CONSTITUTIVELY ACTIVE MUTANTS OF MAP KINASE KINASE (MEK1) INDUCE GROWTH FACTOR-RELAXATION AND ONCOGENICITY WHEN EXPRESSED IN FIBROBLASTS

The Mitogen Activated Protein Kinase (MAPK) module operates downstream of Pas to convey cell surface signals to the nucleus via the nuclear translocation of p42/p44 MAPKs. We have previously established that MA PK activation is obligatory and must persist in the G1 phase to allow resting fibroblasts to exit from G0 (Pages et al., Proc. Natl. Acad. S ci.1993, 90, 8319-8323). It remained to be established whether MAPK ac tivation was sufficient to trigger cell proliferation. To this aim, we generated and expressed in Chinese hamster lung fibroblasts, constitu tively active mutants of hamster MAP kinase kinase (MAPKK). Three muta nts: S218D, S222D and S218D/S222D in which we substituted the Raf1/MAP KKK-dependent regulatory phosphorylation sites by aspartic acid residu es, displayed increased basal activity when expressed in fibroblasts. Two of them, S218D and S218D/S222D which have a basal activity higher than serum-stimulated wild type-MAPKK (respectively 2- and 5-fold), in duced activation of p42 MAPK in growth factor-deprived cells. Interest ingly, only these two mutants led to a growth factor-independent state as judged by early gene transcription (activation of the fos promoter ), increased sensitivity to growth factors for reinitiation of DNA syn thesis, autonomous cell cycling and rapid tumor formation in nude mice . Therefore we conclude that the downstream elements of the growth fac tor signalling cascade, MAPKK-MAPK, are both necessary and sufficient to promote growth factor signals and autonomous cell cycling in fibrob lasts.