Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation.
The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.
Tikhonenko
A T
AT
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Hartman
A R
AR
Linial
M L
ML
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D002467:Cell Nucleus / Q000378:metabolism*; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D005347:Fibroblasts / Q000502:physiology; D016259:Genes, myc; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016274:Oncogene Protein p55(v-myc) / Q000096:biosynthesis* / Q000378:metabolism; D016366:Open Reading Frames; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011784:Quail; D015183:Restriction Mapping; D014157:Transcription Factors; D014162:Transfection
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C067850:Myc associated factor X; D009838:Oligodeoxyribonucleotides; D016274:Oncogene Protein p55(v-myc); D014157:Transcription Factors