Dephosphorylation in Vitro Inhibits Ligand and DNA Binding Activities of Human Estrogen Receptor

Abstract

Phosphorylation and/or dephosphorylation of estrogen receptor (ER) triggered by parallel signalling pathways is likely to be an important mechanism regulating the transcription of estrogen responsive genes (Herschman, 1991). Phosphorylation is implicated in regulating the hormone binding of estrogen, glucocorticoid, as well as other steroid receptors (Moudgil, 1990; Rao et al., 1987). In our laboratory we have studied the molecular polymorphism and structure function relationships of ER in human breast cancer (Hyder et al., 1987). Estrogen receptor bears phosphorylation sites in its hormone binding domain (Migliaccio et al., 1989), which also harbors one of its two transcription activation functions (Gronemeyer, 1992). We have demonstrated that human ER expressed in E. coli, despite being depraved of the phosphorylation reactions of its cell of origin, retains its ligand binding properties (Wittliff et al., 1990). Here we show that both the ligand- and DNA-binding activities of ER can be abolished by in vitro dephosphorylation of ER with alkaline phosphatase (AP), suggesting that dephosphorylation plays a role during and after receptor transformation to a DNA-binding form.