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Regulation of the activity of protein kinases depending on their functional domains

根据蛋白激酶的功能域调节其活性

基本信息

批准号：
07557200
负责人：
KIKKAWA Ushio
金额：
$ 0.9万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

RAC-protein kinase (RAC-PK) and protein kinase C (PKC) have a catalytic domain highly homologous each other in the carboxyl-terminal regions, and the regulatory domain at their amino-terminal regions. RAC-PK and PKC have a PH domain and a Cl region, respectively, in the regulatory domain, and PKC has been shown to associate with PKC through the PH domain. We examined the regulation of these protein kinases through the regulatory domain in this study. In the initial study, the screening of the cDNA library was carried out and a novel clone of RAC-PK gamma was isolated in addition to the previously known RAC-PK alpha and beta. The PH domain of three types of RAC-PK associated with PKC subspecies in vitro. Analysis using deletion mutants revealed that the specific region for the binding with the PH domain was hardly identified in the PKC molecule, suggesting that the whole structure of PKC is important for the interaction with the PH domain. During the analysis of the measurement of RAC-PK activity expressed in cultured cell lines, it was revealed that RAC-PK is activated by cellular stress such as heat shock and hyperosmotic treatment. On the other hand, RAC-PK has been reported to be a downstream target of phosphatidylinositol 3-kinase, that is activated by the growth factor-stimulation. Activation of RAC-PK by cellular stress was, however, not suppressed by wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase. It is plausible that RAC-PK is activated through alternative pathways. Furthermore, the association of RAC-PK and PKC was enhanced by stress treatment. The mutual interaction between RAC-PK and PKC,probably through the protein-protein interaction, may have an important role in the intracellular signal transduction.

RAC-蛋白激酶（RAC-PK）和蛋白激酶C（PKC）在羧基端区域具有彼此高度同源的催化结构域，并且在它们的氨基端区域具有调节结构域。RAC-PK和PKC在调节结构域中分别具有PH结构域和Cl区域，并且已显示PKC通过PH结构域与PKC缔合。在这项研究中，我们研究了这些蛋白激酶通过调节域的调节。在最初的研究中，进行了cDNA文库的筛选，并且除了先前已知的RAC-PK α和β之外，还分离了RAC-PK γ的新克隆。三种RAC-PK的PH结构域在体外与PKC亚型相关。使用缺失突变体的分析表明，与PH结构域的结合的特定区域几乎没有被确定在PKC分子中，这表明PKC的整个结构是重要的与PH结构域的相互作用。在对培养的细胞系中表达的RAC-PK活性的测量进行分析的过程中，揭示了RAC-PK被细胞应激如热休克和高渗处理激活。另一方面，据报道RAC-PK是磷脂酰肌醇3-激酶的下游靶标，其被生长因子刺激激活。然而，激活RAC-PK细胞应激，不抑制渥曼青霉素，磷脂酰肌醇3-激酶的有效抑制剂。RAC-PK可能通过替代途径激活。此外，应激处理增强了RAC-PK和PKC的关联。RAC-PK与PKC之间可能通过蛋白质-蛋白质相互作用，在细胞内信号转导中发挥重要作用。