Protein kinase C regulates transcription of the human guanylate cyclase C gene.
Guanylate cyclase C is the receptor for the bacterial heat-stable enterotoxins and guanylin family of peptides, and mediates its action by elevating intracellular cGMP levels. Potentiation of ligand-stimulated activity of guanylate cyclase C in human colonic T84 cells is observed following activation of protein kinase C as a result of direct phosphorylation of guanylate cyclase C. Here, we show that prolonged exposure of cells to phorbol esters results in a decrease in guanylate cyclase C content in 4beta-phorbol 12-myristate 13-acetate-treated cells, as a consequence of a decrease in guanylate cyclase C mRNA levels. The reduction in guanylate cyclase C mRNA was inhibited when cells were treated with 4beta-phorbol 12-myristate 13-acetate (PMA) in the presence of staurosporine, indicating that a primary phosphorylation event by protein kinase C triggered the reduction in RNA levels. The reduction in guanylate cyclase C mRNA levels was not due to alterations in the half-life of guanylate cyclase C mRNA, but regulation occurred at the level of transcription of guanylate cyclase C mRNA. Expression in T84 cells of a guanylate cyclase C promoter-luciferase reporter plasmid, containing 1973 bp of promoter sequence of the guanylate cyclase C gene, indicated that luciferase activity was reduced markedly on PMA treatment of cells, and the protein kinase C-responsive element was present in a 129-bp region of the promoter, containing a HNF4 binding element. Electrophoretic mobility shift assays using an oligonucleotide corresponding to the HNF4 binding site, indicated a decrease in binding of the factor to its cognate sequence in nuclear extracts prepared from PMA-treated cells. We therefore show for the first time that regulation of guanylate cyclase C activity can be controlled at the transcriptional level by cross-talk with signaling pathways that modulate protein kinase C activity. We also suggest a novel regulation of the HNF4 transcription factor by protein kinase C.
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.
N
N
Roy
M R
MR
Guruprasad
P
P
Kondaiah
E A
EA
Mann
R A
RA
Giannella
S S
SS
Visweswariah
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D015153:Blotting, Western; D020134:Catalytic Domain; D002460:Cell Line; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006162:Guanylate Cyclase / Q000235:genetics* / Q000378:metabolism; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000235:genetics* / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; D012333:RNA, Messenger; D018000:Receptors, Peptide; D014157:Transcription Factors; D011493:Protein Kinase C; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate