Liu Y C, Storm D R
Department of Pharmacology, University of Washington School of Medicine, Seattle 98195.
J Biol Chem. 1989 Aug 5;264(22):12800-4.
Neuromodulin (p57, GAP-43, F1, B-50) is a major neural-specific, calmodulin binding protein found in brain, spinal cord, and retina that is associated with membranes. Phosphorylation of neuromodulin by protein kinase C causes a significant reduction in its affinity for calmodulin (Alexander, K. A., Cimler, B. M., Meirer, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). It has been proposed that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons and that activation of protein kinase C causes the release of free calmodulin at high concentrations near its target proteins. It was the goal of this study to determine whether bovine brain contains a phosphoprotein phosphatase that will utilize phosphoneuromodulin as a substrate. Phosphatase activity for phosphoneuromodulin was partially purified from a bovine brain extract using DEAE-Sephacel and Sephacryl S-200 gel filtration chromatography. The neuromodulin phosphatase activity was resolved into two peaks by Affi-Gel Blue chromatography. One of these phosphatases, which represented approximately 60% of the total neuromodulin phosphatase activity, was tentatively identified as calcineurin by its requirement for Ca2+ and calmodulin (CaM) and inhibition of its activity by chlorpromazine. Therefore, bovine brain calcineurin was purified to homogeneity and examined for its phosphatase activity against bovine phosphoneuromodulin. Calcineurin rapidly dephosphorylated phosphoneuromodulin in the presence of micromolar Ca2+ and 3 microM CaM. The apparent Km and Vmax for the dephosphorylation of neuromodulin, measured in the presence of micromolar Ca2+ and 2 microM CaM, were 2.5 microM and 70 nmol Pi/mg/min, respectively, compared to a Km and Vmax of 4 microM and 55 nmol Pi/mg/min, respectively, for myosin light chain under the same conditions. Dephosphorylation of neuromodulin by calcineurin was stimulated 50-fold by calmodulin in the presence of micromolar free Ca2+. Half-maximal stimulation was observed at a calmodulin concentration of 0.5 microM. We propose that phosphoneuromodulin may be a physiologically important substrate for calcineurin and that calcineurin and protein kinase C may regulate the levels of free calmodulin available in neurons.
神经调节蛋白（p57、GAP - 43、F1、B - 50）是一种主要存在于脑、脊髓和视网膜中的神经特异性钙调蛋白结合蛋白，与细胞膜相关。蛋白激酶C对神经调节蛋白的磷酸化作用会使其对钙调蛋白的亲和力显著降低（亚历山大，K. A.，西姆勒，B. M.，迈勒，K. E.，和斯托姆，D. R.（1987年）《生物化学杂志》262，6108 - 6113）。有人提出，神经调节蛋白可能起到在神经元内特定部位结合并浓缩钙调蛋白的作用，并且蛋白激酶C的激活会导致在其靶蛋白附近释放高浓度的游离钙调蛋白。本研究的目的是确定牛脑中是否含有一种能将磷酸化神经调节蛋白作为底物的磷酸蛋白磷酸酶。利用DEAE - 葡聚糖凝胶和Sephacryl S - 200凝胶过滤色谱法从牛脑提取物中部分纯化了针对磷酸化神经调节蛋白的磷酸酶活性。通过Affi - Gel Blue色谱法将神经调节蛋白磷酸酶活性分离为两个峰。其中一种磷酸酶，约占总神经调节蛋白磷酸酶活性的60%，根据其对Ca2 +和钙调蛋白（CaM）的需求以及氯丙嗪对其活性的抑制作用，初步鉴定为钙调神经磷酸酶。因此，将牛脑钙调神经磷酸酶纯化至同质，并检测其对牛磷酸化神经调节蛋白的磷酸酶活性。在微摩尔浓度的Ca2 +和3微摩尔的CaM存在下，钙调神经磷酸酶能迅速使磷酸化神经调节蛋白去磷酸化。在微摩尔浓度的Ca2 +和2微摩尔的CaM存在下测量神经调节蛋白去磷酸化的表观Km和Vmax分别为2.5微摩尔和70纳摩尔无机磷/毫克/分钟，而在相同条件下肌球蛋白轻链的Km和Vmax分别为4微摩尔和55纳摩尔无机磷/毫克/分钟。在微摩尔浓度的游离Ca2 +存在下，钙调蛋白能将钙调神经磷酸酶对神经调节蛋白的去磷酸化作用刺激50倍。在钙调蛋白浓度为0.5微摩尔时观察到半最大刺激。我们提出，磷酸化神经调节蛋白可能是钙调神经磷酸酶在生理上的重要底物，并且钙调神经磷酸酶和蛋白激酶C可能调节神经元中游离钙调蛋白的水平。
