Directed mutation of the basic domain of v-Jun alters DNA binding specificity and abolishes its oncogenic activity in chicken embryo fibroblasts.
Overexpression of v-Jun in chicken embryo fibroblasts (CEF) leads to oncogenic transformation phenotypically characterized by anchorage independent growth and release from contact inhibition (focus formation). The mechanisms involved in this oncogenic conversion however, are not yet clear. Because Jun is a transcription factor, it has been assumed that oncogenic transformation results directly from deregulated AP-1 target gene expression. However, a number of experimental observations in avian cell culture models fail to correlate oncogenesis with AP-1 activity suggesting that transformation induced by v-Jun may occur through an indirect mechanism. To test this possibility, we introduced point mutations into the basic DNA binding domain of v-Jun and created mutants that exhibit altered binding specificity. When expressed in CEF, these mutants fail to deregulate three known v-Jun target genes (JTAP-1, apolipoprotein A1, c-Jun) thus demonstrating in vivo specificity changes. Each of the binding specificity mutants was also tested for its ability to induce oncogenic transformation. Interestingly, expression of these mutants in CEF results in a phenotype indistinguishable from the vector control with respect to growth rate, focus formation and the ability to form colonies in soft agar. These results are consistent with a model requiring direct AP-1 target deregulation as a prerequisite of v-Jun induced cell transformation. With this in mind, we generated a series of additional mutants that retain the ability to bind AP-1 sequence elements, but vary in their oncogenic potential. We demonstrate the use of these mutants to screen v-Jun induced gene targets for a functional role in cell transformation.
Basso
J
J
Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk 23501, USA.
Briggs
J
J
Findlay
C
C
Bos
T
T
D019943:Amino Acid Substitution; D000818:Animals; D016632:Apolipoprotein A-I / Q000096:biosynthesis / Q000235:genetics; D001358:Avian Sarcoma Viruses / Q000235:genetics / Q000502:physiology; D002472:Cell Transformation, Viral / Q000235:genetics*; D002478:Cells, Cultured; D002642:Chick Embryo; D004247:DNA / Q000378:metabolism*; D005347:Fibroblasts; D015967:Gene Expression Regulation, Viral; D016758:Genes, jun; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D016759:Oncogene Protein p65(gag-jun) / Q000737:chemistry / Q000235:genetics*; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection
D016632:Apolipoprotein A-I; D016759:Oncogene Protein p65(gag-jun); D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D004247:DNA