Yamamoto H, Saitoh Y, Fukunaga K, Nishimura H, Miyamoto E
Department of Pharmacology, Kumamoto University Medical School, Japan.
J Neurochem. 1988 May;50(5):1614-23. doi: 10.1111/j.1471-4159.1988.tb03051.x.
Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.
利用环磷酸腺苷依赖性蛋白激酶（环磷酸腺苷激酶）催化亚基磷酸化的组蛋白H1，从牛脑中纯化出蛋白磷酸酶C，纯化倍数为140倍，产率为8%。基于ATP和抑制剂-2的作用，脑蛋白磷酸酶C被认为分别由蛋白磷酸酶1和2A的催化亚基的10%和90%组成。蛋白磷酸酶C使微管相关蛋白2（MAP2）、tau因子和被多功能Ca2+/钙调蛋白依赖性蛋白激酶（钙调蛋白激酶）及环磷酸腺苷激酶催化亚基磷酸化的微管蛋白去磷酸化。比较了MAP2、tau因子和微管蛋白去磷酸化的特性。MAP2和tau因子的米氏常数（Km值）在1.6 - 2.7微摩尔范围内。在1.0毫摩尔锰离子存在的情况下，微管蛋白的Km值从25微摩尔降至10 - 12.5微摩尔。两种激酶磷酸化的底物之间，去磷酸化的动力学特性未观察到差异。蛋白磷酸酶C不能使天然微管蛋白去磷酸化，但能普遍使两种激酶磷酸化的微管蛋白去磷酸化。猪脑蛋白磷酸酶2A的全酶也能使两种激酶磷酸化的MAP2、tau因子和微管蛋白去磷酸化。钙调蛋白激酶分别对MAP2和tau因子的磷酸化诱导了微管组装的抑制，而蛋白磷酸酶C的去磷酸化消除了其抑制作用。这些数据表明，脑蛋白磷酸酶1和2A参与了Ca2+/钙调蛋白依赖性和环磷酸腺苷依赖性微管形成调节的关闭机制。
