Cyclin-dependent kinase (CDK)-activating kinases (CAKs) carry out essential activating phosphorylations of CDKs such as Cdc2 and Cdk2. The catalytic subunit of mammalian CAK, MO15/Cdk7, also functions as a subunit of the general transcription factor TFIIH. However, these functions are split in budding yeast, where Kin28p functions as the kinase subunit of TFIIH and Cak1p functions as a CAK. We show that Kin28p, which is itself a CDK, also contains a site of activating phosphorylation on Thr-162. The kinase activity of a T162A mutant of Kin28p is reduced by approximately 75 to 80% compared to that of wild-type Kin28p. Moreover, cells containing kin28(T162A) and a conditional allele of TFB3 (the ortholog of the mammalian MAT1 protein, an assembly factor for MO15 and cyclin H) are severely compromised and display a significant further reduction in Kin28p activity. This finding provides in vivo support for the previous biochemical observation that MO15-cyclin H complexes can be activated either by activating phosphorylation of MO15 or by binding to MAT1. Finally, we show that Kin28p is no longer phosphorylated on Thr-162 following inactivation of Cak1p in vivo, that Cak1p can phosphorylate Kin28p on Thr-162 in vitro, and that this phosphorylation stimulates the CTD kinase activity of Kin28p. Thus, Kin28p joins Cdc28p, the major cell cycle Cdk in budding yeast, as a physiological Cak1p substrate. These findings indicate that although MO15 and Cak1p constitute different forms of CAK, both control the cell cycle and the phosphorylation of the C-terminal domain of the large subunit of RNA polymerase II by TFIIH.
SciCrunch is a data sharing and display platform. Anyone can create a custom portal where they can select searchable subsets of hundreds of data sources, brand their web pages and create their community. SciCrunch will push data updates automatically to all portals on a weekly basis. User communities can also add their own data to scicrunch, however this is not currently a free service.