Abstract
The cyclin-dependent kinase (CDK) inhibitor p21 is induced by the tumor suppressor gene product p53 and is thought to be important for the arrest of the cell cycle following DNA damage. Here we have investigated the contribution of p21 in inhibiting different cyclin-CDK complexes that drive different cell cycle transitions following UV irradiation-induced DNA damage in normal human fibroblasts and immortalized rodent fibroblasts. When cells were exposed to a low dose of UV irradiation, both p53 and p21 were induced; the protein kinase activities associated with Cdc2, Cdk2, and Cdk4 were inhibited; and there was a good correlation between their inhibition and binding to p21. p21 alone is likely to be sufficient for the inhibition of Cdk2 because all the cyclin-complexed forms of Cdk2 were associated with p21 after irradiation. In contrast, only a small proportion of Cdk4 and Cdc2 was complexed with p21, although the level of Cdk4 associated with either p21 or p27 was increased after irradiation. Furthermore, recombinant p21 added to an unirradiated cell lysate at the same level as that induced by irradiation damage inhibited only the kinase activity associated with Cdk2. Cdc2 is likely to be inhibited by Thr-14/Tyr-15 phosphorylation after irradiation because Cdc2 was tyrosine-phosphorylated, and recombinant Cdc25 was able to increase its kinase activity significantly. Taken together, these results suggest that different CDKs are inhibited by different mechanisms following UV-induced DNA damage: Cdk2 is inhibited by the elevated level of p21; Cdk4 is inhibited by cooperation of p21 with other CDK inhibitors, like p27, and possibly by phosphorylation; and Cdc2 is inhibited by Thr-14/Tyr-15 phosphorylation. It is likely that these underlying mechanisms that inactivate CDKs are similar for other kinds of DNA damage.
Highlights
Cyclins and cyclin-dependent kinases (CDKs)1 are key regulators of the eukaryotic cell cycle
Residues ϳ10 – 80 of p21, p27, and p57 are related in sequence, and this region has been identified as the cyclin- and CDK-binding region. p21 can associate with PCNA [35] and inhibit DNA synthesis [21, 36, 37], but not DNA repair in vitro [38, 39]
We focus on the role of p21 in cells arrested at two different levels of DNA damage: at a relatively low dose of UV (30 J/m2), when p21 was induced and the level of cyclins and CDKs remained high (Fig. 1), whereas their activities were inhibited; and at a relatively high dose of UV (60 J/m2), when the level of p21 did not increase
Summary
Cyclins and cyclin-dependent kinases (CDKs) are key regulators of the eukaryotic cell cycle. Thr-161 is phosphorylated by the CDK-activating kinase (reviewed in Ref. 4) and, at least in Cdk, can be dephosphorylated by the CDK-interacting phosphatase KAP [5]. The Wee protein kinase can phosphorylate Tyr-15 of Cdc (reviewed in Ref. 4), and the membrane-associated Wee1-related kinase Myt can phosphorylate both Thr-14 and Tyr-15 [6]. Both Thr-14 and Tyr-15 can be dephosphorylated by the Cdc protein phosphatases (reviewed in Ref. 4). The activity of CDKs is negatively regulated by binding to protein inhibitors; these CDK inhibitors fall into two families based on sequence homology. When cellular DNA is damaged, continued progression through the cell cycle and DNA replication are undesirable
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