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目的探讨钙调素-43(CaM)在生长相关蛋白(-43)(GAP-43)调控细胞周期过程中的作用。方法利用反转录病毒体系构建表达不同活性状态的GAP-43(即野生型、非磷酸化型和假磷酸化型GAP-43)NIH3T3细胞模型,并分别命名为表达野生型GAP-43(3T3-G)、表达非磷酸化GAP-43(3T3-A)和表达假磷酸化GAP-43(3T3-D);另设对照组(转染空载体NIH3T3细胞)。通过免疫组织化学及Western blotting鉴定各型细胞模型转入GAP-43的情况;应用免疫共沉淀检测各型GAP-43与CaM的功能关系;应用细胞生长曲线、BrdU累积标记法、BrdU脉冲标记法测定各型转GAP-43细胞的细胞周期。结果免疫组织化学和Western blotting结果显示,NIH3T3细胞能够表达转入的各型GAP-43。其中,3T3-A细胞增殖速度较其他细胞减缓。免疫共沉淀显示,3T3-A中有GAP-43和CaM相互作用表现;3T3-G中GAP-43和CaM有少量相互作用;3T3-D与3T3-P中则相互作用。累积BrdU测定,脉冲BrdU测定M期的实验表明,3T3-P细胞无明显周期改变;3T3-D与3T3-G细胞的周期延长;3T3-A细胞的周期明显延长并显示G1期明显延长。结论表达GAP-43的细胞其增殖表现可能是由于GAP-43结合了CaM,使之与Ca2+的结合减少而引起细胞周期(尤其是G1期)的延长。
Objective To investigate the role of calmodulin-43 (CaM) in the regulation of cell cycle by the growth associated protein (-43) (GAP-43). Methods The NIH3T3 cell model of GAP-43 (wild-type, non-phosphorylated and pseudo- phosphorylated GAP-43) expressing different active states was constructed by retroviral system and named respectively as wild type GAP-43 -G), the expression of non-phosphorylated GAP-43 (3T3-A) and the expression of pseudophosphorylated GAP-43 (3T3-D); another control group (transfected with empty vector NIH3T3 cells). The expression of GAP-43 in GAP-43 cells was detected by immunohistochemistry and Western blotting. The function of GAP-43 and CaM was detected by co-immunoprecipitation. The cell growth curve, BrdU accumulation labeling and BrdU pulse labeling The cell cycle of each type of GAP-43 cells was determined. Results Immunohistochemistry and Western blotting showed that NIH3T3 cells could express various types of GAP-43. Among them, 3T3-A cell proliferation slower than other cells. Co-immunoprecipitation showed that there was interaction between GAP-43 and CaM in 3T3-A, small amount of GAP-43 and CaM in 3T3-G, and interaction between 3T3-D and 3T3-P. Accumulation of BrdU assay and measurement of M phase by pulsed BrdU showed that there was no obvious periodic change in 3T3-P cells; 3T3-D and 3T3-G cells prolonged; 3T3-A cells significantly prolonged and G1 phase significantly prolonged. Conclusion The proliferation of GAP-43-expressing cells may be due to GAP-43 binding to CaM, resulting in a decrease in the binding to Ca2 + resulting in an increase in cell cycle (especially G1 phase).