Purification and mass spectrometric identification of GA-binding protein (GABP) as the functional pituitary Ets factor binding to the basal transcription element of the prolactin promoter.
The Ets-binding site within the basal transcription element (BTE) of the rat prolactin (rPRL) promoter is critical for both basal and growth factor-regulated rPRL gene expression. Here we report the purification and identification of the factor that binds to the BTE. This factor was purified from GH3 pituitary nuclear extracts using ammonium sulfate fractionation, heparin-Sepharose and Mono Q chromatography, and BTE-affinity magnetic beads. We purified two proteins of 57 and 47 kDa and identified the 57-kDa protein by mass spectrometry as the Ets factor GABPalpha. Western blot analysis identified the 47-kDa protein as GABPbeta1. Co-transfection of dominant-negative GABPbeta1 blocks prolactin promoter basal activity by 85-88% in GH3 cells in the presence or absence of FGF-4. Additionally, expression of wild-type GABPalpha/beta1 selectively activates a minimal BTE promoter 24-28-fold in GH3 cells, and this activation is dependent on the Ets-binding site. Finally, small interfering RNA depletion of GABP in GH3 cells results in the loss of prolactin protein. Thus, we have identified GABPalpha/GABPbeta1 as a critical and functionally relevant Ets factor that regulates rPRL promoter activity via the BTE site.
Schweppe
Rebecca E
RE
Department of Medicine, Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Melton
Alexis A
AA
Brodsky
Kelley S
KS
Aveline
Lauren D
LD
Resing
Katheryn A
KA
Ahn
Natalie G
NG
Gutierrez-Hartmann
Arthur
A
D000818:Animals; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D051268:GA-Binding Protein Transcription Factor; D005786:Gene Expression Regulation; D011388:Prolactin / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014157:Transcription Factors / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051268:GA-Binding Protein Transcription Factor; D021122:Protein Subunits; D014157:Transcription Factors; D011388:Prolactin