Novel membrane-targeted ERK1 and ERK2 chimeras which act as dominant negative, isotype-specific mitogen-activated protein kinase inhibitors of Ras-Raf-mediated transcriptional activation of c-fos in NIH 3T3 cells

Expression of constructs encoding fusion proteins of ERK1 and ERK2 containi ng a C-terminal farnesylation motif (CAAX) is predominantly localized at th e cell membrane and was activated by to expression of constitutively active Ha-RasL61 and epidermal growth factor. Both fusion proteins significantly inhibit the transcriptional activation of a c-fos-chloramphenicol acetyltra nsferase reporter induced by RasL61, constitutively active MEK1, or constit utively active RafBXB. The corresponding SAAX chimeras or overexpression of the wild-type ERKs did net interfere with the transcriptional activation o f c-fos. The inhibition of the Ras-mediated c-fos induction by ERK2-CAAX ca n in part be rescued by coexpression of a wild-type ERK2 but not by wild-ty pe ERK1. We find that ERK1-CAAX acts in the same fashion, indicating that m itogen-activated protein kinase (MAPK)-CAAX chimeras interact in an isotype -specific manner. It is demonstrated that both ERK1-CAAX and ERK2-CAAX asso ciate with the corresponding endogenous ERKs, which explains the isotype-sp ecific inhibitory effects of the ERK-CAAX chimeras. Evidence is presented t hat expression of ERK-CAAX fusion proteins inhibits the nuclear translocati on of the corresponding endogenous ERKs. Disruption of MAPK translocation b y membrane targeting provides additional, independent proof that nuclear tr anslocation of ERKs is essential for the transcriptional activation of c-fo s.