CDK7 COMPLEXES IN MAMMALIAN CELL CYCLE REGULATION

描述：拟议的研究计划的目标是描述 细胞周期蛋白依赖性激酶7(CDK7)在哺乳动物细胞调控中的作用 循环递增。含有CDK7及其相关蛋白的复合体， Cyclin H和组装促进亚基MAT1的鉴定基于 它在体外激活其他细胞周期蛋白依赖性蛋白激酶(CDK)的能力。 因为CDK7似乎激活了控制细胞周期的其他CDK 进展，申请人认为CDK7是一个主要的目标 化疗干预对癌细胞增殖的影响。 此外，除了CDK激活能力外，CDK7复合体还具有 也与一般转录因子TFIIH有关，提示 CDK7可能在调节基础转录活性中发挥作用。总的来说， 目前尚不清楚这多个CDK7复合体是否有不同的表现 职能，并受到不同的监管。或许始终如一 根据这一假设，CDK7可以被至少两个不同的 机理，包括a)组装含有以下物质的异三聚体配合物 Cyclin H和MAT1和b)关键苏氨酸的磷酸化 导致二聚体CDK7-Cyclin H组装的CAK激活激酶 复合体。在此背景下，这项建议的具体目标是： 1)使用生化分析鉴定CAK激活的激酶 依赖磷酸化的CDK7体外激活及检测 这些酶是否在体内激活CDK7。2)检查细胞周期 调控CDK7的组装和激活途径，以及 在体内消除磷酸化依赖过程的作用 表达非磷酸化突变体CDK7的细胞系。3)描述 二聚体和三聚体CDK7络合物的酶活性和 底物专一性。4)确定MAT1在调节中的作用 与其他蛋白质、亚细胞室的相互作用，以及在修饰 CDK7活性。

DESCRIPTION: The goal of the proposed research plan is to characterize the role of the cyclin-dependent kinase 7 (CDK7) in controlling mammalian cell cycle progression. A complex containing CDK7 and the associated proteins, cyclin H and the assembly promoting subunit MAT1, was identified based on its ability to activate other cyclin-dependent kinases (CDKs) in vitro. Because CDK7 appears to activate other CDKs that govern cell cycle progression, the applicant feels that CDK7 is a prime target for chemotherapeutic intervention in cases of cancer cell proliferation. Moreover, in addition to their CDK-activating ability, CDK7 complexes are also associated with the general transcription factor TFIIH, suggesting that CDK7 may play a role in modulating basal transcriptional activity. Overall, it is unclear whether these multiple CDK7 complexes perform different functions and are subject to differential regulation. Perhaps consistent with this hypothesis, CDK7 can be activated by at least two distinct mechanisms, including a) the assembly of a heterotrimeric complex containing cyclin H and MAT1 and b) the phosphorylation of a critical threonine by a CAK-activating kinase, which leads to the assembly of dimeric CDK7-cyclin H complexes. Given this background, the Specific Aims of the proposal are: 1) Identify CAK-activating kinases using a biochemical assay for phosphorylation-dependent CDK7 activation in vitro followed by testing whether these enzymes activate CDK7 in vivo. 2) Examine the cell-cycle regulation of the CDK7 assembly and activation pathways, as well as the effects of abrogating the phosphorylation-dependent process in vivo, using cell lines that express a non-phosphorylatable mutant CDK7. 3) Characterize dimeric and trimeric CDK7 complexes with respect to enzymatic activity and substrate specificity. 4) Determine the role of MAT1 in mediating interactions with other proteins, subcellular compartments, and in modifying CDK7 activity.