Kaibuchi K, Fukumoto Y, Oku N, Takai Y, Arai K, Muramatsu M
Department of Biochemistry, Kobe University School of Medicine, Japan.
J Biol Chem. 1989 Aug 15;264(23):13489-96.
We have constructed the expression plasmids harboring protein kinase C (PKC) mutant cDNAs with a series of deletions in the PKC coding region. These plasmids were transfected into COS7 cells to characterize the PKC mutants. Immunoblot analysis using the anti-PKC antibody identified proteins with the Mr values expected from the PKC mutant cDNAs in the extracts from COS7 cells. The wild-type PKC, when expressed in COS7 cells, conferred increased phorbol ester binding activity on intact cells; but the PKC mutants with the deletion around the C1 region did not show this activity. The wild-type PKC showed protein kinase activity dependent on phospholipid, Ca2+, and phorbol ester, whereas these PKC mutants exhibited protein kinase activity independent of the activators in a cell-free system. A PKC mutant cDNA with the deletion in the C2 region gave increased phorbol ester binding activity. Protein kinase activity of this mutant was much less dependent on Ca2+ compared with the wild-type PKC. A PKC mutant cDNA with the deletion in the C3 region conferred increased phorbol ester binding activity, but neither activator-dependent nor -independent protein kinase activity. These results indicate that elimination of the C1 region of PKC gives rise to constitutively active PKC independent of phospholipid, Ca2+, and phorbol ester and that the C1-C3 regions play distinct roles in the regulatory and catalytic function of PKC. In another series of experiments, transfection of some PKC mutant cDNAs with the deletions around the C1 region into Chinese hamster ovary and Jurkat cells activated the activator protein-1-binding element or the c-fos gene enhancer linked to the chloramphenicol acetyltransferase reporter gene in the absence of phorbol ester. Microinjection of these constructs into Xenopus oocytes induced initiation of germinal vesicle breakdown, indicating that they stimulated the PKC pathway in vivo. Thus, the phorbol ester-independent PKC mutant cDNAs could be a powerful tool to investigate the transmembrane signaling pathway mediated by PKC.
我们构建了在蛋白激酶C（PKC）编码区带有一系列缺失的PKC突变体cDNA表达质粒。将这些质粒转染到COS7细胞中以鉴定PKC突变体。使用抗PKC抗体进行的免疫印迹分析在COS7细胞提取物中鉴定出了具有PKC突变体cDNA预期Mr值的蛋白质。野生型PKC在COS7细胞中表达时，可使完整细胞上的佛波酯结合活性增加；但在C1区域周围有缺失的PKC突变体未表现出这种活性。野生型PKC表现出依赖磷脂、Ca2+和佛波酯的蛋白激酶活性，而这些PKC突变体在无细胞体系中表现出不依赖激活剂的蛋白激酶活性。在C2区域有缺失的PKC突变体cDNA可使佛波酯结合活性增加。与野生型PKC相比，该突变体的蛋白激酶活性对Ca2+的依赖性要小得多。在C3区域有缺失的PKC突变体cDNA可使佛波酯结合活性增加，但既无依赖激活剂的蛋白激酶活性，也无不依赖激活剂的蛋白激酶活性。这些结果表明，去除PKC的C1区域会产生不依赖磷脂、Ca2+和佛波酯的组成型活性PKC，且C1 - C3区域在PKC的调节和催化功能中发挥着不同的作用。在另一系列实验中，将一些在C1区域周围有缺失的PKC突变体cDNA转染到中国仓鼠卵巢细胞和Jurkat细胞中，在无佛波酯的情况下激活了与氯霉素乙酰转移酶报告基因相连的激活蛋白-1结合元件或c-fos基因增强子。将这些构建体显微注射到非洲爪蟾卵母细胞中可诱导生发泡破裂的起始，表明它们在体内刺激了PKC途径。因此，不依赖佛波酯的PKC突变体cDNA可能是研究由PKC介导的跨膜信号通路的有力工具。
