Loss of cyclin-dependent kinase 2 (CDK2) inhibitory phosphorylation in a CDK2AF knock-in mouse causes misregulation of DNA replication and centrosome duplication.

Cyclin-dependent kinase 1 (CDK1) inhibitory phosphorylation controls the onset of mitosis and is essential for the checkpoint pathways that prevent the G(2)- to M-phase transition in cells with unreplicated or damaged DNA. To address whether CDK2 inhibitory phosphorylation plays a similar role in cell cycle regulation and checkpoint responses at the start of the S phase, we constructed a mouse strain in which the two CDK2 inhibitory phosphorylation sites, threonine 14 and tyrosine 15, were changed to alanine and phenylalanine, respectively (CDK2AF). This approach showed that inhibitory phosphorylation of CDK2 had a major role in controlling cyclin E-associated kinase activity and thus both determined the timing of DNA replication in a normal cell cycle and regulated centrosome duplication. Further, DNA damage in G(1) CDK2AF cells did not downregulate cyclin E-CDK2 activity when the CDK inhibitor p21 was also knocked down. We were surprised to find that this was insufficient to cause cells to bypass the checkpoint and enter the S phase. This led to the discovery of two previously unrecognized pathways that control the activity of cyclin A at the G(1) DNA damage checkpoint and may thereby prevent S-phase entry even when cyclin E-CDK2 activity is deregulated.

Discovery of an oncogenic activity in p27Kip1 that causes stem cell expansion and a multiple tumor phenotype.

The cell cycle inhibitor p27Kip1 also has cyclin-cyclin-dependent kinase (CDK)-independent functions. To investigate the significance of these functions in vivo, we generated a knock-in mouse in which four amino acid substitutions in the cdkn1b gene product prevent its interaction with cyclins and CDKs (p27CK-). In striking contrast to complete deletion of the cdkn1b gene, which causes spontaneous tumorigenesis only in the pituitary, the p27CK- protein dominantly caused hyperplastic lesions and tumors in multiple organs, including the lung, retina, pituitary, ovary, adrenals, spleen, and lymphomas. Moreover, the high incidence of spontaneous tumors in the lung and retina was associated with amplification of stem/progenitor cell populations. Therefore, independently of its role as a CDK inhibitor, p27Kip1 promoted stem cell expansion and functioned as a dominant oncogene in vivo. Thus, the p27CK- mouse unveils a dual role for p27 during tumorigenesis: It is a tumor suppressor by virtue of its cyclin-CDK regulatory function, and also an oncogene through a cyclin-CDK-independent function. This may explain why the cdkn1b gene is rarely inactivated in human tumors, and the p27CK- mouse in which the tumor suppressor function is lost but the cyclin-CDK-independent-oncogenic-function is maintained may represent a more faithful model for the widespread role of p27 misregulation in human cancers than the p27 null.

A pathway in quiescent cells that controls p27Kip1 stability, subcellular localization, and tumor suppression.

We have created two knock-in mouse models to study the mechanisms that regulate p27 in normal cells and cause misregulation of p27 in tumors: p27(S10A), in which Ser10 is mutated to Ala; and p27(CK-), in which point mutations abrogate the ability of p27 to bind cyclins and CDKs. These two mutant alleles identify steps in a pathway that controls the proteasomal degradation of p27 uniquely in quiescent cells: Dephosphorylation of p27 on Ser10 inhibits p27 nuclear export and promotes its assembly into cyclin-CDK complexes, which is, in turn, necessary for p27 turnover. We further show that Ras-dependent lung tumorigenesis is associated with increased phosphorylation on Ser10 and cytoplasmic mislocalization of p27. Indeed, we find that p27(S10A) is refractory to Ras-induced cytoplasmic translocation and that p27(S10A) mice are tumor resistant. Thus, phosphorylation of p27 on Ser10 is an important event in the regulation of the tumor suppressor function of p27.

p27Kip1 modulates cell migration through the regulation of RhoA activation.

The tumor suppressor p27(Kip1) is an inhibitor of cyclin/cyclin-dependent kinase (CDK) complexes and plays a crucial role in cell cycle regulation. However, p27(Kip1) also has cell cycle-independent functions. Indeed, we find that p27(Kip1) regulates cell migration, as p27(Kip1)-null fibroblasts exhibit a dramatic decrease in motility compared with wild-type cells. The regulation of motility by p27(Kip1) is independent of its cell-cycle regulatory functions, as re-expression of both wild-type p27(Kip1) and a mutant p27(Kip1) (p27CK(-)) that cannot bind to cyclins and CDKs rescues migration of p27(-/-) cells. p27(-/-) cells have increased numbers of actin stress fibers and focal adhesions. This is reminiscent of cells in which the Rho pathway is activated. Indeed, active RhoA levels were increased in cells lacking p27(Kip1). Moreover, inhibition of ROCK, a downstream effector of Rho, was able to rescue the migration defect of p27(-/-) cells in response to growth factors. Finally, we found that p27(Kip1) binds to RhoA, thereby inhibiting RhoA activation by interfering with the interaction between RhoA and its activators, the guanine-nucleotide exchange factors (GEFs). Together, the data suggest a novel role for p27(Kip1) in regulating cell migration via modulation of the Rho pathway.