Requirements for Cdk7 in the Assembly of Cdk1/Cyclin B and Activation of Cdk2 Revealed by Chemical Genetics in Human Cells

Abstract

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.

Hyperphosphorylation of RNA Polymerase II in Response to Topoisomerase I Cleavage Complexes and Its Association with Transcription- and BRCA1-dependent Degradation of Topoisomerase I

“…3e) although it is possible that Ser 2 -P can be compensated by other kinases. Likewise, inhibition of Cdk7 did not decrease steady-state levels of Ser 5 -P, as shown previously, 31 suggesting that other kinases can compensate to maintain those levels under normal growth conditions. Nevertheless, our results demonstrate that increased Ser 5 -P in response to transcription arrest by Top1cc involves Cdk7 activity.…”

“…3). Other Cdks are likely involved as complete and selective inhibition of Cdk7 kinase activity in Cdk7 as/as HCT116 cells 31 only partially prevented Pol II-Ser 5 -P (Fig. 3d) whereas the pan-Cdk inhibitors DRB and FLV completely abrogated Pol II-Ser 5 -P (Fig.…”

“…31 This mutation allows Cdk7 to be specifically and quickly inhibited by ATP analogs without affecting other Cdks. 31 In Cdk7 as/as HCT116 cells, inhibition of Cdk7 with the ATP analog 3-MBPP1 reduced CPTinduced Ser 5 -P and Pol II hyperphosphorylation (Fig. 3d, compare lanes 2 and 4).…”

“…Cdk7 as/as HCT116 cells were generated previously as described. 31 MEF cells of each genotype (Brca1 +/+ , Brca1 −/− , and Brca1 −/− stably expressing human Brca1) were kind gifts from W. H. Lee (University of California, Irvine, CA). All the above cells lines were cultured in Dulbecco's modified Eagle's medium.…”

Papel das proteínas XPD e DNA polimerase eta nas respostas de células humanas a danos no genoma

“…XPD também pode ser encontrada associada à subunidade CAK sem a subunidade core do TFIIH (Reardon et al, 1996); a quinase CAK é necessária para a entrada da célula em mitose através da fosforilação do complexo CDK1/ciclinaB1 (Larochelle et al, 2007). Recentemente foi descrita a participação de XPD em um complexo independente de TFIIH, o complexo MMS19-XPD (composto pelas proteínas MMS19, XPD, MIP18, Ciao1 e ANT2), que se localiza no fuso mitótico e é importante para a correta segregação dos fusos (Ito et al, 2010).…”