Activating phosphorylation of the Kin28p subunit of yeast TFIIH by Cak1p
Kimmelman, Jonathan ; Kaldis, Philipp LU
; Hengartner, Christoph J.
; Laff, Geoffrey M.
; Koh, Sang Seok
; Young, Richard A.
and Solomon, Mark J.
(1999)
In Molecular and Cellular Biology
19(7).
p.4774-4787
- Abstract
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 ~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... (More)
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 ~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.
(Less)
- author
- Kimmelman, Jonathan
; Kaldis, Philipp
LU
; Hengartner, Christoph J.
; Laff, Geoffrey M.
; Koh, Sang Seok
; Young, Richard A.
and Solomon, Mark J.
- publishing date
- 1999-01-01
- type
- Contribution to journal
- publication status
- published
- in
- Molecular and Cellular Biology
- volume
- 19
- issue
- 7
- pages
- 4774 - 4787
- publisher
- American Society for Microbiology
- external identifiers
-
- pmid:10373527
- scopus:0033036182
- ISSN
- 0270-7306
- DOI
- 10.1128/MCB.19.7.4774
- language
- English
- LU publication?
- no
- id
- 5c8a128a-e6cb-4e7d-8ca1-b598b89ba721
- date added to LUP
- 2019-09-18 14:33:55
- date last changed
- 2024-02-15 23:17:27
@article{5c8a128a-e6cb-4e7d-8ca1-b598b89ba721,
abstract = {{<p>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 ~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.</p>}},
author = {{Kimmelman, Jonathan and Kaldis, Philipp and Hengartner, Christoph J. and Laff, Geoffrey M. and Koh, Sang Seok and Young, Richard A. and Solomon, Mark J.}},
issn = {{0270-7306}},
language = {{eng}},
month = {{01}},
number = {{7}},
pages = {{4774--4787}},
publisher = {{American Society for Microbiology}},
series = {{Molecular and Cellular Biology}},
title = {{Activating phosphorylation of the Kin28p subunit of yeast TFIIH by Cak1p}},
url = {{http://dx.doi.org/10.1128/MCB.19.7.4774}},
doi = {{10.1128/MCB.19.7.4774}},
volume = {{19}},
year = {{1999}},
}