GABP MEDIATES INSULIN-INCREASED PROLACTIN GENE-TRANSCRIPTION

The insulin-response element from the prolactin gene is identical to t he Ets-binding site, and dominant-negative Ets protein inhibits insuli n-increased prolactin gene expression. Immunoblotting identified the E ts-related transcription factor GABP in nuclear extracts from GH cells . Expression of GABP alpha and GABP beta 1 squelches insulin-increased prolactin gene expression. GABP alpha and GABP beta 1 bind the insuli n-response element of the prolactin promoter, and anti-GABP alpha and anti-GABP beta 1 antibodies supershift a species seen with nuclear ext racts from GH cells. GABP alpha immunoprecipitated from insulin-treate d, P-32 labeled GH cells was phosphorylated 3-fold more than GABP alph a from control cells. There was no increase in phosphorylation of GABP beta in response to insulin. Mitogen-activated protein (MAP) kinase a ctivity is increased 10-fold in insulin-treated GH4 cells. MAP kinase immunoprecipitated from control cells does not phosphorylate GABP alph a while MAP kinase immunoprecipitated from insulin-treated cells shows substantial phosphorylation of GABP alpha. These studies suggest that GABP mediates insulin-increased transcription of the prolactin gene. GABP may be regulated by MAP kinase phosphorylation.