The defective transforming phenotype of c-Jun Ala(63/73) is rescued by mutation of the C-terminal phosphorylation site

Cotransfection of primary rat embryo fibroblasts (REF) with c-Jun and activ ated Ras leads to oncogenic transformation and this process requires the ph osphorylation of the N-terminal domain of c-Jun. Ras augments this phosphor ylation and, consequently activates the c-Jun transactivation property of T RE (TPA Responsive Element)-dependent promoters. To analyse the role of the c-Jun C-terminal phosphorylation site in oncogenic cooperation we tested t he activities of N-terminal c-Jun Ala(63/73) (named Nt), C-terminal c-Jun A la(234/242/246/252) (named Ct) and (Nt+Ct)-with both mutations-nonphosphory latable c-Jun mutants. In cooperation with Ras, the Ct mutant and wt c-Jun display similar oncogenic properties whereas the Nt form was defective in t ransforming REF cells. Unexpectedly, the Nt+Ct mutant exhibited identical o ncogenic properties to wt c-Jun, demonstrating that the Ct mutation rescues in cis the Nt mutation. The transcriptional activity and the capacity to b ind the c-Jun coactivator CREB Binding Protein (CBP) were enhanced by Ras f or the wt and Ct proteins but not for the Nt mutant. Interestingly, the NtCt mutant presents identical transactivation and CBP binding activities to wt c-Jun. Therefore the rescue in cis of the defective Nt mutation by the C t mutation seems to be due to the recovery of CBP binding. Our results reve aled that the process of oncogenic cooperation can occur between Ras and th e Nt+Ct non-phosphorylatable c-Jun protein.