Interacting proteins of protein kinase A regulatory subunit in Saccharomyces cereuisiae
Interacting proteins of protein kinase A regulatory subunit in Saccharomyces cereuisiae
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DOI：
10.1016/j.jprot.2014.07.008
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发表时间：
2014-09-23
影响因子：
3.3
通讯作者：
Rossi, S.
中科院分区：
文献类型：
--
作者：
Galello, F.;Moreno, S.;Rossi, S.
cAMP依赖性蛋白激酶在真核生物中介导许多细胞外信号。PICA的区室化是控制cAMP介导的信号转导特异性的重要水平。与哺乳动物PICA不同，已经获得了通过锚定蛋白（AKAP）控制其定位的机制的证据见解，在酿酒酵母PICA的情况下，几乎没有可用的信息。在这项工作中，我们目前的结果表明，酵母PICA调节亚基（Bcy 1）相关蛋白的分离和鉴定使用MS为基础的蛋白质组学分析和生物信息学方法。通过免疫沉淀、下拉和亚细胞分级共定位来评估这些相互作用的验证。证明了在所鉴定的蛋白质的相互作用结构域中存在的带正电荷的残基的关键作用。因此，定义的相互作用结构域具有与常规AKAP结构域不同的分子特征。最后，我们评估初步的实验，以可视化的生理相关性的相互作用，Bcy 1的Bcy 2和Hsp 60。Bcy 1与PICA结合，将PICA固定在Ras复合物上，Hsp 60分子伴侣将PICA定位在线粒体上，并对激酶的稳定性起作用。生物学意义本工作对信号转导领域，特别是蛋白激酶A的研究具有重要意义。cAMP信号级联的组分通过名为AKAP的支架蛋白定位在细胞中，AKAP有助于cAMP-PKA信号通路的高水平特异性调节。在单细胞真核生物酿酒酵母中，PICA在细胞中具有多效性作用，因此区室化是实现反应特异性的关键。目前，所有AKAP都已在哺乳动物中被描述，但尚不清楚哺乳动物AKAP的功能同源物是否存在于酵母中。因此，目前还不清楚哺乳动物锚定蛋白的哪些分子特征是通用的，哪些是独特的。通过蛋白质组学和生物信息学的方法，我们鉴定并表征了酿酒酵母中蛋白激酶A调节亚基的相互作用蛋白。Bcy 1连接蛋白具有一个结构域，其中带正电荷的残基是与PICA调节亚基相互作用的关键，Bcy 1 N端在相互作用中起重要作用。在哺乳动物AKAP中，两亲性α-螺旋的疏水氨基酸面对于结合相互作用的高亲和力是必需的。在这项工作中获得的结果似乎表明，在相互作用的Bcy 1蛋白中鉴定的结构域具有不同于哺乳动物AKAPs-R相互作用所定义的相互作用的结构性质。带正电荷的残基不仅作为独特的分子决定簇参与其中，而且它们所处的螺旋的疏水面与Bcy 1的相互作用无关。尽管通常使用非常好的表征模型对于回答问题是必不可少的，在哺乳动物AKAP的情况下，其他替代模型的研究有助于建立更普遍的概念。(C)2014爱思唯尔有限公司版权所有。
cAMP-dependent protein kinase mediates many extracellular signals in eukaryotes. The compartmentalization of PICA is an important level of control of the specificity of signal transduction mediated by cAMP. Unlike mammalian PICA for which proof insights in the mechanism that controls its localization through anchoring proteins (AKAPs) has been obtained, in the case of Saccharomyces cerevisiae PICA there was little information available. In this work, we present results that demonstrate the isolation and identification of yeast PICA regulatory subunit (Bcy1) associated proteins using a MS-based proteomic analysis and a bioinformatic approach. The verification of some of these interactions was assessed by immunoprecipitation, pull down and co-localization by subcellular fractionation. The key role of positively charged residues present in the interaction domain of the identified proteins was demonstrated. The defined interaction domain has therefore different molecular characteristics than conventional AKAP domains. Finally we assess initial experiments to visualize the physiological relevance of the interaction of both Ira2 and Hsp60 with Bcy1. Bcy1 interacts with Ira2 tethering PICA to the Ras complex and Hsp60 chaperone localizes PICA to mitochondria and has a role in the kinase stability.Biological significanceOur work has an important impact in the field of signal transduction especially of protein kinase A. Components of the cAMP signaling cascade are localized in the cell via scaffold proteins named AKAPs that contribute to the high level specific regulation of the cAMP-PKA-signaling pathway. In the unicellular eukaryote Saccharomyces cereuisiae PICA has a pleiotropic role in the cell and the compartmentalization therefore is key to achieve the specificity in the response. At present all AKAPs have been described in mammals and it is unknown whether functional homologs of mammalian AKAPs exist in yeast. Therefore, it is unknown which molecular features of the mammalian anchoring proteins are general and which are distinctive. We have identified and characterized interacting proteins of protein kinase A regulatory subunit in Saccharomyces cereuisiae, through a proteomic and bioinformatic approach. Bcy1 tethering proteins have a domain in which charged positives residues are key for the interaction with regulatory subunit of PICA and Bcy1 N-terminus is important in the interaction. In mammalian AKAPs a hydrophobic amino acid face of an amphipathic a-helix is essential for the high affinity of the binding interaction. The results obtained in this work seem to indicate that the domains identified in the interacting Bcy1 proteins have a structural nature of the interaction different than those defined for mammalian AKAPs-R interaction. Not only positive charged residues are involved as distinctive molecular determinants but also the hydrophobic face of the helix in which they are included was not relevant in the interaction with Bcy1.Even though generally the use of very well characterized models is essential to answer questions, as would be in this case AKAPs from mammals, the study of other alternative models contributes to the building of more universal concepts. (C) 2014 Elsevier B.V. All rights reserved.