Studies on a protein-serine/threonine protein kinase family having a SH2 domain

RAC-蛋白激酶在其氨基和羧基末端分别有一个调节域和一个蛋白-丝氨酸/苏氨酸激酶催化结构域。被认为在其氨基末端含有SH2结构域的调节域，被发现具有Pleckstrin同源(PH)结构域。从大鼠基因文库中克隆了三种类型的RAC蛋白激酶，分别命名为α、β和γ，Northern印迹分析表明，α和β分布广泛，而γ在脑和睾丸中大量表达。生化研究表明，以髓鞘碱性蛋白为磷酸盐受体时，从COS-7细胞中纯化的RAC-蛋白激酶α、β和γ的比活性相似。谷胱甘肽S转移酶的融合蛋白分析表明，这三种类型的Rac-蛋白激酶的PH域与蛋白激酶C亚种相关联。利用缺失突变体进行的结合研究表明，蛋白激酶C与PH域氨基末端的β-折叠结构相互作用。已报道的与β-肾上腺素能受体激酶PH域相关的G蛋白的β-amma亚基与三个RAC-PKs的PH域结合。用共表达这两种蛋白激酶的细胞提取物进行免疫沉淀分析表明，RAC-蛋白激酶和蛋白激酶C在细胞内相互作用。这些结果表明，Rac蛋白激酶和蛋白激酶C之间通过PH结构域的蛋白-蛋白质相互作用可能对这些蛋白激酶的调控起重要作用，Rac蛋白激酶家族的PH结构域通过不同的方式结合多个蛋白质来调节该酶家族的活性和/或细胞内的分布。

RAC-protein kinase has a regulatory domain and a protein-serine/threonine kinase catalytic domain at its amino-and carboxyl-terminal regions, respectively. The regulatory domain, that had been regarded to contain a SH2 domain at its amino-terminal end, was revealed to have a pleckstrin homology (PH) domain. Three types of rat RAC-protein kinase, designated alpha, beta, and gamma, were cloned from rat cDNA libraries, and Northern blot analysis showed that alpha and beta are distributed widely, whereas gamma was expressed abundantly in brain and testis. Biochemical studies indicated that the specific activities of RAC-protein kinase alpha, beta, and gamma purified from transfected COS-7 cells were similar when measured by using myelin basic protein as a phosphate acceptor. Analysis using fusion proteins of glutathione S-transferase revealed that the PH domain of the three types of RAC-protein kinase associates with protein kinase C subspecies. Binding studies using deletion mutants indicated that protein kinase C interacts with the beta-sheet structure in the amino-terminal portion of the PH domain. The betagamma subunits of G proteins, that had been reported to associate with the PH domain of beta-adreenergic receptor kinase, were shown to bind to the PH domain of three RAC-PKs. Immunoprecipitation assay using cell extracts co-expressing these two protein kinases indicated that RAC-protein kinase and protein kinase C associate in cells. These results suggest that the protein-protein interaction between RAC protein kinase and protein kinase C through the PH domain may be important for the regulation of these protein kinases, and the PH domain of RAC protein kinase family associates more than one protein to regulate the activity and/or intracellular distribution of this enzyme family by different ways.