Article

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

Cornell University, Итак, New York, United States
Molecular Cell (Impact Factor: 14.02). 04/2007; 25(6):839-50. DOI: 10.1016/j.molcel.2007.02.003
Source: PubMed

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.

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Available from: Robert P Fisher, Oct 14, 2014
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    • "In vitro, Xrn2-Thr439 is phosphorylated by either Cdk7 or Cdk9 (Fig. 2D; Supplemental Fig. 2D,E), and Cdk7 is also inhibited by FP (but not by DRB or 2-FP-FP) at the concentrations used in the previous experiment. We therefore asked whether Cdk7 contributes to Xrn2 phosphorylation in vivo by immunoblot analysis of Xrn2- T439P after selective inhibition of Cdk7 in CDK7 as/as HCT116 cells (Larochelle et al. 2007). In contrast to results with Cdk9 inhibitors, there was little or no effect on Xrn2-T439P when these cells were treated for 4 h with 10 µM 3-MB-PP1, a bulky adenine analog that inhibits Cdk7 as with an IC 50 of ∼1 nM (Supplemental Fig. 3B), suggesting that the direct contribution of Cdk7 to Xrn2- Thr439 phosphorylation in vivo, if any, is minor. "
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    • ") (Figure S2E), indicating on-target effects of THZ1 on CDK7. CDK7 also stimulates cell-cycle progression by activating CDK1 and CDK2 through its T-loop phosphorylation function (Larochelle et al., 2007). We observed a time-dependent Figure 2. THZ1 Inhibits General Transcription and Cell-Cycle Regulation in MYCN-Amplified Tumor Models (A) Immunoblot analysis of RNA Pol II CTD phosphorylation in MYCN-amplified and nonamplified NB cells treated with DMSO or THZ1 at the indicated concentrations for the indicated times. "
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    • ") (Figure S2E), indicating on-target effects of THZ1 on CDK7. CDK7 also stimulates cell-cycle progression by activating CDK1 and CDK2 through its T-loop phosphorylation function (Larochelle et al., 2007). We observed a time-dependent Figure 2. THZ1 Inhibits General Transcription and Cell-Cycle Regulation in MYCN-Amplified Tumor Models (A) Immunoblot analysis of RNA Pol II CTD phosphorylation in MYCN-amplified and nonamplified NB cells treated with DMSO or THZ1 at the indicated concentrations for the indicated times. "
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