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

rac -蛋白激酶（RAC-PK）和蛋白激酶C （PKC）在羧基端具有高度同源的催化结构域，在其氨基端具有高度同源的调控结构域。RAC-PK和PKC在调控域中分别有一个PH结构域和一个Cl区域，PKC已被证明通过PH结构域与PKC结合。在本研究中，我们通过调控域研究了这些蛋白激酶的调控。在最初的研究中，我们进行了cDNA文库的筛选，并在之前已知的RAC-PK α和β的基础上，分离到了一个新的RAC-PK γ克隆。三种与PKC亚种相关的RAC-PK的PH结构域。通过缺失突变体的分析发现，PKC分子中几乎没有确定与PH结构域结合的特定区域，这表明PKC的整个结构对于与PH结构域的相互作用很重要。通过对培养细胞系中表达的RAC-PK活性的分析，发现RAC-PK在热休克和高渗处理等细胞应激下被激活。另一方面，据报道，RAC-PK是磷脂酰肌醇3-激酶的下游靶点，受生长因子刺激激活。然而，细胞应激对RAC-PK的激活不受wortmannin（一种有效的磷脂酰肌醇3-激酶抑制剂）的抑制。RAC-PK可能是通过其他途径激活的。此外，应力处理增强了RAC-PK和PKC的关联。RAC-PK和PKC之间的相互作用可能通过蛋白-蛋白相互作用，在细胞内信号转导中起重要作用。

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.