Requirements for Cdk7 in the assembly of Cdk1/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells.
Cell division is controlled by cyclin-dependent kinases (CDKs). In metazoans, S phase onset coincides with activation of Cdk2, whereas Cdk1 triggers mitosis. Both Cdk1 and -2 require cyclin binding and T loop phosphorylation for full activity. The only known CDK-activating kinase (CAK) in metazoans is Cdk7, which is also part of the transcription machinery. To test the requirements for Cdk7 in vivo, we replaced wild-type Cdk7 with a version sensitive to bulky ATP analogs in human cancer cells. Selective inhibition of Cdk7 in G1 prevents activation (but not formation) of Cdk2/cyclin complexes and delays S phase. Inhibiting Cdk7 in G2 blocks entry to mitosis and disrupts Cdk1/cyclin B complex assembly, indicating that the two steps of Cdk1 activation-cyclin binding and T loop phosphorylation-are mutually dependent. Therefore, by combining chemical genetics and homologous gene replacement in somatic cells, we reveal different modes of CDK activation by Cdk7 at two distinct execution points in the cell cycle.
Larochelle
Stéphane
S
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Merrick
Karl A
KA
Terret
Marie-Emilie
ME
Wohlbold
Lara
L
Barboza
Nora M
NM
Zhang
Chao
C
Shokat
Kevan M
KM
Jallepalli
Prasad V
PV
Fisher
Robert P
RP
D016203:CDC2 Protein Kinase / Q000235:genetics*; D002453:Cell Cycle; D002455:Cell Division; D002470:Cell Survival; D019926:Cyclin B / Q000235:genetics*; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008938:Mitosis; D016196:S Phase; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D019926:Cyclin B; D016203:CDC2 Protein Kinase; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human