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

RAC-蛋白激酶在其氨基端和羧基端分别具有调节结构域和蛋白丝氨酸/苏氨酸激酶催化结构域。调节结构域，这已被认为是包含在其氨基末端的SH 2结构域，被发现有一个普列克底物蛋白同源（PH）结构域。从大鼠cDNA文库中克隆了三种类型的大鼠RAC蛋白激酶，命名为α、β和γ，并且北方印迹分析显示α和β广泛分布，而γ在脑和睾丸中大量表达。生物化学研究表明，RAC-蛋白激酶α，β，和γ纯化转染COS-7细胞的具体活动是相似的，当使用髓鞘碱性蛋白作为磷酸受体测量。使用谷胱甘肽S-转移酶的融合蛋白的分析表明，PH结构域的三种类型的RAC-蛋白激酶与蛋白激酶C亚种。使用缺失突变体的结合研究表明，蛋白激酶C与PH结构域的氨基末端部分的β-折叠结构相互作用。G蛋白的β-γ亚基与β-肾上腺素能受体激酶的PH结构域结合，并与三种RAC-PK的PH结构域结合。用共表达这两种蛋白激酶的细胞提取物进行的免疫沉淀试验表明，RAC-蛋白激酶和蛋白激酶C在细胞中缔合。这些结果表明RAC蛋白激酶和蛋白激酶C通过PH结构域的蛋白-蛋白相互作用对这些蛋白激酶的调节可能是重要的，RAC蛋白激酶家族的PH结构域通过不同的方式联合多个蛋白来调节该酶家族的活性和/或细胞内分布。

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.