We continued the analysis of the two novel genes that we identified previously and have shown to regulate ETS2 transcription, SBF and ERF. We developed antipeptide polyclonal antibodies against SBF that can discriminate between the splicing isoforms of SBF and we studied its subcellular and tissue distribution. We analyzed the phosphorylation pattern of ERF and we identified specific phosphopeptides that are phosphorylated during cell cycle. By site directed mutagenesis, we identified four of the residues that are phosphorylated in vivo during mitogenic stimulation and in vitro by Erk2 kinase. These data provide further evidence that mitogen associated proteins (MAP)-related kinases, phosphorylate ERF. We analyzed the function of the phosphorylation deficient mutants and we found that phosphorylation decreases the ERF repressor function. We isolated the murine genomic locus of ERF. The mouse and the human gene have 98% amino acid identity and the same genomic organization. We generated constructs that disrupt the ERF murine genomic locus and ERF protein production in order to generate ERF null transgenic mice and better understand ERF function.
