Abstract
Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is induced upon activation of protein kinase A by cAMP and phosphorylation of Ser-133 in the transcription factor, cAMP-response element binding protein (CREB), and this induction is inhibited by insulin. We show here that insulin does not act by dephosphorylating CREB or by affecting heterologous kinases that phosphorylate Ser-129 or Ser-142 in CREB. In addition, insulin inhibition of minimal PEPCK promoter activity induced by CREB-GAL4 + protein kinase A was equivalent to inhibition of basal transcription, and thus cAMP-independent. On the other hand, nearly complete insulin inhibition is observed with the full PEPCK promoter (-600/+69), indicating that other factors are involved. The additional promoter elements required for induction by protein kinase A lie within -271 nucleotides of the start site and correspond to putative binding sites for activator protein-1 and CAAT/enhancer-binding protein (C/EBP), first identified by Roesler et al. (Roesler, W. J., McFie, P. J., and Puttick, D. M., (1993) J. Biol. Chem. 268, 3791-3796). This tripartite array of binding sites for CREB, C/EBP, and activator protein-1 (AP-1) factors forms a cAMP response unit that, together with the minimal promoter, can mediate both induction by cAMP and inhibition by insulin. Thus, for the PEPCK gene with a single CREB site, the CREB.CBP.RNA polymerase II complex cannot mediate either induction by cAMP or inhibition by insulin.
Highlights
Both cAMP and insulin alter the activities of protein kinases and phosphatases, exerting their acute effects through changes in the phosphorylation state of a variety of regulatory molecules in the cell [1,2,3,4,5,6]
We previously showed that multiple cAMP-response element binding protein (CREB) binding sites ligated to a minimal phosphoenolpyruvate carboxykinase (PEPCK) promoter could mediate induction by PKA and that this could be at least partly inhibited by insulin [39]
The data presented here demonstrate that the P-CREB/CBP/ polymerase complex is not sufficient to mediate insulin inhibition of cAMP-induced PEPCK gene transcription, as we [39] and others [40] previously proposed
Summary
P-CREB Analysis by Western Blot—Nuclear extracts were prepared from H4IIe cells after 30 min of hormone treatment, a time at which transcription induction of PEPCK by cAMP in these cells is maximal. 1. The PKA site in CREB remains phosphorylated in H4IIe cells treated with insulin. H4IIe cells were treated with nothing, 8-(4-chlorophenylthio)-cAMP (1 mM), and/or insulin (10 nM), as indicated, for 30 min. Aliquots of the nuclear extracts were loaded onto two gels in parallel, which were electrophoresed and transferred to polyvinylidene difluoride membranes. One of these was probed with anti-PKA-phospho-CREB antibody and the other with anti-CREB antibody, and the blots were developed with chemiluminescent reagents and exposed to film. A promoter fragment containing the desired mutation was sequenced in its entirety and used to replace the corresponding fragment in the wild type reporter. All promoter sequences were verified to be correct by DNA sequencing of the final plasmids
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