PROTEIN-KINASE-C ACTIVATION POTENTLY DOWN-REGULATES THE EXPRESSION OF ITS MAJOR SUBSTRATE, 80K, IN SWISS 3T3-CELLS
PROTEIN-KINASE-C ACTIVATION POTENTLY DOWN-REGULATES THE EXPRESSION OF ITS MAJOR SUBSTRATE, 80K, IN SWISS 3T3-CELLS
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DOI：
10.1002/j.1460-2075.1991.tb07789.x
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发表时间：
1991-09-01
期刊：
影响因子：
11.4
通讯作者：
ROZENGURT, E
中科院分区：
文献类型：
--
作者：
BROOKS, SF;HERGET, T;ROZENGURT, E
从cDNA核苷酸序列推导出Swiss 3 T3成纤维细胞主要的酸性蛋白激酶C（PKC）底物80 K的氨基酸序列。总体而言，25%的预测氨基酸序列得到直接蛋白质序列数据的支持。Southern印迹分析表明，小鼠基因组含有该基因的单拷贝。用北方杂交技术检测到两条80 K mRNA，一条2.25 kb的主带和一条3.9 kb的次带。用具有生物活性的佛波醇酯（包括佛波醇-12，13-二丁酸酯（PDB））刺激PKC，在5-7 h内使80 KmRNA的稳态水平下降到对照的8.8%。这种作用是剂量依赖性的，并通过预先消耗PKC而消除。PDB诱导的80 K mRNA水平下调是短暂的，恢复与PKC活性的消失相一致。在小鼠胚胎成纤维细胞的三级培养物中也证明了80 K mRNA水平的类似瞬时降低。80 K mRNA水平的下调被完全废除放线菌素D，放线菌酮或茴香霉素，如果添加到30分钟后PDB添加。由于PDB处理的80 K基因的转录速率没有改变，我们得出结论，PKC诱导的80 K mRNA的下调是由转录后机制介导的。此外，PDB在14-18 h内瞬时降低80 K蛋白的水平，从而反映了这种佛波酯对mRNA表达的影响。
The amino acid sequence of 80K, the major acidic protein kinase C (PKC) substrate of Swiss 3T3 fibroblasts, was deduced from a cDNA nucleotide sequence. Overall, 25% of the predicted amino acid sequence is supported by direct protein sequence data. Southern blot analysis suggests that the mouse genome contains a single copy of this gene. Two 80K mRNA species, a major band of 2.25 kb and a minor band of 3.9 kb, were detected by Northern blot analysis. Stimulation of PKC by biologically active phorbol esters, including phorbol-12, 13-dibutyrate (PDB), reduced the steady state level of 80K mRNA to 8.8% of control within 5-7 h. This effect was dose-dependent, and was abolished by prior depletion of PKC. The PDB-induced down-regulation of 80K mRNA levels was transient, and recovery coincided with the disappearance of PKC activity. A similar transient decrease in 80K mRNA levels was also demonstrated in tertiary cultures of mouse embryo fibroblasts. The down-regulation of 80K mRNA levels was completely abolished by actinomycin D, cycloheximide or anisomycin if added up to 30 min after PDB addition. Since the rate of transcription of the 80K gene was unaltered by PDB treatment, we concluded that the PKC-induced down-regulation of 80K mRNA is mediated by a posttranscriptional mechanism. In addition, PDB transiently decreased the level of 80K protein within 14-18 h, thus reflecting the effects of this phorbol ester on mRNA expression.