Activating phosphorylation of the Kin28p subunit of yeast TFIIH by the Cdk-activating kinase, Cak1p

摘要

Cdk-Activating Kinases (CAKs) carry out essential activating phosphorylations of cyclin-dependent kinases (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 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 form of Kin28p is reduced ∼ 75--80% compared to wild-type Kin28p. Moreover, cells containing kin28TI62A 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 greatly reduced 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 and that Cak1p can phosphorylate Kin28p on Thr-162 in vitro. Thus, Kin28p joins Cdc28p, the major cell cycle cdk in budding yeast, as a physiological Cak1p substrate. These findings indicate that though 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. We also report several other genetic interactions involving CAK1, and speculate about their implications.