MAP kinase - dependent degradation of p 27 Kip 1 by calpains in choroidal melanoma cells : requirement of p 27

We investigated the status and the regulation of the CDK inhibitor p27 in a choroidal melanoma tumour derived cell line (OCM-1). By contrast to normal choroidal melanocytes, the expression level of p27 was low in these cells and the MAP kinase pathway was constitutively activated. Genetic or chemical inhibition of this pathway induced p27 accumulation, while MAP kinase reactivation triggered a down-regulation of p27 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 expression. In vitro, p27 was degraded by calpains, and OCM-1 cell extracts contained a calciumdependent p27 degradation activity . MAP kinase inhibition partially inhibited both calpain activity and calcium-dependent p27 degradation by cellular extracts. Immunofluorescence labelling and subcellular fractionation revealed that p27 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-1dependent cytoplasmic translocation was necessary for MAP kinaseand calpain-dependent degradation. Taken together, these data suggest that in tumour derived cells, p27 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 into this alternative mechanism of degradation. by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from