Seki K, Chen H C, Huang K P
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Arch Biochem Biophys. 1995 Feb 1;316(2):673-9. doi: 10.1006/abbi.1995.1090.
Neurogranin, neuromodulin, and MARCKS are among the most prominent substrates of protein kinase C (PKC) in the mammalian brain. These phosphoproteins were dephosphorylated by three isoforms of rat brain calcineurin, also known as calmodulin (CaM)-dependent protein phosphatase (CaMPP). The three CaMPP isozymes dephosphorylate neurogranin, the most favorable substrate among the three tested, with subtle differences in their responses to divalent metal ions, Mn2+ and Ni2+. Dephosphorylation of neurogranin by all three CaMPP isozymes, CaMPP-1, -2, and -3, were stimulated to a higher extent by Mn2+ than by Ni2+ in the presence of CaM and Ca2+. The Km values of neurogranin in the presence of Mn2+ were lower than those in the presence of Ni2+ for CaMPP-1 and -2, but that for CaMPP-3 was comparable with either divalent metal ion. The Vmax values were higher in the presence of Mn2+ than those of Ni2+ for all three isozymes. Neurogranin and neuromodulin, both phosphorylated by PKC at a single site, were dephosphorylated completely by CaMPP; however, MARCKS, phosphorylated by PKC at three sites, was partially dephosphorylated by this phosphatase. A higher extent of dephosphorylation of MARCKS could be achieved by the combination of CaMPP and protein phosphatase 2A and a complete dephosphorylation of this protein was observed with protein phosphatase 1. Protein phosphatase 1 and 2A were also effective in a complete dephosphorylation of neurogranin and neuromodulin. Amino acid sequence analysis of the tryptic phosphopeptides derived from MARCKS dephosphorylated by CaMPP and protein phosphatase 2A revealed that the former preferentially dephosphorylated Ser155 and the latter Ser162 of rat brain MARCKS. Both phosphatases dephosphorylated poorly of Ser151. Because of the high concentration of CaMPP in the brain and the colocalization of this phosphatase with major PKC substrates in the various brain regions, it is likely that CaMPP is a phosphatase with potential to reverse the action of PKC.
神经颗粒蛋白、神经调节蛋白和丙酰肌醇富含丙氨酸的蛋白激酶C底物（MARCKS）是哺乳动物大脑中蛋白激酶C（PKC）最主要的底物。这些磷蛋白可被大鼠脑钙调神经磷酸酶的三种同工型去磷酸化，钙调神经磷酸酶也被称为钙调蛋白（CaM）依赖性蛋白磷酸酶（CaMPP）。三种CaMPP同工酶可使神经颗粒蛋白去磷酸化，神经颗粒蛋白是三种受试底物中最适宜的底物，它们对二价金属离子锰离子（Mn2+）和镍离子（Ni2+）的反应存在细微差异。在钙调蛋白（CaM）和钙离子（Ca2+）存在的情况下，三种CaMPP同工酶，即CaMPP-1、-2和-3，对神经颗粒蛋白的去磷酸化作用受Mn2+的刺激程度高于Ni2+。对于CaMPP-1和-2，在Mn2+存在下神经颗粒蛋白的米氏常数（Km）值低于Ni2+存在时的Km值，但CaMPP-3的Km值与任何一种二价金属离子存在时的Km值相当。对于所有三种同工酶，在Mn2+存在下的最大反应速度（Vmax）值均高于Ni2+存在时的Vmax值。神经颗粒蛋白和神经调节蛋白均在单个位点被PKC磷酸化，二者均可被CaMPP完全去磷酸化；然而，在三个位点被PKC磷酸化的MARCKS仅被这种磷酸酶部分去磷酸化。CaMPP与蛋白磷酸酶2A联合作用可使MARCKS的去磷酸化程度更高，而蛋白磷酸酶1可使该蛋白完全去磷酸化。蛋白磷酸酶1和2A对神经颗粒蛋白和神经调节蛋白的完全去磷酸化也有效。对经CaMPP和蛋白磷酸酶2A去磷酸化的MARCKS胰蛋白酶磷酸肽进行氨基酸序列分析发现，前者优先使大鼠脑MARCKS的丝氨酸155（Ser155）去磷酸化，后者优先使丝氨酸162（Ser162）去磷酸化。两种磷酸酶对丝氨酸151（Ser151）的去磷酸化作用均较弱。由于脑中CaMPP浓度较高，且该磷酸酶与不同脑区的主要PKC底物共定位，因此CaMPP很可能是一种具有逆转PKC作用潜力的磷酸酶。
