A highly conserved cysteine in the v-Myb DNA-binding domain is essential for transformation and transcriptional trans-activation.
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.
Grässer
F A
FA
Institut für Medizinische Mikrobiologie und Hygiene, Abteilung Virologie, Homburg, Germany.
LaMontagne
K
K
Whittaker
L
L
Stohr
S
S
Lipsick
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D002471:Cell Transformation, Neoplastic; D002645:Chickens; D002874:Chromosome Mapping; D003545:Cysteine / Q000502:physiology*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020626:Oncogene Proteins v-myb; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics / Q000502:physiology*; D016385:TATA Box; D014158:Transcription, Genetic / Q000502:physiology*; D015533:Transcriptional Activation / Q000235:genetics
D004268:DNA-Binding Proteins; D020626:Oncogene Proteins v-myb; D015682:Retroviridae Proteins, Oncogenic; D004247:DNA; D003545:Cysteine