C/EBPbeta contributes to cAMP-activated transcription of phosphoenolpyruvate carboxykinase in LLC-PK(1)-F+ cells.
Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme in renal gluconeogenesis. Activation of various PEPCK(-2300)Luc reporter constructs in LLC-PK(1)-F+ cells, a gluconeogenic line of porcine renal proximal tubule-like cells, by protein kinase A (PKA) is mediated, in part, through the cAMP-response element (CRE)-1 of the PEPCK promoter. Incubation of a CRE-1 containing oligonucleotide with nuclear extracts from LLC-PK(1)-F+ cells produced multiple bands, all of which were blocked by antibodies that are specific for C/EBPbeta but not for C/EBPalpha or C/EBPdelta. Treatment of cells with cAMP did not affect the expression of C/EBPbeta, but the observed binding activity was increased nearly threefold. Mutation of CRE-1 to a Gal-4 binding site reduced the PKA-dependent activation of PEPCK(-2300)Luc to 40% of that observed with the wild-type construct. Coexpression of a chimeric protein containing a Gal-4 binding domain and the transactivation domain of C/EBPbeta, but not of C/EBPalpha or CRE binding protein (CREB), restored full activation by PKA. A deletion construct that lacks the activation domain of C/EBPbeta functions as a dominant negative inhibitor. Thus the binding of C/EBPbeta to the CRE-1 may contribute to the cAMP-dependent activation of the PEPCK promoter in kidney cells.
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Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA.
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Taylor
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Curthoys
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D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005943:Gluconeogenesis / Q000502:physiology; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D018374:LLC-PK1 Cells; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D011993:Recombinant Fusion Proteins; D013552:Swine; D014158:Transcription, Genetic / Q000502:physiology*
D022763:CCAAT-Enhancer-Binding Protein-alpha; D017362:Cyclic AMP Response Element-Binding Protein; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019764:Phosphoenolpyruvate Carboxykinase (ATP)