MAP kinase-dependent degradation of p27Kip1 by calpains in choroidal melanoma cells -: Requirement of p27Kip1 nuclear export
MAP kinase-dependent degradation of p27Kip1 by calpains in choroidal melanoma cells -: Requirement of p27Kip1 nuclear export
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DOI：
10.1074/jbc.m209523200
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发表时间：
2003-04-04
影响因子：
4.8
通讯作者：
Manenti, S
中科院分区：
文献类型：
--
作者：
Delmas, C;Aragou, N;Manenti, S
我们研究了周期蛋白依赖性激酶（CDK）抑制剂p27（Kip1）在脉络膜黑色素瘤肿瘤来源细胞系（OCM-1）中的状态和调控。与正常脉络膜黑色素细胞相比，p27（Kip1）在这些细胞中的表达水平较低，丝裂原活化蛋白（MAP）激酶途径被组成性激活。该途径的遗传或化学抑制诱导p27（Kip1）积累，而MAP激酶再激活引发p27（Kip1）的下调，可被calpain抑制剂部分逆转。同样，细胞calpain抑制剂calpastatin的异位表达导致内源性p27（Kip1）表达增加。在体外，钙蛋白酶可降解p27(Kip1)， OCM-1细胞提取物具有钙依赖性的p27（Kip1）降解活性。MAP激酶抑制部分抑制calpain活性和细胞提取物对钙依赖性p27（Kip1）的降解。免疫荧光标记和亚细胞分离显示p27（Kip1）部分定位于OCM-1细胞的细胞质室，而不是黑素细胞，并在MAP激酶抑制下积聚到细胞核中。MAP激酶激活触发了该蛋白的细胞质易位，以及其磷酸化状态的变化。这种依赖于crm -1的细胞质易位是MAP激酶和calpain依赖性降解所必需的。综上所述，这些数据表明，在肿瘤来源的细胞中，p27（Kip1）可以通过MAP激酶依赖的过程被钙蛋白酶降解，并且该蛋白的异常胞质定位可能与其磷酸化状态的修饰有关，可能参与了这种降解的替代机制。
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. Immunofluoreseence 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.