Final steps in the feedback regulation of human glucocorticoid receptor gene and role of nuclear protein phosphatase 2A.

The feedback regulation of the human glucocorticoid receptor gene by glucocorticoid receptor-hormone complexes involves the recruitment of the nuclear phosphatase, PP2A. The PP2A holoenzyme consists of a structural subunit A, a catalytic subunit C, and a variable regulatory subunit B or PPP2R3C,with distinct specificities. Our goal was to investigate the nature of the interaction between glucocorticoid receptor-hormone complex and PPP2R3C in the regulation of the glucocorticoid receptor gene involving the feedback regulatory element. Co-immunoprecipitation assays revealed interaction of glucocorticoid receptor with PPP2R3C only in the presence of ligand. In mammalian two-hybrid assays, we expanded our investigation using mutations of specific serines in the AF-1 region of the glucocorticoid receptor and deletion mutants of PPP2R3C. We determined which of the two nuclear recognition motifs in PPP2R3C interacted with glucocorticoid receptor. Following purification of CREB and PPP2R3C, we performed in vitro de-phosphorylation experiments and demonstrated the direct role of glucocorticoid receptor dexamethasone complexes in recruiting nuclear PP2A complexes containing PPP2R3C to dephosphorylate CREB-P. Based on these results, GST-GR proteins were produced and in GST pull-down assays we determined that it was indeed the hormone binding domain as well as amino acids 76-262 in the AF-1 of the GR that interacted with PPP2R3C. Protein-protein interaction assays using GST-GR fusion proteins revealed that Ser 211 and Ser 226 in the AF-1 domain of the GR were contacted by PPP2R3C in the presence of dexamethasone. PPP2R3C mediated suppression of luciferase reporter gene activity in HeLa cells treated with dexamethasone and Okadaic acid. Knocking down PPP2R3C enhanced GR level in Western blots. In immunoprecipitation studies, GR expression was not inhibited in PPP2R3C knockdown cells. Indeed, luciferase reporter activity containing feedback regulatory element of GR gene increased in the presence of cAMP and dexamethasone. This suggested that PPP2R3C prevented CREB-P de-phosphorylation and GR recruitment, a conclusion supported by the fact that rescue of PPP2R3C restored function. In conclusion, our studies show the role of PPP2R3C in GR-mediated negative regulation of target genes where knocking down PPP2R3C increased GR level. Glucocorticoids are widely used in cancer therapy and resistance to treatment is a common occurrence. Our understanding of the data presented here could be useful in designing treatment regimens for patients.