S and G₂ Phase Roles for Cdk2 Revealed by Inducible Expression of a Dominant-Negative Mutant in Human Cells

Abstract

Cyclin-dependent kinase 2 (Cdk2) is essential for initiation of DNA synthesis in higher eukaryotes. Biochemical studies in Xenopus egg extracts and microinjection studies in human cells have suggested an additional function for Cdk2 in activation of Cdk1 and entry into mitosis. To further examine the role of Cdk2 in human cells, we generated stable clones with inducible expression of wild-type and dominant-negative forms of the enzyme (Cdk2-wt and Cdk2-dn, respectively). Both exogenous proteins associated efficiently with endogenous cyclins. Cdk2-wt had no apparent effect on the cell division cycle, whereas Cdk2-dn inhibited progression through several distinct stages. Cdk2-dn induction could arrest cells at the G 1 /S transition, as previously observed in transient expression studies. However, under normal culture conditions, Cdk2-dn induction primarily arrested cells with S and G 2 /M DNA contents. Several observations suggested that the latter cells were in G 2 phase, prior to the onset of mitosis: these cells contained uncondensed chromosomes, low levels of cyclin B-associated kinase activity, and high levels of tyrosine-phosphorylated Cdk1. Furthermore, Cdk2-dn did not delay progression through mitosis upon release of cells from a nocodazole block. Although the G 2 arrest imposed by Cdk2-dn was similar to that imposed by the DNA damage checkpoint, the former was distinguished by its resistance to caffeine. These findings provide evidence for essential functions of Cdk2 during S and G 2 phases of the mammalian cell cycle.

Cyclin A/cdk2 coordinates centrosomal and nuclear mitotic events

“…A number of laboratories have provided evidence that G 2 phase cyclin A/cdk2 has a critical role in the timing of entry into mitosis (Furuno et al, 1999;Goldstone et al, 2001;Hu et al, 2001;Mitra and Enders, 2004;Fung et al, 2007;Gong et al, 2007), which is confirmed by our siRNA and cdk2-specific inhibitor studies. In agreement with these reports, the present study demonstrates that inhibition of G 2 phase cyclin A/cdk2 delays the cdc25-dependent activation (Mitra and Enders, 2004;Fung et al, 2007), and nuclear translocation (Gong et al, 2007) of cyclin B/cdk1.…”

“…The previous studies examining the mechanism by which depletion of G 2 phase cyclin A/cdk2 regulates progression into mitosis have provided evidence for deregulation of Wee1 activity (Fung et al, 2007) and cytoplasmic retention of cyclin B1 (Gong et al, 2007). The mechanism appears independent of ataxia telangiectasia mutated/ATM and Rad3 related (ATM/ATR) checkpoint signalling (Hu et al, 2001;Fung et al, 2007). The mechanism by which cyclin A/cdk2 coordinates activation of the centrosomal and soluble pools is also unclear.…”

“…However, other studies have suggested a function for cyclin A/cdk2 in G 2 phase progression. Microinjection of cyclin A antibodies into tissue culture cells delayed entry into mitosis (Clarke et al, 1992), and induction of a dominant-negative cdk2 resulted in G 2 phase delay (Hu et al, 2001). Furthermore, cells from a Drosophila mutant lacking cyclin A arrest in G 2 after the maternal cyclin A has been exhausted (Knoblich and Lehner, 1993), indicating the importance of the cyclin A/cdk complex.…”

Platonin inhibited PDGF-BB-induced proliferation of rat vascular smooth muscle cells via JNK1/2-dependent signaling

“…These results indicate that JNK1/2-dependent signals may play important roles in the platonin-mediated inhibition of VSMC proliferation. Furthermore, JNK, c-Jun, and p27 activation have also been reported to regulate S and G 2 /M phases of cell-cycle progression [28][29][30][31] . These studies are consistent with our finding that the platonin treatment arrested cell cycle progression in the S and G 2 /M phases, which may have resulted from the suppression of JNK1/2-dependent signals.…”

Review of the Molecular Pathogenesis of Osteosarcoma

“…In the human OSA cell line U-2OS, combined depletion of CDK1 and CDK2 but not anyone alone by RNA silencing has been proved to arrest cells in G2-M phase and to induce apoptosis (Hu et al, 2001;Cai et al, 2006;Wei et al, 2011).…”