CDK7 COMPLEXES IN MAMMALIAN CELL CYCLE REGULATION

描述：拟议研究计划的目标是表征 细胞周期蛋白依赖性激酶7（CDK 7）在哺乳动物细胞调控中的作用 循环进展 一种含有CDK 7和相关蛋白的复合物， 细胞周期蛋白H和组装促进亚基MAT 1，是基于 其在体外激活其他细胞周期蛋白依赖性激酶（CDK）的能力。 因为CDK 7似乎激活了其他控制细胞周期的CDK 申请人认为CDK 7是一个主要目标， 在癌细胞增殖的情况下进行化疗干预。 此外，除了它们的CDK活化能力之外，CDK 7复合物还被 也与一般转录因子TFIIH相关，表明 CDK 7可能在调节基础转录活性方面发挥作用。 总的来说， 目前尚不清楚这些多CDK 7复合物是否具有不同的功能， 功能，并受到不同的监管。 也许是一致的 根据这一假设，CDK 7可以被至少两种不同的 a）异源三聚体复合物的组装，所述异源三聚体复合物含有 细胞周期蛋白H和MAT 1，和B）关键的苏氨酸被一种 CAK激活激酶，导致二聚体CDK 7-细胞周期蛋白H的组装 配合物 鉴于这一背景，该提案的具体目标是： 1)使用生物化学测定法鉴定CAK激活激酶， 体外磷酸化依赖性CDK 7活化，随后测试 这些酶是否在体内激活CDK 7。 2)检查细胞周期 CDK 7组装和活化途径的调节，以及 在体内消除磷酸化依赖性过程的效果，使用 表达不可磷酸化的突变CDK 7的细胞系。 3)表征 二聚和三聚CDK 7复合物的酶活性， 底物特异性 4)确定MAT 1在调解中的作用 与其他蛋白质，亚细胞区室的相互作用，以及在修饰 CDK 7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.