Advanced

Analysis of CAK activities from human cells

Kaldis, Philipp LU and Solomon, Mark J. (2000) In European Journal of Biochemistry 267(13). p.4213-4221
Abstract

The cdk-activating kinase (CAK) activates cyclin-dependent kinases (cdks) that control cell-cycle progression by phosphorylating a threonine residue conserved in cdks. CAK from humans contains p40(MO15) (cdk7), cyclin H and MAT1, which are also subunits of transcription factor IIH where they phosphorylate the C-terminal domain of the large subunit of RNA polymerase II. In contrast, budding yeast Cak1p is a monomeric enzyme without C-terminal domain kinase activity. Here, we analyze CAK activities in HeLa cells using cdk2-affinity chromatography. In addition to MO15, a second CAK activity was detected that runs on gel filtration at 30-40 kDa. This activity phosphorylated and activated cdk2 and cdk6. Furthermore, this 'small CAK' activity... (More)

The cdk-activating kinase (CAK) activates cyclin-dependent kinases (cdks) that control cell-cycle progression by phosphorylating a threonine residue conserved in cdks. CAK from humans contains p40(MO15) (cdk7), cyclin H and MAT1, which are also subunits of transcription factor IIH where they phosphorylate the C-terminal domain of the large subunit of RNA polymerase II. In contrast, budding yeast Cak1p is a monomeric enzyme without C-terminal domain kinase activity. Here, we analyze CAK activities in HeLa cells using cdk2-affinity chromatography. In addition to MO15, a second CAK activity was detected that runs on gel filtration at 30-40 kDa. This activity phosphorylated and activated cdk2 and cdk6. Furthermore, this 'small CAK' activity resembled Cak1p rather than MO15 in terms of substrate specificity, reactivity to antibodies against MO15 and Cak1p, and sensitivity to 5'- fluorosulfonylbenzoyladenosine, an irreversible inhibitory ATP analog. Our findings suggest the presence of at least two different CAK activities in human cells.

(Less)
Please use this url to cite or link to this publication:
author
and
publishing date
type
Contribution to journal
publication status
published
keywords
CAK, Cdk, Cyclin-dependent kinase, P40(MO15) (cdk7)
in
European Journal of Biochemistry
volume
267
issue
13
pages
4213 - 4221
publisher
Wiley-Blackwell
external identifiers
  • scopus:0033919678
  • pmid:10866826
ISSN
0014-2956
DOI
10.1046/j.1432-1327.2000.01455.x
language
English
LU publication?
no
id
6f60fa94-0045-4dcb-bd63-6d26116b0901
date added to LUP
2019-09-18 14:32:36
date last changed
2020-06-10 06:18:39
@article{6f60fa94-0045-4dcb-bd63-6d26116b0901,
  abstract     = {<p>The cdk-activating kinase (CAK) activates cyclin-dependent kinases (cdks) that control cell-cycle progression by phosphorylating a threonine residue conserved in cdks. CAK from humans contains p40(MO15) (cdk7), cyclin H and MAT1, which are also subunits of transcription factor IIH where they phosphorylate the C-terminal domain of the large subunit of RNA polymerase II. In contrast, budding yeast Cak1p is a monomeric enzyme without C-terminal domain kinase activity. Here, we analyze CAK activities in HeLa cells using cdk2-affinity chromatography. In addition to MO15, a second CAK activity was detected that runs on gel filtration at 30-40 kDa. This activity phosphorylated and activated cdk2 and cdk6. Furthermore, this 'small CAK' activity resembled Cak1p rather than MO15 in terms of substrate specificity, reactivity to antibodies against MO15 and Cak1p, and sensitivity to 5'- fluorosulfonylbenzoyladenosine, an irreversible inhibitory ATP analog. Our findings suggest the presence of at least two different CAK activities in human cells.</p>},
  author       = {Kaldis, Philipp and Solomon, Mark J.},
  issn         = {0014-2956},
  language     = {eng},
  month        = {07},
  number       = {13},
  pages        = {4213--4221},
  publisher    = {Wiley-Blackwell},
  series       = {European Journal of Biochemistry},
  title        = {Analysis of CAK activities from human cells},
  url          = {http://dx.doi.org/10.1046/j.1432-1327.2000.01455.x},
  doi          = {10.1046/j.1432-1327.2000.01455.x},
  volume       = {267},
  year         = {2000},
}