Genetic Characterization of the Role of the Cip/Kip Family of Proteins as Cyclin-Dependent Kinase Inhibitors and Assembly Factors

Abstract

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.