钙调素蛋白磷酸酶对Slingshot-1L的调控作用

目的 探讨钙调素依赖蛋白激酶(Calcinenurin/PP2B/CnA/Cn)对Slingshot-1L(SSH-1L)活性的调控作用.方法 用脂质体法将含YFP-SSH-1L的重组质粒转染至MG63细胞,经钙离子载体A23187诱导10 min,进行免疫细胞化学染色,观察细胞形态变化;体内及体外实验检测PP3B对SSH-1L的活性调控作用;应用免疫共沉实验检测PP2B与SSH-1L的相互结合情况.结果 Ca~(2+)信号诱导骨肉瘤细胞形成伪叶,同时SSH-1L与F-actin移位共聚集至细胞膜伪叶,但是被PP2B有效抑制剂Cypermethrin完全阻断;在体外,SSH-1L磷酸酶活性测定实验以及SSH-1L与PP2B结合实验证实SSH-1L与PP2B在细胞内是一对相互结合的蛋白质,同时PP2B具有促进SSH-1L酶活性的作用.结论 PP2B通路对细胞伪叶的形成及细胞运动具有重要调控作用,同时对SSH-1L酶具有促进其活性的作用.

Experimental research for the regulation of calcineurin on Slingshot-1L

Objective To explore the regulation of calcineurin(PP2B/CnA/Cn) on Slingshot-1L(SSH-1L)activity. Methods Recombinant plasmids containing YFP-SSH1L were transfected to cell MG63 through liposome method and incubated for 10 min via calcium ionophore A23187. The changes in modality were observed by immunocytochemical staining. The regulation of PP2B on SSH1L activity was tested in vivo and in vitro and the interaction between them was further examined by co-immunoprecipitation. Results Ca~(2+) signal induced osteosarcoma cell to form lamellipodia, where SSH1L and F-actin moved together, but were entirely blocked by cypermethrin as the effective inhibitor. SSH1L and PP2B were conjugated proteins in cells; furthermore, PP2B promoted the enzyme activity of SSH-1L. Conclusions PP2B pathway can regulation cell motility and lamellipodia production, and promote the activity of SSH-1L.