Mechanisms of hormonal activation of Cdc25A and coactivation of estrogen receptor alpha by protein inhibitor of activated STAT3 (PIAS3)

dc.contributorSafe, Stephen H.
dc.creatorLee, Wan-Ru
dc.description.abstractThe estrogen receptor (ER) is a ligand-activated transcription factor that regulates gene expression. The classical mechanisms of nuclear ER action include ligand-induced dimerization of ER which binds estrogen responsive elements (EREs) in promoters of target genes. In addition, non-genomic pathways of ER action have also been identified in breast cancer cells. Cdc25A is a tyrosine phosphatase that catalyzes dephosphorylation of cyclin/cyclin-dependent kinase complexes to regulate G1- to S-phase cell cycle progression. Cdc25A mRNA levels are induced by 17?-estradiol (E2) in ZR-75 breast cancer cells, and deletion analysis of the Cdc25A promoter identified the -151 to -12 region as the minimal E2-responsive sequence. Subsequent mutation/deletion analysis showed that at least three different cis-elements were involved in activation of Cdc25A by E2, namely, GC-rich Sp1 binding sites, CCAAT motifs, and E2F sites. Studies with inhibitors and dominant negative expression plasmids show that E2 activates Cdc25A expression through activation of genomic ER?/Sp1 and E2F1 and cAMP-dependent activation of NF-YA. Thus, both genomic and non-genomic pathways of estrogen action are involved in induction of Cdc25A in breast cancer cells. The PIAS family was initially identified as cytokine-induced inhibitors of STATs which contain several conserved domains involved in binding to other nuclear coactivators. In this study we have investigated coactivation of ER? by PIAS3 in breast cancer cell lines transiently cotransfected with the pERE3 constructs which contain three tandem EREs linked to a luciferase reporter gene. PIAS3 coactivated ER?-mediated transactivation in cells cotransfected with pERE3 and wild-type ER?. In contrast to many other coactivators, PIAS3 also enhanced transactivation of ER? when cells were cotransfected with the TAF1 ER? mutant. In addition, PIAS3 does not interact with activation function 2 (AF2) domain of ER? in a mammalian two-hybrid assay. These data indicate that coactivation of ER? by PIAS3 was AF2-domain independent. Analysis of several PIAS3 deletion mutants showed that the region containing amino acids 274 to 416 of PIAS3 are required for coactivation suggesting that the RING finger domain and acidic region of PIAS3 are important for interactions with wild-type ER?. These results demonstrate that PIAS3 coactivated ER? and this represents a non-classical LXXLL-independent coactivation pathway.
dc.subjectEstrogen receptor
dc.titleMechanisms of hormonal activation of Cdc25A and coactivation of estrogen receptor alpha by protein inhibitor of activated STAT3 (PIAS3)