65. progestin. CDK2 induced nuclear localization of unliganded wt but not S400A PR; liganded S400A PR exhibited delayed nuclear accumulation. These studies demonstrate that CDK2 regulates PR in the absence of progestins via phosphorylation of Ser400, thus revealing a novel mechanism for upregulated PR transcriptional activity in human breast cancer cells expressing altered cell cycle regulatory molecules. The steroid hormones estrogen and progesterone regulate breast development (29, 89) and contribute to breast cancer progression (29, 54). Breast cancer cell lines are used to model the effects of steroid hormones on cell proliferation and survival. Steroidal control of cell cycle progression takes place at defined points in the G1 phase of the cell cycle (52). Progesterone has either stimulatory (25, 47) or biphasic (11, 23, 53) effects on human T47D breast cancer cell growth, dependent in part upon cell culture conditions and the presence of estrogenic stimuli. Cell cycle analyses of biphasic cell growth patterns indicate that following a single dose of progesterone, cyclin D1 and cyclin E levels initially increase as cells undergo S-phase entry. Cyclin-dependent protein kinase 2 (CDK2) activity peaks at approximately 16 h. Coincident with increased CDK2 activity, progesterone receptor (PR) protein levels begin to decline. The CDK inhibitors p21 and p27 are then induced after this early proliferative phase, leading to G1 arrest, and PR levels slowly recover as cells exit the mitotic cell cycle. Cells are further growth inhibited in the presence of additional progesterone treatments (23, 53), but progestin-primed cells can be induced to grow by administration of growth factors (23). These studies demonstrate a complex interplay between PR and cell cycle regulators. Several studies have demonstrated that cyclin D1 and p27 play important roles in normal mammary gland development (14, 49, 81, 82). Cyclin D1?/? mice have a deficiency in pregnancy-associated mammary gland development (16, 80). In addition, overexpression of cyclins D1 and E and decreased expression of the CDK inhibitor p27 are associated with the high growth rates seen in human breast cancers. For example, approximately 45 to 50% of breast cancers overexpress cyclin D1 (5, 21). Furthermore, progression from LY2365109 hydrochloride normal breast tissue through invasive ductal carcinoma (77), high-grade ductal carcinoma relative to low grade (77), and late-stage lesions (34) are all associated with increased expression of cyclin E. In addition, decreased expression of p27 occurs in 30% of breast cancers and is correlated with poor prognosis in primary breast cancers (7, 63, 87). Mouse models of breast cancer support a role for alterations in cell cycle molecules in progression of mammary epithelial cells to preneoplastic stages (69). Deregulated cell cycle molecules are predicted to augment breast cancer progression in part as a result of increased CDK activity. The relevant CDK targets in breast cancers remain unknown. The PR is highly phosphorylated, primarily on serine residues, by multiple kinases in a manner similar to other steroid hormone receptor family members (41, 85, 93). While the role of phosphorylation of steroid receptors is not fully understood, phosphorylation may influence promoter specificity (65), cofactor interaction (19), ligand-dependent (78) and ligand-independent (39) transcriptional activities, receptor turnover (43), and nuclear association (66). In addition, Rabbit polyclonal to ABCA5 steroid hormone receptor phosphorylation may serve to integrate signals initiated by growth factors in tissues under steroidal control. A number of endogenously regulated phosphorylation sites on human PR have been well defined (41, 93). LY2365109 hydrochloride For example, serines at positions 294 and 345 in PR are predominantly phosphorylated following treatment of cells with progestin (96). Ser400 is both basally phosphorylated and regulated by ligand in cells; Ser400 is a basally phosphorylated site in vivo LY2365109 hydrochloride (96, 97) and phosphorylated by CDK2 in vitro (95). Of the 14 identified phosphorylation sites, 8 are known to be phosphorylated by CDK2 in vitro (36, 95). The consequence of PR phosphorylation by CDK2 is unknown LY2365109 hydrochloride but suggests a mechanism for cell cycle-dependent regulation of PR. We therefore investigated the role of direct regulation of PR by LY2365109 hydrochloride CDK2 in breast cancer cells by.