IS CDK7 CYCLIN-H MAT1 THE GENUINE CDK ACTIVATING KINASE IN CYCLING XENOPUS EGG EXTRACTS

Formation of active cdk (cyclin dependent kinase)/ cyclin kinases invo lves phosphorylation of a conserved threonine residue in the T loop of the cdk catalytic subunit by CAK (Cdk Activating Kinase). CAK was fir st purified biochemically from higher eukaryotes and identified as a t rimeric complex containing a cdk7 catalytic subunit, cyclin H and MAT1 (Menage a trois), a member of the RING finger family. The same trimer ic complex is also part of basal transcription factor TFIIH. In buddin g yeast, the closest homologs of cdk7 and cyclin H, KIN28 and CCL1, re spectively, also associate with TFIIH. However, the KIN28/CCL1 complex does not display CAK activity and a distinct protein kinase able to p hosphorylate monomeric CDC28 and GST-cdk2 was recently identified, cha llenging the identification of cdk7 as the physiological CAK in higher eukaryotes. Here we demonstrate that immunodepletion of cdk7 suppress es CAK activity from cycling Xenopus egg extracts, and arrest them bef ore M-phase. We also show that specific translation of mRNAs encoding Xenopus cdk7 and its associated subunits restores CAK activity in cdk7 -immunodepleted Xenopus egg extracts. Hence, the cdk7 complex is neces sary and sufficient for activation of cdk-cyclin complexes in cycling Xenopus egg extracts.