Dominant negative mutants of Myc inhibit cooperation of both Myc and adenovirus serotype-5 E1a with Ras.

We have used dominant negative Myc mutants to analyze the Myc and E1a mechanisms of cooperation with Ras. We show that mutants of Myc with an altered basic region (BR; RR366, 367EE) or deletion of the leucine zipper (LZ; delta aa 414-439), changes which modify the DNA binding domain, or with deletions in the Myc amino terminal conserved regions box 1 (dlMB1; delta aa 46-55) and box 2 (dlMB2; delta aa 132-140) inhibit cooperation of wt Myc and activated Ras to transform rat embryo fibroblasts (REF). Expression of the amino terminal 104 aa had no effect whereas wt Myc stimulated focus formation. Mutant dlMB1 cooperated with Ras with one half wt efficiency while dlMB2 was inactive. No mutant tested was toxic during neomycin cotransformation of REF to G418 resistance. Interestingly, these Myc mutants exerted a parallel inhibition of E1a-Ras cooperation to transform REF. This suggests that the Myc-Ras and E1a-Ras cooperation pathways intersect and require common protein factors. A Myc box 2 deletion mutant which is a wt transactivator of the Myc responsive ornithine decarboxylase promoter, but unlike the wt does not repress the adenovirus-2 core promoter (Li et al., 1994, EMBO J., 13:4070-4079), inhibits Myc-Ras and E1a-Ras cooperation. This suggests that a box 2-dependent step, potentially gene repression, is required for both the E1a- and Myc-Ras cooperation mechanisms.