Oncogenic point mutations induce altered conformation, redox sensitivity, and DNA binding in the minimal DNA binding domain of avian myeloblastosis virus v-Myb.
c-Myb is the founder member of a class of transcription factors with tryptophan-rich repeats responsible for DNA binding. Activated oncogenic forms of Myb are encoded by the avian retroviruses, avian myeloblastosis virus (AMV) and E26. AMV v-Myb encodes a truncated protein with 11 point mutations relative to c-Myb. The mutations in the DNA binding domain (DBD) were reported to impose distinct phenotypes of differentiation on transformed myeloid cells (Introna, M., Golay, J., Frampton, J., Nakano, T., Ness, S. A., and Graf, T. (1990) Cell 63, 1287-1297). The molecular mechanism operating has remained elusive since no change in sequence specificity has been found. We introduced AMV-specific point mutations in the minimal DBD of chicken c-Myb and studied their effect on structure and function of the purified protein. Fluorescence emission spectra and fluorescence quenching experiments showed that the AMV-specific point mutations had a significant effect on the conformation of the DBD, giving rise to a more compact structure, a change that was accompanied by a reduced sensitivity toward cysteine-specific alkylation and oxidation. The DNA binding properties were also altered by the AMV-specific point mutations, leading to protein-DNA complexes with highly reduced stability. This reduction in stability was, however, more severe with certain subtypes of binding sequences than with others. This differential behavior was also observed in an in vivo model system where DBD-VP16 fusions were coexpressed with various reporters. These findings imply that different subsets of Myb-responsive promoters may react differentially toward the AMV-specific mutations, a phenomenon that could contribute to the altered patterns of gene expression induced by the AMV v-Myb relative to wild type c-Myb.
Brendeford
E M
EM
Department of Biochemistry, University of Oslo, N-0316 Oslo 3, Norway.
Myrset
A H
AH
Hegvold
A B
AB
Lundin
M
M
Gabrielsen
O S
OS
D009189:Avian Myeloblastosis Virus / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D016297:Mutagenesis, Site-Directed; D020626:Oncogene Proteins v-myb; D010084:Oxidation-Reduction; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D015682:Retroviridae Proteins, Oncogenic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004268:DNA-Binding Proteins; D020626:Oncogene Proteins v-myb; D015682:Retroviridae Proteins, Oncogenic