Study of multifunctional signalling proteins implicated in cell proliferation

细胞增殖中多功能信号蛋白的研究

基本信息

批准号：
08454241
负责人：
OHYA Yoshikazu
金额：
$ 5.7万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08454241/
关键词：
Calm odulin Rholp yeast cell wall GTPase グルカン細胞周期細胞壁合成形態形成

项目摘要

We study functions of calmodulin and rho-type GTPase. Recent genetic studies of yeast calmodulin (yCaM) have shown that alternations 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). Our results indicated that each target protein requires a specific subset of Phe residues of yCaM for target binding and activation, and that the subsets of Phe residues are required differently among various target proteins. One of four intragenic complementing groups of temperature-sensitive yeast calmodulin mutations, cmd1A, results in a characteristic functional defects in actin organization. We report that among the complementing mutations, a represe … More ntative mutant of cmd1A (cmd1-226 : F92A) is synthetically-lethal with a mutation in MYO2, which encodes a class V unconventional myosin with calmodulin binding domains. Gel-overlay assay shows that a mutant calmodulin with the F92A alteration has severely reduced binding affinity to a GST-Myo2p fusion protein. Random replacement and site-directed mutagenesis at position 92 of calmodulin indicate that hydrophobic and aromatic residues are allowed at this position, suggesting an importance of hydrophobic interaction between calmodulin and Myo2p.1,3-beta-glucan synthase is a multi-enzyme complex that catalyzes the synthesis of 1,3-beta-linked glucan, a major structural component of the yeast cell wall. We found that Rho1 is a regulatory subunit of 1,3-beta-glucan synthase. Temperature-sensitive mutants in the essential Rho-type guanosine triphosphatase, Rho1, displayed thermolabile glucan synthase activity, which was restored by the addition of recombinant Rho1. Glucan synthase from mutants expressing constitutively active Rho1 did not require exogenous guanosine triphosphate for activity. Rho1 copurified with 1,3-beta-glucan synthase and associated with the Fks1 subunit of this complex in vivo. Both proteins were localized predominantly at sites of cell wall remodeling. Less

我们研究钙调蛋白和 rho 型 GTP 酶的功能。最近对酵母钙调蛋白 (yCaM) 的遗传学研究表明，不同 Phe 残基组的交替会导致明显的功能缺陷（Ohya, Y. 和 Botstein, D. (1994) Science）。 263, 963-966）为了检查 Phe 残基对于靶标结合和激活的重要性，我们纯化了含有单 Phe 或双 Phe 至 Ala 的突变 yCaM。我们的结果表明，每种靶蛋白都需要 yCaM 的 Phe 残基的特定子集来进行靶标结合和激活，并且这些子集是结合和激活两种靶蛋白的能力。不同靶蛋白之间需要 Phe 残基。温度敏感酵母钙调蛋白突变的四个基因内互补组之一 cmd1A 会导致肌动蛋白组织中不同的特征性功能缺陷。突变，cmd1A 的典型突变体（cmd1-226：F92A）具有合成致死性，MYO2 中的突变编码具有钙调蛋白结合域的 V 类非常规肌球蛋白，凝胶覆盖分析表明，具有钙调蛋白结合域的突变钙调蛋白。 F92A 改变严重降低了与 GST-Myo2p 融合蛋白的结合亲和力。钙调蛋白的第 92 位表明该位置允许存在疏水性和芳香族残基，这表明钙调蛋白和 Myo2p 之间疏水性相互作用的重要性。1,3-β-葡聚糖合酶是一种多酶复合物，可催化 1,3- 的合成β-连接葡聚糖，酵母细胞壁的主要结构成分我们发现 Rho1 是 1,3-β-葡聚糖的调节亚基。必需的 Rho 型鸟苷三磷酸酶 Rho1 中的温度敏感突变体表现出不耐热的葡聚糖合酶活性，通过添加表达组成型活性 Rho1 的重组葡聚糖合酶即可恢复该活性，不需要外源三磷酸鸟苷即可发挥活性。 Rho1 与 1,3-β-葡聚糖合酶共纯化并与该复合物的 Fks1 亚基在体内主要定位于细胞壁重塑位点。