Barrett J, Birrer M J, Kato G J, Dosaka-Akita H, Dang C V
Hematology Division, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Mol Cell Biol. 1992 Jul;12(7):3130-7. doi: 10.1128/mcb.12.7.3130-3137.1992.
Members of the Myc family of proteins share a number of protein motifs that are found in regulators of gene transcription. Conserved stretches of amino acids found in the N-terminal transcriptional activation domain of c-Myc are required for cotransforming activity. Most of the Myc proteins contain the basic helix-loop-helix zipper (bHLH-Zip) DNA-binding motif which is also required for the cotransforming activity of c-Myc. L-Myc, the product of a myc family gene that is highly amplified in many human lung carcinomas, was found to cotransform primary rat embryo cells with an activated ras gene. However, L-Myc cotransforming activity was only 1 to 10% of that of c-Myc (M. J. Birrer, S. Segal, J. S. DeGreve, F. Kaye, E. A. Sausville, and J. D. Minna, Mol. Cell. Biol. 8:2668-2673, 1988). We sought to determine whether functional differences between c-Myc and L-Myc in either the N-terminal or the C-terminal domain could account for the relatively diminished L-Myc cotransforming activity. Although the N-terminal domain of L-Myc could activate transcription when fused to the yeast GAL4 DNA-binding domain, the activity was only 5% of that of a comparable c-Myc domain. We next determined that the interaction of the C-terminal bHLH-Zip region of L-Myc or c-Myc with that of a Myc partner protein, Max, was equivalent in transfected cells. A Max expression vector was found to augment the cotransforming activity of L-Myc as well as that of c-Myc. In addition, a bacterially synthesized DNA-binding domain of L-Myc, like that o c-Myc, heterodimerizes with purified Max protein to bind the core DNA sequence CACGTG. To determine the region of L-Myc responsible for its relatively diminished cotransforming activity, we constructed chimeras containing exons 2 (constituting activation domains) and 3 (constituting DNA-binding domains) of c-Myc fused to those of L-Myc. The cotransforming potencies of these chimeras were compared with those of full-length L-Myc of c-Myc in rat embryo cells. The relative cotransforming activities suggest that the potencies of the activation domains determine the cotransforming efficiencies for c-Myc and L-Myc. This correlation supports the hypothesis that the Myc proteins function in neoplastic cotransformation as transcription factors.
Myc 蛋白家族的成员具有一些在基因转录调节因子中发现的蛋白质基序。在 c-Myc 的 N 端转录激活域中发现的保守氨基酸序列是共转化活性所必需的。大多数 Myc 蛋白含有碱性螺旋-环-螺旋拉链（bHLH-Zip）DNA 结合基序，这也是 c-Myc 共转化活性所必需的。L-Myc 是 myc 家族基因的产物，在许多人类肺癌中高度扩增，发现它能与激活的 ras 基因共转化原代大鼠胚胎细胞。然而，L-Myc 的共转化活性仅为 c-Myc 的 1%至 10%（M. J. Birrer、S. Segal、J. S. DeGreve、F. Kaye、E. A. Sausville 和 J. D. Minna，《分子细胞生物学》8:2668 - 2673，1988 年）。我们试图确定 c-Myc 和 L-Myc 在 N 端或 C 端结构域的功能差异是否可以解释 L-Myc 相对较低的共转化活性。尽管 L-Myc 的 N 端结构域与酵母 GAL4 DNA 结合结构域融合时可以激活转录，但其活性仅为可比的 c-Myc 结构域的 5%。接下来我们确定，在转染细胞中，L-Myc 或 c-Myc 的 C 端 bHLH-Zip 区域与 Myc 伴侣蛋白 Max 的相互作用是相同的。发现一个 Max 表达载体可以增强 L-Myc 以及 c-Myc 的共转化活性。此外，与 c-Myc 一样，L-Myc 的细菌合成 DNA 结合结构域与纯化的 Max 蛋白异源二聚化以结合核心 DNA 序列 CACGTG。为了确定 L-Myc 中负责其相对较低共转化活性的区域，我们构建了嵌合体，其中包含 c-Myc 的外显子 2（构成激活结构域）和外显子 3（构成 DNA 结合结构域）与 L-Myc 的外显子 2 和 3 融合。在大鼠胚胎细胞中比较了这些嵌合体与全长 L-Myc 或 c-Myc 的共转化能力。相对共转化活性表明激活结构域的能力决定了 c-Myc 和 L-Myc 的共转化效率。这种相关性支持了 Myc 蛋白作为转录因子在肿瘤共转化中发挥作用的假设。
