摘要
目的探讨钙(Ca2+)/钙调素依赖蛋白激酶Ⅱ(CaMKⅡ)对Slingshot-1L(SSH-1L)活性的调控作用。方法用脂质体法将含Myc-SSH-1L的重组质粒转染至MG63细胞,经不同浓度的钙离子载体A23187诱导10min,蛋白印迹实验,观察P-cofilin、P-SSH1L、P-CaMKⅡ水平的变化;体内及体外实验检测CaMKⅡ对SSH-1L的磷酸化及活性调控作用。结果当A23187浓度为1μmol/L时CaMKⅡ活性达到了最大,同时此浓度下P-cofilin水平以及SSH-1L的978位点丝氨酸的磷酸化水平升高。体外实验证实CaMKⅡ可使SSH-1L(WT)磷酸化,但是对其突变体SSH-1L(2SA)无作用;同时CaMKⅡ明显抑制了SSH1L(WT)的活性,但对SSH-1L(2SA)的活性无作用。结论CaMKⅡ对SSH-1L的活性具有明显调控作用,且与SSH-1L的丝氨酸位点密切相关。
Objective To investigate the regulation of CaMKⅡ on Slingshot-1L（SSH-1L）activity. Methods The recombinant plasmids containing Myc-SSH-1L was transfected to cell MG63 through liposome method and incubate for ten min via calcium ionophore A23187 of different concentrations;then the changes of P-cofilin,P-SSH-1L and P-CaMK Ⅱ were observed by Western blot,and the phosphorylation and activity regulation of CaMKⅡon SSH-1L were tested by in vivo and in vitro assays.Results When the concentration of A23187 was 1 μmol/L,the activity of CaMK Ⅱ was the highest and the levels of P-cofilin and phosphorylation on Ser 978 of SSH1L were increased too. In vitro assays had proved that CaMK Ⅱ could phosphorylate SSH-1L（WT and NP）,SSH-1L（C）,but had no effect on its mutant SSH-1L （2SA））;otherwise,CaMK Ⅱ inhibited the activity of SSH-1L （WT） obviously,but had no effect on SSH-1L（2SA）. Conclusions CaMKⅡ can regulate the activity of SSH-1L.
出处
《中国老年学杂志》
CAS
CSCD
北大核心
2010年第4期474-476,共3页
Chinese Journal of Gerontology
基金
国家自然科学基金资助项目(No30772488)
