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.

The generation of neurons by progenitor cells involves the tight coordination of multiple cellular activities, including cell cycle exit, initiation of neuronal differentiation, and cell migration. The mechanisms that integrate these different events into a coherent developmental program are not well understood. Here we show that the cyclin-dependent kinase inhibitor p27^Kip1 plays an important role in neurogenesis in the mouse cerebral cortex by promoting the differentiation and radial migration of cortical projection neurons. Importantly, these two functions of p27^Kip1 involve distinct activities, which are independent of its role in cell cycle regulation. p27^Kip1 promotes neuronal differentiation by stabilizing Neurogenin2 protein, an activity carried by the N-terminal half of the protein. p27^Kip1 promotes neuronal migration by blocking RhoA signaling, an activity that resides in its C-terminal half. Thus, p27^Kip1 plays a key role in cortical development, acting as a modular protein that independently regulates and couples multiple cellular pathways contributing to neurogenesis.

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.

The cell cycle inhibitor p27^Kip1also 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 thecdkn1bgene product prevent its interaction with cyclins and CDKs (p27CK⁻). In striking contrast to complete deletion of thecdkn1bgene, 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, p27^Kip1promoted 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 thecdkn1bgene 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.