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 oncoge nic transformation phenotypically characterized by anchorage independent gr owth and release from contact inhibition (focus formation). The mechanisms involved in this oncogenic conversion however, are not yet clear. Because J un is a transcription factor, it has been assumed that oncogenic transforma tion results directly from deregulated AP-1 target gene expression. However , a number of experimental observations in avian cell culture models fail t o correlate oncogenesis with AP-I activity suggesting that transformation i nduced by v-Jun may occur through an indirect mechanism, To test this possi bility, we introduced point mutations into the basic DNA binding domain of v-Jun and created mutants that exhibit altered binding specificity, When ex pressed in CEF, these mutants fail to deregulate three known v-Jun target g enes (JTAP-1, apolipoprotein Al,: c-Jun) thus demonstrating in vivo specifi city changes, Each of the binding specificity mutants was also tested for i ts ability to induce oncogenic transformation. Interestingly, expression of these mutants in CEF results in a phenotype indistinguishable from the vec tor control with respect to growth rate, focus formation and the ability to form colonies in soft agar, These results are consistent with a model requ iring direct AP-1 target deregulation as a prerequisite of v-Jun induced ce ll transformation. With this in mind, we generated a series of additional m utants that retain the ability to bind AP-1 sequence elements, but vary in their oncogenic potential. We demonstrate the use of these mutants to scree n v-Jun induced gene targets for a functional role in cell transformation.