Abstract
The insulin-response element from the prolactin gene is identical to the Ets-binding site, and dominant-negative Ets protein inhibits insulin-increased prolactin gene expression. Immunoblotting identified the Ets-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 insulin-response element of the prolactin promoter, and anti-GABP alpha and anti-GABP beta 1 antibodies supershift a species seen with nuclear extracts from GH cells. GABP alpha immunoprecipitated from insulin-treated, 32P-labeled GH cells was phosphorylated 3-fold more than GABP alpha from control cells. There was no increase in phosphorylation of GABP beta in response to insulin. Mitogen-activated protein (MAP) kinase activity is increased 10-fold in insulin-treated GH4 cells. MAP kinase immunoprecipitated from control cells does not phosphorylate GABP alpha 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.
Highlights
GABP Mediates Insulinincreased Prolactin Gene Transcription*From the Departments of Medicine and Pharmacology, New York University Medical Center, New York, New York 10016
The activation of gene transcription by hormones that function through protein-tyrosine kinase receptors is not well understood in comparison with that mediated by other classes of hormones
The fusion protein consists of the GST protein (26 kDa) and GABP␣-(76 –336) (30 kDa) that contains the three potential Mitogen-activated protein (MAP) kinase phosphorylation sites
Summary
From the Departments of Medicine and Pharmacology, New York University Medical Center, New York, New York 10016. The insulin-response element from the prolactin gene is identical to the Ets-binding site, and dominant-negative Ets protein inhibits insulin-increased prolactin gene expression. MAP kinase immunoprecipitated from control cells does not phosphorylate GABP␣ while MAP kinase immunoprecipitated from insulin-treated cells shows substantial phosphorylation of GABP␣ These studies suggest that GABP mediates insulin-increased transcription of the prolactin gene. Neither the hormone-responsive DNA element nor the transcription factors activated by protein-tyrosine kinase receptors are known. The increase in the transcription of these genes in insulin-treated cells was inhibited by expression of a dominant-negative Ets protein (5). These studies identify the predominant Ets-related protein of GH4 cells, GABP␣, and suggest that GABP mediates insulinincreased prolactin gene expression. Phosphorylation of GABP by MAP1 kinase may regulate its activity
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