RAS-MEDIATED PHOSPHORYLATION OF A CONSERVED THREONINE RESIDUE ENHANCES THE TRANSACTIVATION ACTIVITIES OF C-ETS1 AND C-ETS2

The Ras oncogene products regulate the expression of genes in transfor med cells, and members of the Ets family of transcription factors have been implicated in this process. To determine which Ets factors are t he targets of Ras signaling pathways, the abilities of several Ets fac tors to activate Ras-responsive enhancer (RRE) reporters in the presen ce of oncogenic Ras were examined. In transient transfection assays, r eporters containing RREs composed of Ets-AP-1 binding sites could be a ctivated 30-fold in NIH 3T3 fibroblasts and 80-fold in the macrophage- like line RAW264 by the combination of Ets1 or Ets2 and Res but not by several other Ets factors that were tested in the assay. Ets2 and Ras also superactivated an RRE composed of Ets-Ets binding sites, but the Ets-responsive promoter of the c-fms gene was not superactivated. Mut ation of a threonine residue to alanine in the conserved amino-termina l regions of Ets1 and Ets2 (threonine 38 and threonine 72, respectivel y) abrogated the ability of each of these proteins to superactivate re porter gene expression. Phosphoamino acid analysis of radiolabeled Ets 2 revealed that Ras induced normally absent threonine-specific phospho rylation of the protein, The Ras-dependent increase in threonine phosp horylation was not observed in Ets2 proteins that had the conserved th reonine (sic) residue mutated to alanine or serine. These data indicat e that Ets1 and Ets2 are specific nuclear targets of Ras signaling eve nts and that phosphorylation of a conserved threonine residue is a nec essary molecular component of Ras-mediated activation of these transcr iption factors.