Yu J S, Yang S D
Institute of Biomedical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
J Neurochem. 1993 May;60(5):1714-21. doi: 10.1111/j.1471-4159.1993.tb13395.x.
Mg-ATP-dependent protein phosphatase activating factor [kinase FA/glycogen synthase kinase 3 (GSK-3)] has been identified in highly purified clathrin-coated vesicles (CCVs) isolated from pig brain. Kinase FA was found to exist in an inactive state but can be activated by 1% Triton X-100 or 1 M Tris-HCl extraction in brain CCVs. Activation of kinase FA in CCVs is due to disassociation of the kinase from CCVs as demonstrated on sucrose density-gradient ultracentrifugation and Sepharose CL-4B gel filtration. Using purified brain CCVs as substrates, kinase FA enhanced the endogenous phosphorylation of assembly protein complexes in the molecular weight range of 100,000-130,000 severalfold, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Comparisons with well-defined brain CCV-associated endogenous protein kinases such as pp50 kinase/AP50 and casein kinase 2 provide evidence that kinase FA/GSK-3 represents a third potent and unique CCV-associated protein kinase distinctly different from the previously described CCV protein kinases, suggesting the possible involvement of kinase FA in the regulation of CCV functions in the brain. The results also support the notion that protein kinase FA is involved in cell surface signal transduction in the CNS.
镁 - 三磷酸腺苷（Mg-ATP）依赖性蛋白磷酸酶激活因子[激酶FA/糖原合酶激酶3（GSK-3）]已在从猪脑中分离出的高度纯化的网格蛋白包被小泡（CCV）中被鉴定出来。发现激酶FA以无活性状态存在，但在脑CCV中可被1% Triton X-100或1 M Tris-HCl提取激活。CCV中激酶FA的激活是由于激酶与CCV解离，这在蔗糖密度梯度超速离心和琼脂糖CL-4B凝胶过滤实验中得到证实。以纯化的脑CCV为底物，激酶FA使分子量在100,000 - 130,000范围内的装配蛋白复合物的内源性磷酸化增强了数倍，这通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳随后进行放射自显影得以证明。与明确定义的脑CCV相关内源性蛋白激酶如pp50激酶/AP50和酪蛋白激酶2进行比较，结果表明激酶FA/GSK-3代表了第三种强效且独特的CCV相关蛋白激酶，与先前描述的CCV蛋白激酶明显不同，这表明激酶FA可能参与调节脑中CCV的功能。这些结果也支持了蛋白激酶FA参与中枢神经系统细胞表面信号转导的观点。
