Thyrotropin releasing hormone stimulates transient phosphorylation of the tissue-specific transcription factor, Pit-1.
The hypothalamic hormone, thyrotropin releasing hormone (TRH), stimulates prolactin (PRL) secretion and gene transcription in the GH3 pituitary cell line. Several studies have provided indirect evidence that phosphorylation of the pituitary-specific transcription factor Pit-1 may mediate TRH effects on PRL transcription. In the present study we have investigated the ability of TRH to alter the phosphorylation of Pit-1. In vivo 32P labeling experiments demonstrated that TRH stimulated a transient phosphorylation of Pit-1, reaching a maximum in 5 min and returning to basal levels within 30 min. Phosphopeptide mapping experiments demonstrated that TRH induced the transient phosphorylation of specific sites in Pit-1. TRH-stimulated phosphorylation of Pit-1 was blocked by treatments that deplete the cellular content of protein kinase C. Metabolic labeling and Western blot analysis demonstrate that TRH does not alter the total cellular content or nuclear concentration of Pit-1. TRH-mediated stimulation of a PRL promoter-luciferase fusion gene occurred under conditions that blocked the transient phosphorylation of Pit-1. These studies suggest that phosphorylation of Pit-1 may not be necessary for TRH mediated enhancement of PRL gene transcription.
Howard
P W
PW
Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland 97201.
Maurer
R A
RA
D001483:Base Sequence; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011388:Prolactin / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004268:DNA-Binding Proteins; D010748:Phosphopeptides; D013873:Thionucleotides; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate