Domain shuffling as a tool for investigation of protein function: substitution of the cysteine-rich region of Raf kinase and PKC eta for that of yeast Pkc1p.
Domain shuffling as a tool for investigation of protein function: substitution of the cysteine-rich region of Raf kinase and PKC eta for that of yeast Pkc1p.
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结构域改组作为研究蛋白质功能的工具：用 Raf 激酶和 PKC eta 的富含半胱氨酸区域替换酵母 Pkc1p 的半胱氨酸区域。
DOI：
10.1006/jmbi.2001.4848
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发表时间：
2001
影响因子：
5.6
通讯作者：
Jürgen J. Heinisch
中科院分区：
文献类型：
--
作者：
H. Schmitz;J. Jöckel;Christoph Block;Jürgen J. Heinisch
随着整个基因组序列的完成，对蛋白质及其结构域的功能表征的需求变得迫切。蛋白质中的保守区域通常具有重叠的功能，但尽管如此，在不同的物种中可能会完成完全不同的任务。本研究以酵母蛋白激酶C（Pkc 1 p）的富含半胱氨酸基序（C1结构域）为模型，建立了一个结构域功能检测系统。C1结构域通过结合二酰基甘油和/或磷脂酰丝氨酸（如蛋白激酶C（PKC）家族的许多成员）或通过结合小GTP酶（如Raf激酶）激活激酶。与蛋白激酶C超家族的其他成员相比，酿酒酵母的Pkc 1 p通过小G蛋白Rho 1 p与其C1结构域的结合而被激活。我们开发了一个系统的结构域改组，以建立在酵母中的人Raf激酶和大鼠PKC eta的C1结构域的功能。只有来自Raf激酶的C1结构域使嵌合酶能够在取代天然酵母结构域时结合Rho 1 p。因此，嵌合Pkc 1 p携带的C1从Raf激酶，但不是从PKC eta，能够部分地补充酵母pkc 1缺失突变体的表型。我们解释这些数据作为进一步的证据表明，与一个小的GTdR的相互作用是在酵母中的C1结构域的主要调控功能。
With the completion of the sequences of entire genomes, the need for functional characterisation of proteins and their domains is becoming acute. Conserved regions within proteins often share overlapping functions but despite this conservation may fulfil quite different tasks in different species. In this work, we investigated the cysteine-rich motif (C1 domain) of yeast protein kinase C (Pkc1p) as a model to establish a test system for domain function. C1 domains activate kinases through binding of either diacylglycerol and/or phosphatidylserine, as in many members of the protein kinase C (PKC) family, or by binding small GTPases, as in Raf kinase. In contrast to other members of the protein kinase C superfamily, Pkc1p of Saccharomyces cerevisiae is activated via binding of the small G-protein Rho1p to its C1 domain. We developed a system for domain shuffling to establish the function of C1 domains from human Raf kinase and rat PKC eta in yeast. Only the C1 domain from Raf kinase enabled the chimeric enzyme to bind Rho1p when substituted for the native yeast domain. Accordingly, a chimeric Pkc1p carrying the C1 from Raf kinase, but not that from PKC eta, was able to partially complement the phenotypes of a yeast pkc1 deletion mutant. We interpret these data as further evidence that interaction with a small GTPase is the main regulatory function of the C1 domain in yeast.
酵母蛋白激酶 C 与寡糖基转移酶的几个亚基相互作用的证据。
影响因子：
4.3
作者：
Park,H;Lennarz,WJ
通讯作者：
Lennarz,WJ
影响因子：
8
作者：
Kuniyoshi Iwabuchi;Bin Li;P. Bartel;S. Fields
通讯作者：
Kuniyoshi Iwabuchi;Bin Li;P. Bartel;S. Fields