Analysis of Cyclin D3-cdk4 Complexes in Fibroblasts Expressing and Lacking p27^kip1 and p21^cip1

Abstract

Our studies examined the effects of p27 kip1 and p21 cip1 on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27 kip1 -p21 cip1-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27 kip1 and/or p21 cip1 removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27 kip1 interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27 kip1 -p21cip1 -deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27 kip1 or p21 cip1 or were monomeric. We conclude that neither p27 kip1 nor p21 cip1 is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27 kip1 or p21 cip1 are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27 kip1 and p21 cip1.Cell cycle progression is regulated by an ordered sequence of events that includes the activation of the cyclin-dependent kinases (cdk's) (37, 43). Activation of the cdk's requires both their association with cyclins, whose levels fluctuate during the cell cycle, and their phosphorylation at specific threonine residues by cdk-activating kinase, a constitutively expressed enzyme (46). cdk's also interact with a group of proteins collectively termed cdk inhibitors (CKIs); CKI levels, like cyclin levels, vary during the cell cycle and thus contribute to the timing of cdk activation (44,45). Traverse of G 0 /G 1 and entry into S phase is controlled by the sequential activation of complexes containing the D cyclins and cdk4 or cdk6, cyclin E and cdk2, and cyclin A and cdk2. This series of events is initiated by mitogen-induced increases in the expression of the D cyclins (D1, D2, and D3) and the formation of active cyclin D-cdk4 (or cdk6) complexes in mid-G 1 (30,50). D cyclin-containing complexes phosphorylate the antioncogene Rb, as do cyclin E-cdk2 complexes, which become active in late G 1 due, at least in part, to decreases in CKI levels (16,20,22,29,49). When phosphorylated by these kinases, Rb no longer represses the activity of the E2F transcription factors, and a variety of E2F target genes, including those encoding cyclin E, cyclin A, and several DNA replication enzymes, are expressed (6,13,48). At this point, cells pass through the restriction point in late G 1 and, in a manner dependent on cyclin E-cdk2 and cyclin A-cdk2 activity, enter and traverse S phase (36).Two classes of CKIs have been defined: the INK pro...

Activation of cyclin D1-kinase in murine fibroblasts lacking both p21Cip1 and p27Kip1

“…Therefore, it is likely that p21 Cip1 and p27 Kip1 are implicated in assembly factor(s) of cyclin D-CDK4/6 complexes. However, the idea that p21 Cip1 /p27 Kip1 are essential activators of cyclin D-kinases conflicts with recent observations made by Bagui et al (2000). Although Cheng et al (1999) were able to remove virtually all of the CDK4 kinase activity from the extracts of wild type MEFs by immunodepletion of p21/p27-complexes, Bagui et al (2000) failed to reproduce these results.…”

“…However, the idea that p21 Cip1 /p27 Kip1 are essential activators of cyclin D-kinases conflicts with recent observations made by Bagui et al (2000). Although Cheng et al (1999) were able to remove virtually all of the CDK4 kinase activity from the extracts of wild type MEFs by immunodepletion of p21/p27-complexes, Bagui et al (2000) failed to reproduce these results. Moreover, Bagui et al (2000) showed the presence of cyclin D3-CDK4 kinase activity in p21/p27-null MEFs.…”

“…Although Cheng et al (1999) were able to remove virtually all of the CDK4 kinase activity from the extracts of wild type MEFs by immunodepletion of p21/p27-complexes, Bagui et al (2000) failed to reproduce these results. Moreover, Bagui et al (2000) showed the presence of cyclin D3-CDK4 kinase activity in p21/p27-null MEFs. Therefore, it is vital to resolve these paradoxes regarding the complexity of regulation of cyclin D-kinase activity.…”

Induction of p21 by p53 following DNA damage inhibits both Cdk4 and Cdk2 activities

“…To account for previously reported preferential binding of p21 to the Cdk2 complex upon UV irradiation of mammalian fibroblast cells , it is possible that majority of the pre-existing Cdk4 complex has already been associated with and inactivated by p27 (Polyak et al, 1994;Toyoshima and Hunter, 1994;Bagui et al, 2000;Bagui et al, 2003) and thus does not need p21 binding for inactivation. In this context, it is noteworthy that DAP treatment of A2780 cells induces a great increase in the amount of the Cdk4 and Cdk2 complexes (Figure 2), making the pre-existing p27-containing Cdk4 complex quantitatively less significant in the final population of the inhibited Cdk4 complex.…”

Loss of the cell cycle inhibitors p21Cip1 and p27Kip1 enhances tumorigenesis in knockout mouse models

“…CDK activation requires cyclin association, and cyclin-CDK activity is negatively regulated by a group of proteins termed CDK inhibitors (CKIs). The CKI family includes p21 Cip1 and p27 Kip1 , both of which interact with and inhibit the activity of complexes containing the D cyclins and cdk4 or cdk6, cyclin E and cdk2, and cyclin A and cdk2 (Bagui et al, 2000;Sherr and Roberts, 1995). When active, these complexes implement G0/G1 progression and S phase entry.…”