First identified as cell cycle inhibitors mediating the growth inhibitory cues of upstream signaling pathways, the cyclin-CDK inhibitors of the Cip/Kip family p21Cip1, p27Kip1, and p57Kip2 have emerged as multifaceted proteins with functions beyond cell cycle regulation. In addition to regulating the cell cycle, Cip/Kip proteins play important roles in apoptosis, transcriptional regulation, cell fate determination, cell migration and cytoskeletal dynamics. A complex phosphorylation network modulates Cip/Kip protein functions by altering their subcellular localization, protein-protein interactions, and stability. These functions are essential for the maintenance of normal cell and tissue homeostasis, in processes ranging from embryonic development to tumor suppression.

The tumor suppressor p27Kip1 is an inhibitor of cyclin/cyclin-dependent kinase (CDK) complexes and plays a crucial role in cell cycle regulation. However, p27Kip1 also has cell cycle-independent functions. Indeed, we find that p27Kip1 regulates cell migration, as p27Kip1-null fibroblasts exhibit a dramatic decrease in motility compared with wild-type cells. The regulation of motility by p27Kip1 is independent of its cell-cycle regulatory functions, as re-expression of both wild-type p27Kip1 and a mutant p27Kip1 (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 p27Kip1. 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 p27Kip1 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 p27Kip1 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 p27Kip1 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. p27Kip1 promotes neuronal migration by blocking RhoA signaling, an activity that resides in its C-terminal half. Thus, p27Kip1 plays a key role in cortical development, acting as a modular protein that independently regulates and couples multiple cellular pathways contributing to neurogenesis.[Keywords: Neurogenesis; neurogenin; radial migration; RhoA; electroporation; RNA interference] Supplemental material is available at http://www.genesdev.org.

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.[Keywords: p27 Kip1 ; cyclin; CDK; tumor suppressor; tumorigenesis; Ras; lung cancer] Supplemental material is available at http://www.genesdev.org.

The cell cycle inhibitor p27 Kip1 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 (p27 CK − ). In striking contrast to complete deletion of the cdkn1b gene, which causes spontaneous tumorigenesis only in the pituitary, the p27 CK − 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 Kip1 promoted stem cell expansion and functioned as a dominant oncogene in vivo. Thus, the p27 CK − 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 p27 CK − 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.[Keywords: p27 Kip1 ; lung tumor; oncogene; retina; bronchioalveolar stem cell; desquamative interstitial pneumonitis] Supplemental material is available at http://www.genesdev.org.

Cyclin-dependent kinase inhibitors (CKIs) are well known inhibitors of cell proliferation. Their activity is disrupted in many tumour types. Recent studies show that some of these proteins have interesting alternative functions, acting in the cytoplasm to regulate Rho signalling and thereby controlling cytoskeletal organization and cell migration. The upregulation of CKIs in the cytoplasm of many cancer cells indicates that although loss of nuclear CKIs is important for cancer cell proliferation, gain of cytoplasmic CKI function might be involved in tumour invasion and metastasis.

Integrin affinity is modulated by intracellular signaling cascades, in a process known as "inside-out" signaling, leading to changes in cell adhesion and motility. Protein kinase C (PKC) plays a critical role in integrinmediated events; however, the mechanism that links PKC to integrins remains unclear. Here, we report that PKC⑀ positively regulates integrin-dependent adhesion, spreading, and motility of human glioma cells. PKC⑀ activation was associated with increased focal adhesion and lamellipodia formation as well as clustering of select integrins, and it is required for phorbol 12-myristate 13-acetate-induced adhesion and motility. We provide novel evidence that the scaffolding protein RACK1 mediates the interaction between integrin ␤ chain and activated PKC⑀. Both depletion of RACK1 by antisense strategy and overexpression of a truncated form of RACK1 which lacks the integrin binding region resulted in decreased PKC⑀-induced adhesion and migration, suggesting that RACK1 links PKC⑀ to integrin ␤ chains. Altogether, these results provide a novel mechanistic link between PKC activation and integrin-mediated adhesion and motility.

The migration of cortical interneurons is characterized by extensive morphological changes that result from successive cycles of nucleokinesis and neurite branching. Their molecular bases remain elusive, and the present work describes how p27(Kip1) controls cell-cycle-unrelated signaling pathways to regulate these morphological remodelings. Live imaging reveals that interneurons lacking p27(Kip1) show delayed tangential migration resulting from defects in both nucleokinesis and dynamic branching of the leading process. At the molecular level, p27(Kip1) is a microtubule-associated protein that promotes polymerization of microtubules in extending neurites, thereby contributing to tangential migration. Furthermore, we show that p27(Kip1) controls actomyosin contractions that drive both forward translocation of the nucleus and growth cone splitting. Thus, p27(Kip1) cell-autonomously controls nucleokinesis and neurite branching by regulating both actin and microtubule cytoskeletons.