title: A Pit-1 threonine 220 phosphomimic reduces binding to monomeric DNA sites to inhibit Ras and estrogen stimulation of the prolactin gene promoter.
abstract: Pit-1 is a POU-homeodomain transcription factor that dictates the ontogeny of pituitary somatotrophs, lactotrophs, and thyrotrophs through regulation of their respective protein hormone genes: GH, prolactin (PRL), and TSHbeta. Although Pit-1 threonine 220 (T220) and serine 115 are protein kinase phospho-acceptor sites, the transcriptional role of Pit-1 phosphorylation remains unclear. In the rat PRL promoter (rPRL), Ras-stimulated transcription is mediated by binding of Ets-1 and Pit-1 at a composite site (FPIV). Ets-1 and Pit-1 physically interact, and Pit-1 T220 is a major Ets-1 contact point. T220 was mutated to aspartic acid (D, to mimic phosphorylation) or a neutral alanine (A), and DNA binding and transcriptional activity were tested. The Pit-1 T220D mutation reduced binding at monomeric Pit-1 sites (FPIV, PRL-1d), but not dimeric Pit-1 sites (FPI). Pit-1 T220A bound all sites with wild-type (WT) affinity. In transfections of HeLa cells, each Pit-1 mutant transcriptionally activated the -425rPRL promoter and cooperated with Ets-1 to WT levels. In contrast, Pit-1-mediated Ras activation of the -425 rPRL promoter was significantly inhibited by T220D. Finally, Pit-1 synergistic activation of the 2500-bp rPRL promoter with estrogen receptor was reduced by T220D compared with T220A and WT Pit-1. Thus, phosphorylation of Pit-1 T220 reduces binding to monomeric sites blunting Ras and estrogen/estrogen receptor stimulation of the rPRL gene promoter. Consequently, T220 phosphorylation of Pit-1 by protein kinase A, protein kinase C, or cell cycle-dependent kinases appears to serve as a regulatory switch, inhibiting Ras and estrogen/estrogen receptor regulatory pathways, while enhancing the cAMP/protein kinase A response, thus allowing a more precise integration of pituitary responses to distinct signaling stimuli.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Anschutz Medical Center, Aurora, Colorado 80045, USA.
authors.forename: Annie
authors.initials: A
authors.lastname: Jean
authors.forename: Arthur
authors.initials: A
authors.lastname: Gutierrez-Hartmann
authors.forename: Dawn L
authors.initials: DL
authors.lastname: Duval
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D010766:Phosphorylation; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011485:Protein Binding; D050784:Proto-Oncogene Protein c-ets-1 / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020218:Response Elements; D050817:Transcription Factor Pit-1 / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection / Q000379:methods
chemical_list: D004268:DNA-Binding Proteins; D050505:Mutant Proteins; D050784:Proto-Oncogene Protein c-ets-1; D011993:Recombinant Fusion Proteins; D050817:Transcription Factor Pit-1; D001224:Aspartic Acid; D004958:Estradiol; D011388:Prolactin; D015689:Oncogene Protein p21(ras)