Cytoplasmic Localization of Human cdc25C during Interphase Requires an Intact 14-3-3 Binding Site

cdc25C induces mitosis by activating the cdc2-cyclin B complex. The intracellular localization of cyclin B1 is regulated in a cell cycle-specific manner, and its entry into the nucleus may be required for the initiation of mitosis. To determine the cellular localization of cdc25C, monoclonal antibodies specific for cdc25C were developed and used to demonstrate that in human cells, cdc25C is retained in the cytoplasm during interphase. A deletion analysis identified a 58-amino-acid region (amino acids 201 to 258) in cdc25C that was required for the cytoplasmic localization of cdc25C. This region contained a specific binding site for 14-3-3 proteins, and mutations in cdc25C that disrupted 14-3-3 binding also disrupted the cytoplasmic localization of cdc25C during interphase. cdc25C proteins that do not contain a binding site for 14-3-3 proteins showed a pancellular localization and an increased ability to induce premature chromosome condensation. The cytoplasmic localization of cdc25C was not altered by γ irradiation or treatment with the nuclear export inhibitor leptomycin B. These results suggest that 14-3-3 proteins may negatively regulate cdc25C function by sequestering cdc25C in the cytoplasm.

cdc25C通过激活cdc2-细胞周期蛋白B复合物（cdc2-cyclin B complex）诱导有丝分裂。细胞周期蛋白B1（cyclin B1）的细胞内定位受细胞周期特异性调控，其向细胞核内转运可能是有丝分裂起始的必要条件。为明确cdc25C的细胞定位，研究者制备了针对cdc25C的单克隆抗体（monoclonal antibodies），并通过实验证实：在人类细胞中，cdc25C在间期（interphase）全程滞留于细胞质内。通过缺失分析（deletion analysis），研究者鉴定出cdc25C中一段含58个氨基酸残基的区域（氨基酸201至258位），该区域是cdc25C实现细胞质定位的必需元件。此区域包含14-3-3蛋白（14-3-3 proteins）的特异性结合位点；若cdc25C发生破坏14-3-3结合能力的突变，则同样会导致间期cdc25C的细胞质定位异常。不携带14-3-3蛋白结合位点的cdc25C蛋白，会呈现全细胞分布特征，且诱导早熟染色体凝缩的能力显著增强。γ射线照射（γ irradiation）或核输出抑制剂 leptomycin B处理，均不会改变cdc25C的细胞质定位模式。上述结果表明，14-3-3蛋白可能通过将cdc25C隔离于细胞质中，负向调控cdc25C的生物学功能。