Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit

Abstract

The major events of the cell division cycle are triggered by periodic changes in the activity of cyclin-dependent protein kinases (CDKs). In mammals, the members of the CDK family include CDK2 and CDC2, which are thought to be involved in the control of DNA replication and mitosis, respectively. The protein kinase activity of these enzymes is controlled by a complex array of mechanisms. Activation of the CDK catalytic subunit requires association with a positive regulatory subunit (cyclin) and phosphorylation (at Thr 160 in CDK2). This activated complex can be inhibited by additional phosphorylation at Thr 14 and Tyr 15. Here we report the identification of a new mechanism for the regulation of CDK2 activity. We find that CDK2/cyclin complexes in mouse fibroblasts associate tightly with a 20K protein (CAP20). Complexes containing CAP20 were isolated from cell lysates and found to have negligible kinase activity, indicating that CAP20 association in vivo may inhibit CDK2 activity. We purified CAP20 from 3T3 cells and found that low concentrations of the protein completely inhibit the kinase activity of CDK2 in vitro. Thus CAP20 represents a new negative regulatory subunit that inhibits the activity of CDK2/cyclin complexes in mammalian cells.

Identification of a p130 domain mediating interactions with cyclin A/cdk 2 and cyclin E/cdk 2 complexes

“…Several other cellular proteins which have been shown to stably interact with cyclin/cdk complexes also contain a region that is homologous to the p130 cyclin binding region. Included among these are the highly related p107 protein, the cdk inhibitors p21 cip1 , p27 kip1 , p57 kip2 (ElDeiry et al, 1993;Gu et al, 1993;Harper et al, 1993;Xiong et al, 1993;Polyak et al, 1994;Toyoshima and Hunter, 1994;Lee et al, 1995;Matsuoka et al, 1995) and the transcription factor E2F-1 (Helin et al, 1992;Kaelin et al, 1992;Shan et al, 1992). A similar sequence is found in E2F-2 and -3; however, these proteins have not been demonstrated to interact with the cyclin/cdk complexes (Ivey-Hoyle et al, 1993;Lees et al, 1993).…”

“…Studies on E2F-1 interactions with cyclins indicate that although E2F-1 contains a cyclin binding motif that is similar to p130, it is capable of interacting only with cyclin A and not cyclin E (Dynlacht et al, 1994;Krek et al, 1994;Xu et al, 1994;Kitagawa et al, 1995). Further diversity of cyclin interaction is observed with the cyclin binding regions of p21 cip1 , p27 kip1 and p57 kip2 (El-Deiry et al, 1993;Gu et al, 1993;Harper et al, 1993;Xiong et al, 1993;Polyak et al, 1994;Toyoshima and Hunter, 1994;Lee et al, 1995;Matsuoka et al, 1995). These proteins have a broader speci®city range and can interact with complexes containing cyclins A, B and E and the D cyclins.…”

“…An examination of the sequences of various other cyclin/cdk interacting proteins revealed sequences similar to the p130 RRRLFVE motif in several other proteins including E2F-1, 2 and 3, p21 cip1 , p27 kip1 and p57 kip2 (Helin et al, 1992;Kaelin et al, 1992;Shan et al, 1992;El-Deiry et al, 1993;Gu et al, 1993;Harper et al, 1993;Ivey-Hoyle et al, 1993;Lees et al, 1993;Xiong et al, 1993;Polyak et al, 1994;Toyoshima and Hunter, 1994;Lee et al, 1995;Matsuoka et al, 1995) (Figure 5a). As discussed below, each of these proteins has been shown to be present in complexes containing cyclin A or E. In some cases, this particular region has been implicated in the cyclin interactions.…”

p21WAF1 induces permanent growth arrest and enhances differentiation, but does not alter apoptosis in PC12 cells

“…Additionally, induction may result from exogenous signals, such as inducers of differentiation, leading to the inhibition of proliferation (Sherr and Roberts, 1995;Harper and Elledge, 1996). p21 (known as WAF1, SDI1, MDA-6, CIP1 and CAP20) was the ®rst CDKI isolated, based on interactions with CDK2 (Gu et al, 1993;Harper et al, 1993) and PCNA (Xiong et al, 1992), as well as induction associated with p53 tumor-suppressor expression (El-Deiry et al, 1993), terminal di erentiation of human melanoma cells (Jiang and Fisher, 1993), and senescence of human diploid ®broblasts (Noda et al, 1994). p21 has not only been shown to inhibit the activity of a wide array of CDK-cyclin complexes (Xiong et al, 1993;Harper et al, 1995), but also inhibits DNA replication through its interaction with PCNA (Waga et al, 1994;Flores-Rozas et al, 1994).…”

Expression of a novel form of p21Cip1/Waf1 in UV-irradiated and transformed cells

“…The other CDK inhibitor family includes p21(Cip1/Waf1), p27 Kip1 (Polyak et al, 1994;Toyoshima and Hunter, 1994), and p57 Kip2 (Lee et al, 1995;Matsuoka et al, 1995). The archetypal member of this family, p21, was isolated as a Cdk2-associated protein and an inhibitor of Cdk2 (Gu et al, 1993;Harper et al, 1993;Xiong et al, 1993a), a gene induced by the tumor suppressor p53 (El-Deiry et al, 1993) and a gene whose RNA expression is increased in senescent cells (Noda et al, 1994). In addition to being induced by p53, p21 is also induced by p53-independent pathways (Guo et al, 1995;Halevy et al, 1995;Jiang et al, 1994;Macleod et al, 1995;Michieli et al, 1994;Missero et al, 1995;Parker et al, 1995;Sheikh et al, 1994;Steinman et al, 1994;Zhang et al, 1995).…”