4和CDK6组成的G1/S调控网络；2.探讨CDK7和CDK2在DNA损伤反应中的具体功能；3.通过小分子抑制剂靶向CDK2激活途径，我们的初步研究揭示了一个单一的CAK-CDK网络，该网络通过不同的激酶激活和失活模式实现调节灵活性。我现在提议探索CAK和下游CDK在协调构成细胞周期事件和确保对基因毒性侮辱的有效反应中的具体功能；并测试 通过选择性靶向激活途径抑制特定CDK的新范式。这些研究有望促进对细胞周期控制的基本了解，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.