SH_2A基因对细胞信号转导的影响及其亚细胞定位

Effect ofSH_2 A gene in cell signal transduction and its subcellular locolization

目的 研究 Src同源域 2 (src homology2 ,SH2 ) A基因在细胞信号转导中的作用并进行亚细胞定位。方法 通过 RT- PCR方法扩增 SH2 A c DNA编码序列 ,构建真核重组表达载体 pc DNA3.1-SH2 A,利用脂质体转染肝癌 Bel74 0 2细胞、COS7细胞 ,检测蛋白酶 C(protein kinase C,PKC)、酪氨酸蛋白激酶 (tyrosine protein kinase,TPK)、丝裂原激活蛋白激酶 (mitogen activated protein kinase,MAPK)活性的改变 ;另构建 p EGEP- SH2 A,转染同前 ,荧光显微镜观察荧光定位。结果 测序结果显示真核重组表达载体 pc DNA3.1- SH2 A及 p EGFP- SH2 A中均含有 SH2 Ac DNA编码序列 ;肝癌 Bel74 0 2细胞、COS7细胞转染pc DNA3.1- SH2 A后 ,胞浆 PKC的活性下降了 4 0 %左右 ,MAPK和 TPK活性未见明显改变。荧光显微镜观察发现 SH2 A基因在细胞质中表达。结论 SH2 A基因编码蛋白在 PKC信号转导通路中起抑制作用 ;SH2 A基因编码蛋白定位于细胞质。

Objective To examine the effect of SH 2 A gene in cell signal transduction and its subcellular localization.Methods RT-PCR method was used to amplify the coding squence of SH 2A gene. Eukaryotic recombined expression vector pcDNA 3.1-SH 2A was constructed, and then Bel7402 cell and COS7 cell transfected by liposome. Multiple kinase assay was performed to examine the activity of protein kinase(PKC), mitogen activated protein kinase(MAPK), tyrosine protein kinase(TPK) in the transfected cells. Meantime, pEGFP-SH 2A vector was also constructed and the cells transfected with it were examined by fluorescent microscopy.Results Recombined expression vector pcDNA3.2-SH 2A and pEGFP-SH 2A contained the coding sequence ofSH 2 A cDNA. In both cell lines expressingSH 2 A gene, the cytoplasm PKC activity decreased by 40% or so, but no apparent alteration was found in MAPK and TPK activity. SH 2A gene was found localized in the cytoplasm of transfected cells under fluorescent microscope.Conclusion SH 2 A gene may act as an inhibiting factor in PKC signal transduction, and it is localized in cytoplasm.