Generation of truncated C/EBPβ isoforms by in vitro proteolysis
Generation of truncated C/EBPβ isoforms by in vitro proteolysis
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DOI：
10.1074/jbc.m004268200
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发表时间：
2000-08-25
影响因子：
4.8
通讯作者：
Johnson, PF
中科院分区：
文献类型：
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作者：
Baer, M;Johnson, PF
在细胞提取物中观察到多种形式的转录调节因子CCAAT/增强子结合蛋白β（C/EBP β），分子量约为38、34、20和14 kDa。已经提出，这些蛋白质通过在框内AUG密码子处的选择性起始而产生。截短的C/EBP β亚型（p14和p20/LIP）缺乏反式激活结构域，但保留DNA结合和二聚化序列，因此被认为是体内C/EBP介导的转录的竞争性抑制剂。通过比较各种提取方法来分析内源性和过表达的C/EBP β蛋白，我们确定p20-C/EBP β主要通过从细胞分离期间的体外蛋白水解裂解产生，并且p14-C/EBP β仅通过该机制产生。在转染的细胞中，全长（p34和p38）亚型，但不是截短的蛋白质在细胞质中检测，表明后者不是主要的翻译产物。此外，C/EBP β亮氨酸拉链二聚化结构域对于截短物质的出现是必不可少的，表明蛋白质折叠或二聚化是蛋白水解敏感性的关键决定因素。我们的研究结果表明，在细胞提取物中截短的C/EBP β蛋白的存在必须谨慎解释，这些异构体的体内相关性的假设应重新评估。
Multiple forms of the transcriptional regulator CCAAT/enhancer-binding protein beta (C/EBP beta) with molecular masses of approximately 38, 34, 20, and 14 kDa have been observed in cell extracts. It has been proposed that these proteins arise by alternative initiation at in-frame AUG codons. The truncated C/EBP beta isoforms (p14 and p20/LIP) lack transactivation domains but retain DNA-binding and dimerization sequences and are therefore assumed to function as competitive inhibitors of C/EBP-mediated transcription in vivo. By comparing various extraction procedures to analyze endogenous and overexpressed C/EBP beta proteins, we determined that p20-C/EBP beta is generated predominantly by in vitro proteolytic cleavage during isolation from cells and that p14-C/EBP beta is produced exclusively by this mechanism. In transfected cells, the full-length (p34 and p38) isoforms but not the truncated proteins were detectable in the cytoplasm, indicating that the latter are not primary translation products. In addition, the C/EBP beta leucine zipper dimerization domain was essential for the appearance of the truncated species, demonstrating that protein folding or dimerization are critical determinants of proteolytic sensitivity. Our findings suggest that the presence of truncated C/EBP beta proteins in cell extracts must be interpreted with caution and that assumptions about the in vivo relevance of these isoforms should be re-evaluated.