Grässer F A, LaMontagne K, Whittaker L, Stohr S, Lipsick J S
Institut für Medizinische Mikrobiologie und Hygiene, Abteilung Virologie, Homburg, Germany.
Oncogene. 1992 May;7(5):1005-9.
The v-Myb protein is nuclear, binds to DNA in a sequence-specific fashion, regulates the transcription of various reporter gene and transforms myelomonocytic cells. Cysteine is one of the most conserved residues during protein evolution and has been implicated in DNA binding, protein-protein interaction and redox regulation of various proteins. Therefore, we have now individually substituted each of the seven cysteines of v-Myb with a serine. All seven mutant proteins bound to DNA when they were expressed in E. coli. However, mutant C65S neither trans-activated transcription in vivo nor transformed myeloid cells, although it was transported into the nucleus. This cysteine is conserved in the Myb-related proteins of animals, plants, yeast and the cellular slime mold Dictyostelium discoideum. The C65S mutation and a nearby codon insertion mutation also abolished trans-activation by fusion proteins containing the v-Myb DNA-binding domain and the strong constitutive activation domain of herpes simplex virus (HSV) VP16. Because this domain of VP16 appears to activate transcription whenever it is bound upstream of an appropriate promoter, these results imply that C65 may be required for high-affinity DNA binding in vivo. In support of this hypothesis, we have also shown that, in contrast to wild-type v-Myb, mutant C65S is unable to block transcription from a reporter gene in which Myb binding sites overlap the initiation site.
v-Myb蛋白定位于细胞核，以序列特异性方式与DNA结合，调节各种报告基因的转录并转化骨髓单核细胞。半胱氨酸是蛋白质进化过程中最保守的残基之一，与DNA结合、蛋白质-蛋白质相互作用以及各种蛋白质的氧化还原调节有关。因此，我们现在已将v-Myb的七个半胱氨酸逐一替换为丝氨酸。当这七种突变蛋白在大肠杆菌中表达时，它们均能与DNA结合。然而，突变体C65S尽管被转运到细胞核中，但在体内既不能反式激活转录，也不能转化髓样细胞。这种半胱氨酸在动物、植物、酵母以及细胞黏菌盘基网柄菌的Myb相关蛋白中是保守的。C65S突变以及附近密码子插入突变也消除了含有v-Myb DNA结合结构域和单纯疱疹病毒（HSV）VP16的强组成型激活结构域的融合蛋白的反式激活作用。由于VP16的这一结构域似乎只要结合在合适启动子的上游就能激活转录，这些结果表明C65可能是体内高亲和力DNA结合所必需的。为支持这一假设，我们还表明，与野生型v-Myb不同，突变体C65S无法阻断报告基因的转录，该报告基因中Myb结合位点与起始位点重叠。
