Effect of Glycoprotein ⅢaT1565C Mutation on FAK Phosphorylation

Objective To study the effect of Glycoprotein Ⅲ aT1565C mutation on the phosphorylation of focal adhesion kinase (FAK). Methods The recombinants of pcDNA3.1( + ) Ⅱb and pcDNA3.1( + )Ⅲa or pcDNA3.1( + )ⅢaT1565C were transfected into CHO cells by Lipofectamine 2000. Cell lines expressing wild-type and mutational 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. lmmunoprecipitation, 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 Ⅱb/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 GPⅡb/ⅢaT1565C were constructed successfully. GPⅢaT1565C mutation could enhance the serine 910 and tyrosine phosphorylation of FAK. Increased phosphorylation of FAK may enhance GPⅡb/Ⅲ a-mediated outside-in signal transduction.