ACTIVATED HA-RAS BUT NOT TPA INDUCES TRANSCRIPTION THROUGH BINDING-SITES FOR ACTIVATING TRANSCRIPTION FACTOR 3 JUN AND A NOVEL NUCLEAR FACTOR/

We report the identification of a 20-base pair sequence mediating indu ced transcription in response to an activated Ha-ras gene and epiderma l growth factor (EGF) but not 12-O-tetradecanoylphorbol-13-acetate sti mulation. This signal-specific nuclear target is present in the long t erminal repeat of a mouse VL30 retrotransposon expressed in epidermis. Functional studies and in vitro binding analyses using cultured kerat inocytes (Balb/MK) reveal that the response element is composed of two cooperating sequence motifs in juxtaposed position, both of which are targets for induced binding activity 1-2 h after EGF stimulation. Of many different activating transcription factor/cAMP-responsive element binding protein/activating protein 1 factors tested, one part of the sequence selectively binds endogenous proteins immunologically related to activating transcription factor 3 (ATF3) and Jun isotypes. The oth er sequence is a target for a nuclear factor showing binding specifici ty unrelated to factors known to mediate EGF- or ras-induced transcrip tion as determined by its sequence specificity and by antibody experim ents. This component has been characterized and partially purified by gel filtration chromatography and velocity centrifugation revealing a Stokes radius of 43.6 Angstrom and a sedimentation coefficient of 9.7 S in solution. Based on these parameters, a molecular mass of 178,000 Da was calculated. The results indicate that the specific binding of A TF3/Jun and a previously uncharacterized factor account for signal-spe cific transcription in response to EGF or an activated Ha-ras gene in a cell type in which the cooperative action of an activated Ha-ras gen e and 12-O-tetradecanoylphorbol-13-acetate cause tumor growth.