Phosphorylation of ETS transcription factor ER81 in a complex with its coactivators CREB-binding protein and p300

The ETS protein ER81 is a DNA-binding factor capable of enhancing gene tran scription and is implicated in cellular transformation, but presently the m echanisms of its actions are unclear. In this report, ER81 is shown to coim munoprecipitate with the transcriptional coactivator CREB-binding protein ( CBP) and the related p300 protein (together referred to as CBP/p300). Moreo ver, confocal laser microscopic studies demonstrated that ER81 and p300 col ocalized to nuclear speckles. In vitro and in vivo interaction studies reve aled that ER81 amino acids 249 to 429, which encompass the ETS DNA-binding domain, are responsible for binding to CBP/p300. However, mutation of a put ative protein-protein interaction motif, LXXLL, in the ETS domain of ER81 d id not affect interaction with CBP/p300, whereas DNA binding of ER81 was ab olished. Furthermore, two regions within CBP, amino acids 451 to 721 and 18 91 to 2175, are capable of binding to ER81. Consistent with the physical in teraction between ER81 and the coactivators CBP and p300, ER81 transcriptio nal activity was potentiated by CBP/p300 overexpression. Moreover, an ER81- associated protein kinase activity was enhanced upon p300 overexpression. T his protein kinase phosphorylates ER81 on serines 191 and 216, and mutation of these phosphorylation sites increased ER81 transcriptional activity in Mv1Lu cells but not in HeLa cells. Altogether, our data elucidate the mecha nism of how ER81 regulates gene transcription, through interaction with the coactivators CBP and p300 and an associated kinase that may cell type spec ifically modulate the ability of ER81 to activate gene transcription.