Inhibition of protein phosphatase PP1 in GH3B6, but not in GH3 cells, activates the MEK/ERK/c-fos pathway and the human prolactin promoter, involving the coactivator CPB/p300.
The human (hPRL) PRL gene proximal promoter (-164/+15) is the target for numerous signal transduction pathways involving protein kinases. The inhibitor of Ser/Thr-protein phosphatases okadaic acid (OA) was shown to induce this promoter in rat pituitary GH3B6 through a synergism between increased amounts of the ubiquitous factor AP-1 and the pituitary-specific factor Pit-1. Here we show that this activation results mainly from transcriptional stimulation of the c-fos promoter leading to increased AP-1 activity. We report the surprising absence of the hPRL and c-fos promoter stimulation by OA in GH3 cells, closely related to GH3B6 cells, and we use this discrepancy to dissect the precise mechanism of action. c-fos gene activation involves the mitogen-activated kinase (MAPK)-ternary complex factor (TCF) pathway and can be obtained by expressing active V12ras in both cell lines. We show that OA acts by inhibiting protein phosphatase PP1, thereby protecting MAPK kinase (MEK)1/2 and/or a MEK1/2-kinase from dephosphorylation. PP1 inhibition of MEK activation by V12ras does not occur in GH3 cells, indicating that a distinct, PP1-sensitive phosphorylation site is used in GH3B6 cells to activate the TCF pathway in GH3B6 cells. Finally, we show that the synergistic OA activation of the hPRL promoter by Pit-1 and AP-1 is independent of the Pit-1 transactivation domain and is mediated by the general coactivator (CRE-binding protein)-binding protein (CBP)/p300.
Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Institut de Chimie B6, B-4000 Sart-Tilman, Belgique.
I
I
Manfroid
J A
JA
Martial
M
M
Muller
D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D050881:E1A-Associated p300 Protein; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes; D048728:MAP Kinase Kinase Kinase 1; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010911:Pituitary Neoplasms; D011388:Prolactin / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007527:Isoenzymes; D009687:Nuclear Proteins; C494187:POU1F1 protein, human; C494186:Pou1f1 protein, rat; D016760:Proto-Oncogene Proteins c-fos; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D019319:Okadaic Acid; D011388:Prolactin; D050881:E1A-Associated p300 Protein; C494362:Ep300 protein, rat; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D010749:Phosphoprotein Phosphatases; D018631:ras Proteins; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one