Banks G C, Li Y, Reeves R
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660, USA.
Biochemistry. 2000 Jul 18;39(28):8333-46. doi: 10.1021/bi000378+.
The HMGI(Y) family of "high mobility group" nonhistone proteins are architectural transcription factors whose overexpression is highly correlated with both cancerous transformation and increased malignancy and metastatic potential of tumors in vivo. Here we report on the types of posttranslational modifications found in vivo on the HMG-I and HMG-Y proteins isolated from two human breast epithelial cell lines, MCF-7 and MCF-7/PKC-alpha, that represent different stages of neoplastic progression. The MCF-7 cell line exhibits many characteristics of normal breast epithelial cells and does not form tumors when injected into nude mice, whereas the MCF-7/PKC-alpha cell line, a derivative of MCF-7 that expresses a transgene coding for the enzyme protein kinase C-alpha (PKC-alpha), is both malignant and highly metastatic. Using MALDI mass spectrometry, we show that the HMG-Y protein is more highly modified than the HMG-I protein in both the MCF-7 and the MCF-7/PKC-alpha cells. Significantly, the HMG-Y protein isolated from the highly metastatic MCF-7/PKC-alpha cells possesses a unique constellation of phosphorylations, methylations, and acetylations not found on the HMG-I protein isolated from either the MCF-7 or MCF-7/PKC-alpha cells. We further demonstrate that some of the same amino acid residues phosphorylated on recombinant HMGI(Y) proteins by purified PKC in vitro are also phosphorylated on the HMG-I(Y) proteins isolated from MCF-7/PKC-alpha cells, suggesting that PKC phosphorylates these proteins in vivo. Quantitative substrate binding analyses indicate that the biochemical modifications present on the HMG-I and HMG-Y proteins differentially influence the ability of these proteins to interact with both A.T-rich DNA substrates and nucleosome core particles in vitro, suggesting a similar modulation of such binding affinities in vivo. To our knowledge, this is the first demonstration of differences in the types of in vivo biochemical modifications found on the HMG-I and HMG-Y proteins in cells and also the first experimental evidence suggesting a possible linkage between such posttranslational modifications and the neoplastic potential of cells.
“高迁移率族”非组蛋白的HMGI(Y)家族是结构转录因子，其过表达与体内肿瘤的癌变转化以及恶性程度和转移潜能的增加高度相关。在此，我们报告了从两个人类乳腺上皮细胞系MCF-7和MCF-7/PKC-α中分离出的HMG-I和HMG-Y蛋白在体内发现的翻译后修饰类型，这两个细胞系代表了肿瘤进展的不同阶段。MCF-7细胞系表现出许多正常乳腺上皮细胞的特征，注射到裸鼠体内不会形成肿瘤，而MCF-7/PKC-α细胞系是MCF-7的衍生物，表达编码酶蛋白激酶C-α（PKC-α）的转基因，具有恶性且高度转移的特性。使用基质辅助激光解吸电离质谱法，我们表明在MCF-7和MCF-7/PKC-α细胞中，HMG-Y蛋白比HMG-I蛋白有更高程度的修饰。值得注意的是，从高转移性的MCF-7/PKC-α细胞中分离出的HMG-Y蛋白具有一组独特的磷酸化、甲基化和乙酰化修饰，而从MCF-7或MCF-7/PKC-α细胞中分离出的HMG-I蛋白上未发现这些修饰。我们进一步证明，在体外由纯化的PKC对重组HMGI(Y)蛋白进行磷酸化的一些相同氨基酸残基，在从MCF-7/PKC-α细胞中分离出的HMG-I(Y)蛋白上也被磷酸化，这表明PKC在体内对这些蛋白进行磷酸化。定量底物结合分析表明，HMG-I和HMG-Y蛋白上存在的生化修饰在体外对这些蛋白与富含A.T的DNA底物和核小体核心颗粒相互作用的能力有不同影响，这表明在体内也有类似的结合亲和力调节。据我们所知，这是首次证明细胞中HMG-I和HMG-Y蛋白在体内生化修饰类型上的差异，也是首次有实验证据表明这种翻译后修饰与细胞的肿瘤潜能之间可能存在联系。
