). 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 ligandindependent 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.Steroid receptors are members of a superfamily of ligandactivated transcription factors that bind to sequence-specific DNA binding sites in the promoter of target genes to regulate transcription. In addition to the regulation by ligand, steroid receptor action is modulated by cellular signaling pathways that activate intracellular kinases that, in turn, target receptors or other proteins relevant to the receptor activation process. Steroid receptors are phosphoproteins, and receptor phosphorylation has been shown to regulate the activity of the progesterone receptor (PR) (9-11, 13, 21), estrogen receptor (ER) (7,15,16,20,23,34), the glucocorticoid receptor (GR) (5,29,36,56), and the androgen receptor (AR) (62).In some cases, activation of a cellular signaling pathway is sufficient to activate a receptor in the absence of hormone. Elevation of intracellular cyclic AMP (cAMP), a common second messenger for a number of hormones and a direct activator of protein kinase A (PKA), can induce a ligand-independent activation of chicken PR (cPR) (22), AR (44), and the ER (8), as well as enhance steroid-dependent activation of a broader range of receptors, including PR (13, 35), ER (8), GR (45), AR (19,30), and the mineralocorticoid receptor (41). Previously, we found that activation of cAMP-dependent signaling pathways with the agent 8-bromo-cAMP caused no change in receptor phosphorylation during ligand-independent activation of cPR (9) or cAMP enhancement of steroid-dependent activation of human PR (hPR) (13). This suggested that other receptor-associated proteins, such as the recently discovered coactivators for the steroid receptor superfamily,...