CYCLIC AMP-DEPENDENT PROTEIN-KINASE CONTROLS BASAL GENE ACTIVITY AND STEROIDOGENESIS IN Y1 ADRENAL-TUMOR CELLS
CYCLIC AMP-DEPENDENT PROTEIN-KINASE CONTROLS BASAL GENE ACTIVITY AND STEROIDOGENESIS IN Y1 ADRENAL-TUMOR CELLS
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DOI：
10.1021/bi00129a023
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发表时间：
1992-04-14
期刊：
影响因子：
2.9
通讯作者：
MCKNIGHT, GS
中科院分区：
文献类型：
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作者：
CLEGG, CH;ABRAHAMSEN, MS;MCKNIGHT, GS
用编码突变型I型调节亚基的DNA转染小鼠Y1肾上腺肿瘤细胞产生稳定的转化体，其中cAMP依赖性蛋白激酶的基础活性被抑制。正如预期的那样，类固醇激素在这些激酶缺陷的细胞不再刺激促肾上腺皮质激素或cAMP类似物，和三个cAMP调节基因（鸟氨酸脱羧酶，尿激酶型纤溶酶原激活剂，和P450侧链裂解）的表达不能再诱导。然而，除了激素反应性的损失，类固醇的基础水平和这些cAMP诱导基因的组成型表达也被抑制在激酶缺陷型突变克隆。为了验证功能性cA-PK是否会逆转这种受抑制的表型，我们用编码cAMP依赖性蛋白激酶的C-α和C-β亚基的DNA转染了Y1细胞的cA-PK缺陷亚克隆Kin 8。在稳定的转化体中，类固醇产生的基础水平恢复到正常，并且在诱导C亚基表达载体后激酶活性的升高引起类固醇生成反应。基因转录也被证明是由C-α或C-β调节，如通过纤溶酶原激活剂和鸟氨酸脱羧酶mRNA水平和转录速率的诱导所测量的。cAMP依赖性蛋白激酶在这些肾上腺细胞中所起的主导作用通过显示蛋白激酶C对鸟氨酸脱羧酶基因表达的调节需要基础cAMP依赖性蛋白激酶活性的实验来证明。
Transfection of mouse Y1 adrenal tumor cells with DNA encoding mutant type I regulatory subunit generated stable transformants in which the basal activity of cAMP-dependent protein kinase was repressed. As expected, steroidogenesis in these kinase-deficient cells was no longer stimulated by corticotropin or cAMP analogues, and the expression of three cAMP-regulated genes (ornithine decarboxylase, urokinase-type plasminogen activator, and P450 side-chain cleavage) could no longer be induced. However, in addition to the loss of hormone responsiveness, the basal level of steroidogenesis and the constitutive expression of these cAMP-inducible genes was also repressed in kinase-defective mutant clones. To verify that functional cA-PK would revert this repressed phenotype, we transfected a cA-PK defective subclone of Y1 cells, Kin 8, with DNA encoding the C-alpha and C-beta subunits of cAMP-dependent protein kinase. Basal levels of steroid production were restored to normal in stable transformants, and the elevation of kinase activity following induction of the C-subunit expression vectors elicited a steroidogenic response. Gene transcription was also shown to be regulated by either C-alpha or C-beta as measured by the induction of plasminogen activator and ornithine decarboxylase mRNA levels and transcription rates. The dominant role played by cAMP-dependent protein kinase in these adrenal cells was demonstrated by experiments showing the regulation of ornithine decarboxylase gene expression by protein kinase C requires basal cAMP-dependent protein kinase activity.