8-Bromo-Cyclic AMP Induces Phosphorylation of Two Sites in SRC-1 That Facilitate Ligand-Independent Activation of the Chicken Progesterone Receptor and Are Critical for Functional Cooperation between SRC-1 and CREB Binding Protein

Abstract

ABSTRACT Elevation of intracellular 8-bromo-cyclic AMP (cAMP) can activate certain steroid receptors and enhance the ligand-dependent activation of most receptors. During ligand-independent activation of the chicken progesterone receptor (cPR A ) with the protein kinase A (PKA) activator, 8-bromo-cAMP, we found no alteration in cPR A phosphorylation (W. Bai, B. G. Rowan, V. E. Allgood, B. W. O'Malley, and N. L. Weigel, J. Biol. Chem. 272:10457–10463, 1997). To determine if other receptor-associated cofactors were targets of cAMP-dependent signaling pathways, we examined the phosphorylation of steroid receptor coactivator 1 (SRC-1). We detected a 1.8-fold increase in SRC-1 phosphorylation in transfected COS-1 cells incubated with 8-bromo-cAMP. Phosphorylation was increased on two mitogen-activated protein kinase (MAPK) sites, threonine 1179 and serine 1185. PKA did not phosphorylate these sites in vitro. However, blockage of PKA activity in COS-1 cells with the PKA inhibitor (PKI) prevented the 8-bromo-cAMP-mediated phosphorylation of these sites. Incubation of COS-1 cells with 8-bromo-cAMP resulted in activation of the MAPK pathway, as determined by Western blotting with antibodies to the phosphorylated (active) form of Erk-1/2, suggesting an indirect pathway to SRC-1 phosphorylation. Mutation of threonine 1179 and serine 1185 to alanine in COS-1 cells coexpressing cPR A and the GRE 2 E1bCAT reporter resulted in up to a 50% decrease in coactivation during both ligand-independent activation and ligand-dependent activation. This was due, in part, to loss of functional cooperation between SRC-1 and CREB binding protein for coactivation of cPR A . This is the first demonstration of cross talk between a signaling pathway and specific phosphorylation sites in a nuclear receptor coactivator that can regulate steroid receptor activation.