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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 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.

Pubmed ID: 10373527

Authors

  • Kimmelman J
  • Kaldis P
  • Hengartner CJ
  • Laff GM
  • Koh SS
  • Young RA
  • Solomon MJ

Journal

Molecular and cellular biology

Publication Data

July 22, 1999

Associated Grants

  • Agency: NIGMS NIH HHS, Id: GM34365
  • Agency: NIGMS NIH HHS, Id: GM47830

Mesh Terms

  • Animals
  • Cell Cycle
  • Cyclin-Dependent Kinases
  • Enzyme Activation
  • Phosphorylation
  • Point Mutation
  • Protein-Serine-Threonine Kinases
  • Rabbits
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins
  • TATA-Binding Protein Associated Factors
  • Threonine
  • Transcription Factor TFIID
  • Transcription Factor TFIIH
  • Transcription Factors
  • Transcription Factors, TFII