Genetic characterization of the role of the Cip/Kip family of proteins as cyclin-dependent kinase inhibitors and assembly factors

Abstract

The Cip/Kip family, namely, p21(Cip1), p27(Kip1), and p57(Kip2), are stoichiometric cyclin-dependent kinase inhibitors (CKIs). Paradoxically, they have been proposed to also act as positive regulators of Cdk4/6-cyclin D by stabilizing these heterodimers. Loss of p21(Cip1) and p27(Kip1) reduces Cdk4/6-cyclin D complexes, although with limited phenotypic consequences compared to the embryonic lethality of Cdk4/6 or triple cyclin D deficiency. This milder phenotype was attributed to Cdk2 compensatory mechanisms. To address this controversy using a genetic approach, we generated Cdk2(-/-) p21(-/-) p27(-/-) mice. Triple-knockout mouse embryonic fibroblasts (MEFs) displayed minimal levels of D-type cyclins and Cdk4/6-cyclin D complexes. p57(Kip2) downregulation in the absence of p21(Cip1) and p27(Kip1) aggravated this phenotype, yet MEFs lacking all Cip/Kip proteins exhibited increased retinoblastoma phosphorylation, together with enhanced proliferation and transformation capacity. In vivo, Cdk2 ablation induced partial perinatal lethality in p21(-/-) p27(-/-) mice, suggesting partial Cdk2-dependent compensation. However, Cdk2(-/-) p21(-/-) p27(-/-) survivors displayed all phenotypes described for p27(-/-) mice, including organomegalia and pituitary tumors. Thus, Cip/Kip deficiency does not impair interphasic Cdk activity even in the absence of Cdk2, suggesting that their Cdk-cyclin assembly function is dispensable for homeostatic control in most cell types.

FIG 1

Stability of Cdk-cyclin D complexes in the absence of Cip/Kip inhibitors. (a) Early-passage primary MEFs were infected with lentiviral particles expressing either an shRNA against p57^Kip2 (p57) or a scramble control (Ct). Protein extracts were prepared, and the levels of a panel of cell cycle regulators were analyzed by immunoblotting with antibodies elicited against the indicated proteins. Expression of β-actin served as a loading control. Results from two independent MEF cultures are shown for p21^−/− p27^−/− (DKO) and Cdk2^−/− p21^−/− p27^−/− (TKO) mice. Cyc, cyclin. (b) Wild-type, p21^−/− p27^−/− (DKO), and Cdk2^−/− p21^−/− p27^−/− (TKO) primary MEFs were infected with lentiviral particles expressing either an shRNA against p57^Kip2 (p57) or a scramble control (Ct). Exponentially growing cells were used to prepare whole-cell extracts, and the amount of Cdk4-cyclin D complexes was estimated by coprecipitation of cyclin D1 with anti-Cdk4 antibodies. The levels of Cdk4, used as a loading control, are shown. WB, Western blotting. (c) The amount of starting whole-cell extract from both p21^−/− p27^−/− (DKO) and Cdk2^−/− p21^−/− p27^−/− (TKO) MEFs was augmented to equal the cyclin D1 levels in the wild-type controls. The amount of Cdk4 (top) and Cdk6 (middle) that was coimmunoprecipitated with cyclin D1 antibodies was detected by Western blotting. Cyclin D1 levels (bottom) are shown to demonstrate equal estimation of the amount of starting material. Results from two independent MEF cultures are shown. (d) Western blotting of Cdk1 protein levels in whole-cell extracts from wild-type, p21^−/− p27^−/− (DKO), and Cdk2^−/− p21^−/− p27^−/− (TKO) MEFs. Expression of β-actin, which served as a loading control, is shown. Results from two independent MEF cultures are shown. (e) In vitro kinase activity associated with Cdk1 immunoprecipitates obtained from wild-type, p21^−/− p27^−/− (DKO), and Cdk2^−/− p21^−/− p27^−/− (TKO) MEFs. Histone H1 was used as the substrate (S). Results from two independent MEF cultures are shown. Lanes WCE, whole-cell extract at a 1:10 dilution before immunoprecipitation (IP); lanes M, mock immunoprecipitate.

