GPⅢa^T1565C点突变对FAK磷酸化的影响

Effect of Glycoprotein Ⅲa^T1565C Mutation on FAK Phosphorylation

目的建立稳定表达野生型CPⅡ b／Ⅲa和突变型GPⅡb／Ⅲa^T1565C的中国仓鼠卵巢（CHO）细胞系，探讨GPⅡb／Ⅲa^T1565C点突变对黏着斑激酶（FAK）磷酸化的影响。方法采用脂质体介导基因转移法将真核表达载体pcDNA3．1（＋）Ⅱb分别与pcDNA3．i（＋）Ⅲa或pcDNA3．1（＋）Ⅲa^T1565C共转染到CHO细胞中，经C,418筛选出稳定表达野生型GPⅡb／Ⅲa和突变型GPⅡb／Ⅲa^T1565C的CHO细胞系。流式细胞术（Flowcytom—etry，FCM）检测野生型和突变型GPⅡb／ⅢaCHO细胞表面CD41和CD61的表达。免疫沉淀、免疫印迹（Westernblot）和FCM细胞内染色等方法检测野生型和突变型GPⅡb／ⅢaCHO细胞经纤维蛋白原（Fbg）激活后发生的FAK酪氨酸磷酸化和丝氨酸磷酸化。结果野生型和突变型GPⅡb／ⅢaCHO细胞表面CD41和CD61均高效表达，分别为97．19％和99．71％。免疫沉淀、Western blot检测结果显示，野生型和突变型GPⅡb／ⅢaCHO细胞经Fbg激活90min后，分别有16．24％和20．44％的FAK发生酪氨酸磷酸化；FCM细胞内染色检测结果显示，经Fbg激活90min后，野生型和突变型GPⅡb／ⅢaCHO细胞均不发生FAK丝氨酸磷酸化；激活48h后，野生型和突变型GPⅡb／ⅢaCHO细胞分别有34．89％和73．84％发生FAK丝氨酸磷酸化。结论成功构建稳定表达野生型GPⅡb／Ⅲa和突变型GPⅡb／ⅢaaT1565C托的CIIO细胞系。GPIU／ⅢaaT1565C点突变能够增强FAK酪氨酸磷酸化和丝氨酸磷酸化，因此可能增强GPⅡb／Ⅲa介导的细胞外向内信号转导功能。

Objective To study the effect of Glycoprotein Ⅲa^T1565C mutation on the phosphorylation of focal adhesion kinase （FAK）. Methods The recombinants of pcDN＇A3. 1 （ ＋ ） Ⅱ b and pcDNA3. 1 （ ＋ ） Ⅲ a or pcDNA3.1 （ ＋ ） Ⅲa^T1565C were transfected into CHO cells by Lipofectamine 2000. Cell lines expressing wild-type and mu- tational GP Ⅱ b/Ⅲ a were screened by G418. The constructed CHO cell lines were examed through flow cytometry （FCM） to detect the expression of CD41 and CD61. Immunoprecipitation, Western blot and FCM were employed to detect the tyrosine and serine 910 phosphorylation of FAK in CHO cells stimulated by fibrinogen （Fbg）. Results CD41 and CD61 were highly expressed in both CHO cell lines detected by FCM, which was 97.19% and 99.71% , respectively. The tyrosine phosphorylation of FAK was detected in CHO cells expressing wild-type and mutational GP Ⅱ b/Ⅲa stimulated by Fbg for 90min, which was 16.24% and 20.44% , respectively. There was no serine 910 phosphorylation of FAK observed in both CHO cell lines stimulated by Fbg for 90min. However, serine 910 phosphorylation of FAK in the two cell lines was increased after 48h of stimulation to 34.89% and 73.84%, respectively. Conclusions CHO cell lines stably expressing wild-type GP Ⅱ b/Ⅲ a and mutational GP Ⅱ b/Ⅲ aTI565c were construc- ted successfully. GPⅢaT156sc mutation could enhance the serine 910 and tyrosine phosphorylation of FAK. Increased phosphorylation of FAK may enhance GP Ⅱ b/Ⅲ a-mediated outside-in signal transduction.