We investigated the status and the regulation of the cyclin-dependent kinases (CDK) inhibitor p27Kip1 in a choroidal melanoma tumor-derived cell line (OCM-1). By contrast to normal choroidal melanocytes, the expression level of p27Kip1 was low in these cells and the mitogen-activated protein (MAP) kinase pathway was constitutively activated. Genetic or chemical inhibition of this pathway induced p27Kip1 accumulation, whereas MAP kinase reactivation triggered a down-regulation of p27Kip1 that could be partially reversed by calpain inhibitors. In good accordance, ectopic expression of the cellular calpain inhibitor calpastatin led to an increase of endogenous p27 Kip1 expression. In vitro, p27 Kip1 was degraded by calpains, and OCM-1 cell extracts contained a calcium-dependent p27Kip1 degradation activity. MAP kinase inhibition partially inhibited both calpain activity and calcium-dependent p27 Kip1 degradation by cellular extracts. Immunofluorescence labeling and subcellular fractionation revealed that p27 Kip1 was in part localized in the cytoplasmic compartment of OCM-1 cells but not of melanocytes, and accumulated into the nucleus upon MAP kinase inhibition. MAP kinase activation triggered a cytoplasmic translocation of the protein, as well as a change in its phosphorylation status. This CRM-1-dependent cytoplasmic translocation was necessary for MAP kinase-and calpain-dependent degradation. Taken together, these data suggest that in tumor-derived cells, p27Kip1 could be degraded by calpains through a MAP kinase-dependent process, and that abnormal cytoplasmic localization of the protein, probably linked to modifications of its phosphorylation state, could be involved in this alternative mechanism of degradation.Progression through the cell cycle involves the sequential activation and inhibition of cyclin-dependent kinases (CDKs).

The p42/p44 mitogen-activated protein (MAP) kinase is stimulated by various mitogenic stimuli, and its sustained activation is necessary for cell cycle G 1 progression and G 1 /S transition. G 1 progression and G 1 /S transition also depend on sequential cyclin-dependent kinase ( Kip1 expression in fibroblasts essentially through a degradation mechanism, independently of p27 Kip1 phosphorylation by CDK2. This strengthens the role of this CDK inhibitor as a key effector of G 1 growth arrest, whose expression can be controlled by extracellular stimuli-dependent signaling pathways.Progression through the cell cycle involves the sequential activation and inhibition of cyclin-dependent kinases (CDKs).

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