Howard P, Day K H, Kim K E, Richardson J, Thomas J, Abraham I, Fleischmann R D, Gottesman M M, Maurer R A
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
J Biol Chem. 1991 Jun 5;266(16):10189-95.
The mechanisms responsible for decreased levels of cAMP-dependent protein kinase activity in a mutant Chinese hamster ovary cell line have been examined. The cAMP-resistant Chinese hamster ovary 10260 cell line was found to possess only 20% of the cAMP-dependent protein kinase activity found in wild-type cells. The presence of decreased concentrations of the catalytic subunit in these cells was confirmed through binding studies using a radiolabeled, heat-stable inhibitor of the kinase. Cloned Chinese hamster ovary catalytic subunit cDNAs were isolated, characterized, and used as hybridization probes to examine the relative concentrations of catalytic subunit mRNAs in the wild-type and 10260 cell lines. A 40-50% decrease in the concentration of the mRNA for the C alpha isozyme of the catalytic subunit was observed in 10260 cells, as compared with wild-type. This decrease in catalytic subunit mRNA concentration probably accounts for a portion of the decreased kinase activity in the mutant cells. Further analysis of C alpha mRNA by polymerase chain reaction confirmed the decreased expression of C alpha mRNA in 10260 cells and further demonstrated the presence of two different species of C alpha mRNA in the 10260 cells. One species of C alpha cDNAs was indistinguishable from the wild-type cDNA, but the other species was shorter. Nucleotide sequence analysis of the amplified cDNAs led to the identification of a 191-base pair deletion in the shorter cDNA. Gene transfer studies using wild-type and 10260 C alpha cDNAs demonstrated that the longer cDNA from the 10260 cells produced wild-type activity, but the shorter cDNA was inactive. These studies suggest that at least two alterations in gene expression are responsible for decreased cAMP-dependent protein kinase activity in the 10260 cell line. One alteration results in an approximately 2-fold decrease in the concentrations of C alpha mRNA in the cells. The other change produces two species of C alpha mRNA; one of the C alpha mRNAs does not encode an active kinase.
对一种突变的中国仓鼠卵巢细胞系中cAMP依赖性蛋白激酶活性水平降低的机制进行了研究。发现对cAMP有抗性的中国仓鼠卵巢10260细胞系的cAMP依赖性蛋白激酶活性仅为野生型细胞的20%。通过使用激酶的放射性标记热稳定抑制剂进行结合研究，证实了这些细胞中催化亚基浓度降低。分离、鉴定了克隆的中国仓鼠卵巢催化亚基cDNA，并将其用作杂交探针，以检测野生型和10260细胞系中催化亚基mRNA的相对浓度。与野生型相比，在10260细胞中观察到催化亚基Cα同工酶的mRNA浓度降低了40 - 50%。催化亚基mRNA浓度的这种降低可能是突变细胞中激酶活性降低的部分原因。通过聚合酶链反应对Cα mRNA的进一步分析证实了10260细胞中Cα mRNA表达降低，并进一步证明了10260细胞中存在两种不同的Cα mRNA。一种Cα cDNA与野生型cDNA无法区分，但另一种较短。对扩增的cDNA进行核苷酸序列分析，发现在较短的cDNA中有一个191个碱基对的缺失。使用野生型和10260 Cα cDNA进行的基因转移研究表明，来自10260细胞的较长cDNA产生野生型活性，但较短的cDNA无活性。这些研究表明，基因表达的至少两种改变导致了10260细胞系中cAMP依赖性蛋白激酶活性降低。一种改变导致细胞中Cα mRNA浓度降低约2倍。另一个变化产生了两种Cα mRNA；其中一种Cα mRNA不编码活性激酶。
