Activating phosphorylation of the Kin28p subunit of yeast TFIIH by Cak1p

Cyclin-dependent kinase (CDK)-activating kinases (CAKs) carry out essential activating phosphorylations of CDKs such as Cdc2 and Cdk2. The catalytic s ubunit of mammalian CAK, MO15/Cdk7, also functions as a subunit of the gene ral transcription factor TFIIH. However, these functions are split in buddi ng yeast, where Kin28p functions as the kinase subunit of TFIIH and Cak1p f unctions as a CAK. We show that Kin28p, which is itself a CDK, also contain s a site of activating phosphorylation on Thr-162. The kinase activity of a T162A mutant of Kin28p is reduced by similar to 75 to 80% compared to that of wild-type Kin28p. Moreover, cells containing kin28(T162A) and a conditi onal allele of TFB3 (the ortholog of the mammalian MAT1 protein, an assembl y factor for MO15 and cyclin H) are severely compromised and display a sign ificant further reduction in Kin28p activity. This finding provides in vivo support for the previous biochemical observation that MO15 cyclin H comple xes can be activated either by activating phosphorylation of MO15 or by bin ding to MAT1. Finally, we show that Kin28p is no longer phosphorylated on T hr-162 following inactivation of Cak1p in vivo, that Cak1p can phosphorylat e Kin28p on Thr-162 in vitro, and that this phosphorylation stimulates the CTD kinase activity of Kin28p. Thus, Kin28p joins Cdc28p, the major cell cy cle Cdk in budding yeast, as a physiological Cak1p substrate. These finding s indicate that although MO15 and Cak1p constitute different forms of CAK, both control the cell cycle and the phosphorylation of the C-terminal domai n of the large subunit of RNA polymerase II by TFIIH.