Wong M, Krolczyk A J, Schimmer B P
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Mol Endocrinol. 1992 Oct;6(10):1614-24. doi: 10.1210/mend.6.10.1333050.
The Y1 adrenocortical tumor cell mutants, Kin-7 and Kin-8, harbor point mutations in the regulatory subunit (RI) of the type 1 cAMP-dependent protein kinase (cAMPdPK) that render the enzyme resistant to activation by cAMP. These mutants also are resistant to many of the regulatory effects of ACTH and cAMP. In order to examine the causal relationships between the mutations in cAMPdPK and the resistance to ACTH and cAMP, the Kin mutants were transfected with expression vectors encoding wild type subunits of cAMPdPK in order to restore cAMP-responsive protein kinase activity. The transformants then were screened for the concomitant recovery of cellular responsiveness to ACTH and cAMP. In the mutant Kin-7, cAMP-responsive protein kinase activity was recovered after transfection with an expression vector encoding wild type mouse RI. Protein kinase activity in the mutant Kin-8 remained largely cAMP-resistant after transfection with the RI expression vector but could be rendered cAMP-responsive by transfection with an expression vector encoding the wild type catalytic subunit. The recovery of cAMP-responsive protein kinase activity was accompanied by the recovery of steroidogenic and morphological responses to ACTH and cAMP, suggesting that the cAMP-dependent signaling cascade plays an obligatory role in these actions of ACTH. The growth-regulatory effects of cAMP were not reversed with the recovery of cAMP-responsive protein kinase activity, suggesting that cAMP-resistant growth regulation results from second-site, adaptive mutations either in the original Kin mutant population or in the transformants. Studies on the conversion of 22(R)-hydroxycholesterol into steroid products in parent and mutant cells indicate that the Kin mutations reduce the steroidogenic capacity of the cell as well as inhibit the hormone- and cyclic nucleotide-dependent mobilization of substrate cholesterol.
Y1肾上腺皮质肿瘤细胞突变体Kin-7和Kin-8在1型环磷酸腺苷（cAMP）依赖性蛋白激酶（cAMPdPK）的调节亚基（RI）中存在点突变，使该酶对cAMP激活产生抗性。这些突变体对促肾上腺皮质激素（ACTH）和cAMP的许多调节作用也具有抗性。为了研究cAMPdPK突变与对ACTH和cAMP抗性之间的因果关系，将Kin突变体用编码cAMPdPK野生型亚基的表达载体进行转染，以恢复cAMP反应性蛋白激酶活性。然后筛选转化体，看其对ACTH和cAMP的细胞反应性是否同时恢复。在突变体Kin-7中，用编码野生型小鼠RI的表达载体转染后，恢复了cAMP反应性蛋白激酶活性。用RI表达载体转染后，突变体Kin-8中的蛋白激酶活性在很大程度上仍对cAMP有抗性，但用编码野生型催化亚基的表达载体转染可使其对cAMP产生反应。cAMP反应性蛋白激酶活性的恢复伴随着对ACTH和cAMP的类固醇生成及形态学反应的恢复，这表明cAMP依赖性信号级联在ACTH的这些作用中起必不可少的作用。cAMP的生长调节作用并未随着cAMP反应性蛋白激酶活性的恢复而逆转，这表明cAMP抗性生长调节是由原始Kin突变群体或转化体中的第二位点适应性突变导致的。对亲本细胞和突变体细胞中22(R)-羟基胆固醇转化为类固醇产物的研究表明，Kin突变降低了细胞的类固醇生成能力，并抑制了激素和环核苷酸依赖性的底物胆固醇动员。
