Protein Phosphatase 5 Is a Negative Regulator of Estrogen Receptor-Mediated Transcription

Estrogen receptors (ERs) are transcription factorsthat can be modulated by both estrogen-dependentand growth factor-dependent phosphorylation.A yeast two-hybrid screening identified aserine/threonine protein phosphatase (PP5) as aninteractant of ER? (1–481), a dominant negativeER? mutant. Glutathione S-transferase pull-downassays, mammalian two-hybrid assays, and immunoprecipitationstudies showed that PP5 directlybinds to both ER? and ER? via its tetratricopeptiderepeat domain. E domains of ER? and ER?, withoutcontaining activation domain core regions in transcriptionactivation function 2, were required forthe binding to PP5. In ER?-positive breast cancerMCF7 cells, estrogen- and epidermal growth factordependentphosphorylation of ER? on serine residue118, a major phosphorylation site of the receptor,was reduced by expressing PP5 but enhanced byPP5 antisense oligonucleotide. Estrogen-inducedtranscriptional activities of both ER? and ER? andmRNA expression of estrogen-responsive genes,including pS2, c-myc, and cyclin D1, were suppressedby PP5 but enhanced by PP5 antisenseoligonucleotide. A truncated PP5 mutant consistingonly of its tetratricopeptide repeat domainacted as a dominant negative PP5 that enhancedserine residue 118 phosphorylation of ER? andtransactivations by ER? and ER?. We present thefirst evidence that PP5 functions as an inhibitoryregulator of ER phosphorylation and transcriptionalactivation in vivo. (Molecular