Targeted Disruption of CDK4 Delays Cell Cycle Entry with Enhanced p27^Kip1 Activity

Abstract

The mechanism by which cyclin-dependent kinase 4 (CDK4) regulates cell cycle progression is not entirely clear. Cyclin D/CDK4 appears to initiate phosphorylation of retinoblastoma protein (Rb) leading to inactivation of the S-phase-inhibitory action of Rb. However, cyclin D/CDK4 has been postulated to act in a noncatalytic manner to regulate the cyclin E/CDK2-inhibitory activity of p27Kip1 by sequestration. In this study we investigated the roles of CDK4 in cell cycle regulation by targeted disruption of the mouse CDK4 gene. CDK4؊/؊ mice survived embryogenesis and showed growth retardation and reproductive dysfunction associated with hypoplastic seminiferous tubules in the testis and perturbed corpus luteum formation in the ovary. These phenotypes appear to be opposite to those of p27-deficient mice such as gigantism and gonadal hyperplasia. A majority of CDK4 ؊/؊ mice developed diabetes mellitus by 6 weeks, associated with degeneration of pancreatic islets. Fibroblasts from CDK4 ؊/؊ mouse embryos proliferated similarly to wild-type embryonic fibroblasts under conditions that promote continuous growth. However, quiescent CDK4 ؊/؊ fibroblasts exhibited a substantial (ϳ6-h) delay in S-phase entry after serum stimulation. This cell cycle perturbation by CDK4 disruption was associated with increased binding of p27 to cyclin E/CDK2 and diminished activation of CDK2 accompanied by impaired Rb phosphorylation. Importantly, fibroblasts from CDK4 ؊/؊ p27 ؊/؊ embryos displayed partially restored kinetics of the G 0 -S transition, indicating the significance of the sequestration of p27 by CDK4. These results suggest that at least part of CDK4's participation in the rate-limiting mechanism for the G 0 -S transition consists of controlling p27 activity.In mammalian cells, the balance of growth-stimulatory and -inhibitory signals regulates the transition between proliferation and quiescence (42). Cyclin-dependent kinases (CDKs) activated by the regulatory subunits, cyclins, control cell cycle progression in all eukaryotes (21, 48, 52). Among several cyclin-CDK complexes, cyclin D-and cyclin E-dependent kinases play critical roles in regulating G 1 progression and entry into S phase. D-type cyclins bind to and activate CDK4 during early to mid-G 1 . This is followed by activation of CDK2 in complex with cyclin E during late G 1 . These two types of CDKs seem to collaborate to determine the rate of the G 1 to S transition (1,40,45). After cells enter S phase, cyclin A binds to and activates CDK2, which is required for maintenance of DNA replication (41). Three D-type cyclins (D1, D2, and D3) are expressed in tissue-specific but overlapping manners (32), whereas CDK4 and CDK2 and cyclins E and A are ubiquitously expressed. D-type cyclins also activate CDK6, a kinase closely related to CDK4 (2, 35). Although CDK6 and CDK4 are coexpressed in many cell types, it is unclear whether these two CDKs have completely overlapping functions.Cyclin D-CDK4 plays an important role in inactivating the S-phase-inhibitory action of the retinobla...

Cdk6-Dependent Regulation of G1 Length Controls Adult Neurogenesis

“…In addition, Cdk4 is dispensable for liver regeneration following partial hepatectomy [64], an assay considered to be one of the most stringent to evaluate the proliferative capacity of an adult tissue [65]. Altogether, these data support that Cdk4 is, with the exception of certain cell types [9,19,66,67], overall dispensable for adult tissue homeostasis.…”

“…Interestingly, our results support the idea that the specific involvement of Cdk6 in neural precursors may partially rely on its unique capacity to phosphorylate pRb on Ser780 in these cells. Cdk4 À/À embryonic fibroblasts have been previously shown to display reduced levels of pRbSer780 while expressing Cdk6 [9], suggesting that the main D-type Cdk responsible for phosphorylation of this residue could be cell-type dependent. Alternatively, it might be conceivable that Cdk4/6 activity itself depends on the cell type [72].…”

“…Cdk4 À/À [9] and Cdk6 À/À [10] colonies were maintained on a mixed CD1 Â C57BL/6J Â FVB Â S129/sv and a mixed CD1 Â C57BL/6J background, respectively. Wild type (WT) and knockout mice used in all experiments were obtained from heterozygous breedings and genotype was determined by polymerase chain reaction as described previously.…”

“…As most Cdk4 À/À mice survive until adulthood [9,19] and Cdk6 À/À develop normally [10], we took advantage of these genetic models to examine the contribution of Cdk4/6 to adult NPC proliferation. In agreement with previous studies reporting the dwarfism-like phenotype of Cdk4-deficient mice [9,19], we first observed that Cdk4 À/À brains were smaller and lighter than their WT counterparts at every postnatal stage analyzed, suggesting an important role for Cdk4 during brain development (Supporting Information Fig. 3).…”

Des oncogènes aux régulateurs de la mitose : un changement de perspective dans notre vision des processus cancéreux

“…Ce dernier point est illustré par les expériences d'invalidation des gènes codant pour la cycline D1 ou la kinase Cdk4. Les souris déficientes en cycline D1 ou en Cdk4 viennent à terme, mais avec des problèmes de développement tels qu'une déficience oculaire, une taille réduite et une absence de maturation lobulo-alvéolaire terminale des glandes mammaires [7,8]. Les animaux cycline D1 -/-, chez lesquels la cycline E est réexprimée après insertion de son gène dans le locus cycline D1 (knock-in), ont un phénotype quasi normal.…”