Mihajlovic V, Krolczyk A J, Auerbach W, Buchwald M, Schimmer B P
Banting and Best Department of Medical Research, University of Toronto, Ont., Canada.
Biochem Cell Biol. 1992 Oct-Nov;70(10-11):1039-46. doi: 10.1139/o92-148.
Caco-2 human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of RI (regulatory subunit of the type 1 cAMP-dependent protein kinase), driven by the metallothionein 1 promoter. A stable transformant was isolated that expressed the mutant RI gene in a Zn(2+)-inducible manner. The consequences of the RI mutation on cAMP-dependent protein kinase activity, cell division, and regulation of chloride efflux were examined. When grown in the absence of ZnSO4, protein kinase activity in the transformant was stimulated 2.5-fold by cAMP and approached the levels of cAMP-dependent protein kinase activity seen in parental Caco-2 cells; when treated with ZnSO4, cAMP-dependent protein kinase activity in the transformant was inhibited by 60%. In the absence of ZnSO4 the transformant grew with the same doubling time and to the same saturation density as the untransformed parent. In the presence of ZnSO4 the transformant exhibited a cAMP-reversible inhibition of cell division, indicating that a functional cAMP-dependent protein kinase was required for the growth of these cells in culture. Induction of the mutant RI gene also abolished forskolin-stimulated chloride efflux from these cells, suggesting obligatory roles for cAMP and cAMP-dependent protein kinase in forskolin's actions on chloride channel activity. We anticipate that this transformant will be useful for further studies on the roles of cAMP and cAMP-dependent protein kinase in the regulation of intestinal epithelial cells, including regulation of cell proliferation and differentiation, and regulation of chloride channel activity by neurohormones and neurotransmitters.
将编码RI（1型环磷酸腺苷依赖性蛋白激酶的调节亚基）突变形式的表达载体，由金属硫蛋白1启动子驱动，转染至Caco-2人结肠癌细胞中。分离出一个稳定的转化体，其以锌离子（Zn²⁺）诱导的方式表达突变的RI基因。研究了RI突变对环磷酸腺苷依赖性蛋白激酶活性、细胞分裂以及氯离子外流调节的影响。在无硫酸锌（ZnSO₄）的情况下培养时，转化体中的蛋白激酶活性被环磷酸腺苷刺激了2.5倍，接近亲本Caco-2细胞中所见的环磷酸腺苷依赖性蛋白激酶活性水平；用硫酸锌处理时，转化体中环磷酸腺苷依赖性蛋白激酶活性被抑制了60%。在无硫酸锌的情况下，转化体的倍增时间和饱和密度与未转化的亲本相同。在有硫酸锌的情况下，转化体表现出对细胞分裂的环磷酸腺苷可逆性抑制，表明功能性环磷酸腺苷依赖性蛋白激酶是这些细胞在培养中生长所必需的。突变RI基因的诱导也消除了毛喉素刺激的这些细胞的氯离子外流，提示环磷酸腺苷和环磷酸腺苷依赖性蛋白激酶在毛喉素对氯离子通道活性的作用中起必不可少的作用。我们预期这个转化体将有助于进一步研究环磷酸腺苷和环磷酸腺苷依赖性蛋白激酶在肠上皮细胞调节中的作用，包括细胞增殖和分化的调节，以及神经激素和神经递质对氯离子通道活性的调节。
