The transcriptional ETS2 repressor factor associates with active and inactive Erks through distinct FXF motifs

The transcriptional ETS2 repressor factor (ERF) is phosphorylated by Erks both in vivo and in vitro. This phosphorylation determines the subcellular localization and biological function of ERF. Here, we show that active and inactive Erk2 proteins bind ERF with high affinity through a hydrophobic pocket formed by the αF and αG helices and the activation loop of Erk2. We have identified two FXF motifs on ERF that mediate the specific interaction with Erks. One of these motifs is utilized only by active Erks, whereas the other mediates the association with inactive Erks but also contributes to interaction with active Erks. Mutation of the phenylalanines of these motifs to alanines resulted in decreased association and phosphorylation of ERF by Erks both in cells and in vitro. ERF proteins carrying these mutations exhibited increased nuclear accumulation and increased inhibition of cellular proliferation. Expression of ERF regions harboring these motifs could inhibit Erk activity in cells. Our data suggest that, in the proper context, FXF motifs can mediate a strong and specific interaction not only with active but also inactive Erks and that these interactions determine protein function in vivo.