FIG 2

Proliferation of primary MEFs in the absence of Cip/Kip inhibitors. (a) The proliferation of wild-type (Wt), p21^−/− p27^−/− (DKO), and Cdk2^−/− p21^−/− p27^−/− (TKO) primary MEFs in 10% fetal bovine serum (FBS) was assessed upon infection with lentiviral particles expressing an shRNA against p57^Kip2 (sh p57) or a scramble control (sh Ctrl). Data are shown as means ± SDs (n = 3). (b) Same as for panel a, but MEFs were maintained under limiting serum conditions (2% fetal bovine serum). arb., arbitrary.

FIG 3

Role of Cip/Kip proteins in cell cycle reentry. (a) Percentage of wild-type (Wt), p21^−/− p27^−/− (DKO), and Cdk2^−/− p21^−/− p27^−/− (TKO) primary MEFs in S phase at the indicated times following serum stimulation from quiescence. Data are shown as means ± SDs (n = 3). (b) Whole-cell extracts from cells for which the results are shown in panel a were prepared at the times (in hours) following serum stimulation from quiescence indicated above each lane. The presence of cyclin D1 and cyclin A2 and the phosphorylation of the Ser807/811 residues of the retinoblastoma protein (pRb) were detected by Western blotting. Expression of β-actin, which served as a loading control, is shown. (c) p57^Kip2 was depleted in wild-type, p21^−/− p27^−/− (DKO), and Cdk2^−/− p21^−/− p27^−/− (TKO) primary MEFs by lentivirus-mediated shRNA delivery. Infected MEFs were subsequently made quiescent by serum deprivation. The percentage of S-phase cells at the indicated times (in hours) following serum stimulation from quiescence is shown. Data are shown as means ± SDs (n = 3).

FIG 4

Cip/Kip factors and cellular transformation. (a) Equal amounts of wild-type, p21^−/−, p27^−/−, as well as compound mutant p21^−/− p27^−/− (DKO) and Cdk2^−/− p21^−/− p27^−/− (TKO) MEFs were infected with retroviral particles coexpressing H-Ras and E1a oncogenes. (Left) At 3 weeks after infection, the number of detectable foci was scored for the different genotypes. Data are shown as means ± SDs (n = 3). (Right) A representative image upon crystal violet staining is shown. (b) Immortalization of wild-type, p21^−/− and p27^−/− single-knockout, p21^−/− p27^−/− (DKO), and Cdk2^−/− p21^−/− p27^−/− (TKO) primary MEFs following a classical 3T3 protocol. Data are shown as means ± SDs (n = 3).

FIG 5

Influence of p21^Cip1 and p27^Kip1 deficiency in adult homeostasis. (a) Total body weight and the weight of the indicated individual organs of mice at 11 weeks of age. Values for p27^−/−, p21^−/− p27^−/− (DKO), and Cdk2^−/− p21^−/− p27^−/− (TKO) mice normalized to those for wild-type controls of the same age are represented. Data are shown as means ± SDs (n = 4). (b) Representative images of hematoxylin-eosin-stained liver sections from 11-week-old p21^−/− p27^−/− (DKO) and Cdk2^−/− p21^−/− p27^−/− (TKO) mice. Bars, 20 μm. (c) Quantification of the total number of hepatocytes in four randomly selected ×400 microscope fields of liver sections from p21^−/− p27^−/− (DKO) and Cdk2^−/− p21^−/− p27^−/− (TKO) mice. Data are shown as means ± SDs (n = 7).

FIG 6

Cooperation between p21^Cip1 and p27^Kip1 tumor suppressors. Survival curve of p21^−/−, p27^−/−, p21^−/− p27^−/− (DKO), and Cdk2^−/− p21^−/− p27^−/− (TKO) mice.