CONSERVED MECHANISMS OF RAS REGULATION OF EVOLUTIONARY RELATED TRANSCRIPTION FACTORS, ETS1 AND POINTED P2

Cell transformation by the Pas oncogene is mediated by members of the ets gene family, To analyse the mechanisms of regulation, we have stud ied activation of several ets factors by Pas expression, We show that expression of Ha-Pas strongly activates the Ets1 p68 and p54 isoforms and Ets2 in F9 EC cells. We have mapped the Pas responsive elements of Ets1 p68 to two domains, RI+II and RIII. Mutation of threonine 82 to alanine in RI+II abolishes both Pas activation and phosphorylation by MAP kinase. Threonine 82 is part of a sequence that is conserved in Dr osophila Pointed P2, an ets protein that has been shown both genetical ly and biochemically to mediate Pas signalling in Drosophila cells. We extend the comparison of these evolutionary related proteins by showi ng that Pointed P2 is activated by Pas in mammalian cells and mutation of the homologous threonine abolishes activation. Furthermore, we sho w that Pointed P2 resembles Ets1, in that it has conserved sequences i n a similar position adjacent to the ets DNA binding domain that negat ively auto-regulates DNA binding. These results go towards showing tha t the Drosophila Pointed and vertebrate Ets1 are evolutionary related proteins that have remarkably conserved Pas regulatory mechanisms down stream from MAP kinase.