MAP kinase-dependent degradation of p27Kip1 by calpains in choroidal melanoma cells. Requirement of p27Kip1 nuclear export.
We investigated the status and the regulation of the cyclin-dependent kinases (CDK) inhibitor p27(Kip1) in a choroidal melanoma tumor-derived cell line (OCM-1). By contrast to normal choroidal melanocytes, the expression level of p27(Kip1) was low in these cells and the mitogen-activated protein (MAP) kinase pathway was constitutively activated. Genetic or chemical inhibition of this pathway induced p27(Kip1) accumulation, whereas MAP kinase reactivation triggered a down-regulation of p27(Kip1) 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 p27(Kip1) 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, p27(Kip1) 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.
Delmas
Christelle
C
Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération, CNRS UMR 5088, IFR 109, Université Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Cedex, France.
Aragou
Nathalie
N
Poussard
Sylvie
S
Cottin
Patrick
P
Darbon
Jean-Marie
JM
Manenti
Stéphane
S
D002154:Calpain / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D002830:Choroid Neoplasms; D050760:Cyclin-Dependent Kinase Inhibitor p27; D006801:Humans; D066298:In Vitro Techniques; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008544:Melanocytes / Q000166:cytology / Q000201:enzymology; D008545:Melanoma; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D002154:Calpain