蛋白激酶A催化微管相关蛋白tau磷酸化的研究

目的：研究蛋白激酶A（PKA）对微管相关蛋白tau的磷酸化作用。方法：采用免疫印迹技术，利用位点特异性、磷酸化依赖的tau蛋白抗体，检测PKA对tau蛋白磷酸化作用的位点特异性及磷酸化作用动力学。用双倒数作图，计算PKA催化tau磷酸化以及各个位点磷酸化的Km值，并结合培养细胞的实验，研究PKA对tau蛋白磷酸化作用的位点特异性。结果：PKA催化tau蛋白在Ser214，Ser262，Ser409三个位点磷酸化，而且各位点磷酸化作用的Km值不同，PKA对Ser214的Km值最低，即对Ser214位点的亲和性最高，催化其磷酸化的能力最强。在细胞实验中，也显示了PKA引起Ser214位点的磷酸化最明显。结论：PKA催化tau蛋白在Ser214，Ser262，Ser409三个位点磷酸化，其中Ser214是PKA催化tau磷酸化反应的最好位点。

Study on the site-specific phosphorylation of tau by PKA

Objective:To study the site-specific phosphorylation of tau by PKA in vitro and in cultured cells.Methods: Tau_(441) was phosphorylated by PKA in vitro.The phosphorylation sites were detected by Western blots using sitespecific and phsophoarylation-dependent tau antibodies.The site-specific phosphorylation level of tau by PKA was measured by immuno-dot blots using site-specific and phosphorylation dependent tau antibodied.The Km values of PKA toward total tau and toward individual site were calculated by using Lineweaver-Burk double-reciprocal method.The site-specific phosphorylation of tau by PKA was further determined in cultured PC12/tau_(441) cells by activating PKA with bd-cAMP. Results: We found that PKA phosphorylated tau had several sites, including Ser214,Ser262,and Ser409.The Km value of PKA to tau was 30.9 M.The Km value of PKA to different site was different.Among all the phosphorylation sites detected here,Ser214 phosphorylation catalyzed by PKA shown the lowest Km,only 16.4 M.This result suggested that the affinity of PKA to Ser214 of tau was highest and the PKA catalyzed tau phosphorylation most efficiently at Ser214 in vitro.In cultured cells,activated PKA also induced tau phosphorylation most dramatically at Ser214. Conclusion: PKA catalyzed tau phosphorylation at Ser214,Ser262, Ser409 sites with different efficiency.Among them,Ser214 was the most favorite site for PKA.