THE INHIBITORY ACTIVITY OF A TRANSDOMINANT C-JUN MUTANT FUSED TO THE LIGAND-BINDING DOMAIN OF THE ESTROGEN-RECEPTOR

Tam-67 is an amino-terminal deletion mutant of c-Jun (Delta 3-122) lac king most of the c-Jun transactivation domain, which has been shown pr eviously to function in a transdominant fashion to inhibit c-Jun-induc ed transactivation and cellular transformation. In order to create a l igand-dependent dominant negative repressor of AP-1, we have construct ed a fusion of the TAM-67 gene with the ligand binding domain of the e strogen receptor. Fusion of TAM-67 with the ligand binding domain of t he estrogen receptor produced a 68 kD protein (TAM-67ER) which was imm unoprecipitated by c-Jun-specific and estrogen receptor-specific antis era and shown by gel retardation assay to bind oligonucleotides contai ning an AP-1 sequence. Cotransfection of TAM-67ER and an AP-1-dependen t reporter construct into rat embryo cells demonstrated ligand specifi c inhibition of AP-1 transactivation. In the absence of hormone, TAM-6 7ER produced complete inhibition of cJun-induced AP-1 transactivation. This inhibition was relieved by treatment with estradiol but not by t reatment with tamoxifen. In addition, TAM-67ER inhibited activated c-H a-ras- or c- raf-induced transformation of NTH3T3 cells. However, this inhibition of transformation was not relieved by the addition of estr ogen. Thus, TAM-67ER inhibits transactivation in a ligand-dependent ma nner, but inhibits transformation in a ligand-independent manner. The results suggest that the ligand-dependent transactivation domain of th e estrogen receptor (TAF-2) can substitute for the cJun transactivatio n domain absent in TAM-67 to stimulate transactivation. However, TAF-2 cannot substitute for the missing c-Jun transactivation domain to ind uce cellular transformation.