Okano H, Cyert M S, Ohya Y
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
J Biol Chem. 1998 Oct 9;273(41):26375-82. doi: 10.1074/jbc.273.41.26375.
Recent genetic studies of yeast calmodulin (yCaM) have shown that alterations of different sets of Phe residues result in distinct functional defects (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). To examine the importance of Phe residues for target binding and activation, we purified mutant yCaMs containing single or double Phe to Ala substitutions and determined their ability to bind and activate two target proteins, calcineurin and CaM-dependent protein kinase (CaMK). Binding assays using the gel overlay technique and quantitative analyses using surface plasmon resonance measurements indicated that the binding of yCaM to calcineurin is impaired by either double mutations of F16A/F19A or a single mutation of F140A, while binding to CaMK is impaired by F89A, F92A, or F140A. These same mutant yCaMs fail to activate calcineurin and CaMK, respectively, in vitro. In addition, F19A exhibited a severe defect in activation of both enzymes. F12A activated calcineurin to only 50% of the level achieved by wild-type calmodulin but fully activated CaMK. These results suggest that each target protein requires a specific and distinct subset of Phe residues in yCaM for target binding and activation.
最近对酵母钙调蛋白（yCaM）的遗传学研究表明，不同组苯丙氨酸（Phe）残基的改变会导致不同的功能缺陷（Ohya, Y., and Botstein, D. (1994) Science 263, 963 - 966）。为了研究Phe残基对靶标结合和激活的重要性，我们纯化了含有单个或双个Phe到Ala取代的突变型yCaM，并测定了它们结合和激活两种靶蛋白——钙调神经磷酸酶和钙调蛋白依赖性蛋白激酶（CaMK）的能力。使用凝胶覆盖技术的结合测定以及使用表面等离子体共振测量的定量分析表明，F16A/F19A的双突变或F140A的单突变会损害yCaM与钙调神经磷酸酶的结合，而F89A、F92A或F140A会损害与CaMK的结合。这些相同的突变型yCaM在体外分别无法激活钙调神经磷酸酶和CaMK。此外，F19A在激活这两种酶方面表现出严重缺陷。F12A激活钙调神经磷酸酶的水平仅为野生型钙调蛋白的50%，但能完全激活CaMK。这些结果表明，每种靶蛋白在yCaM中需要特定且不同的Phe残基子集来进行靶标结合和激活。
