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.
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