Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C.
The heat-stable enterotoxin STa of E. coli causes diarrhea by binding to and stimulating intestinal membrane-bound guanylyl cyclase, triggering production of cyclic GMP. Agents which stimulate protein kinase C (PKC), including phorbol esters, synergistically enhance STa effects on cGMP and secretion. We investigated whether PKC causes phosphorylation of the STa receptor in vivo and in vitro. Immunoprecipitation of the STa receptor-guanylyl cyclase was carried out from extracts of T84 colon cells metabolically labelled with [32P]-phosphate using polyclonal anti-STa receptor antibody. The STa receptor was phosphorylated in its basal state, and 32P content in the 150 kDa holoreceptor band increased 2-fold in cells exposed to phorbol ester for 1 h. In vitro, immunopurified STa receptor was readily phosphorylated by purified rat brain PKC. Phosphorylation was inhibited 40% by 5 microM of a synthetic peptide corresponding to the sequence around Ser1029 of the STa receptor, a site previously proposed as a potential PKC phosphorylation site. Treatment of the immunopurified STaR/GC with purified PKC increased STa-stimulated guanylyl cyclase activity 2-fold. We conclude that PKC phosphorylates and activates the STa receptor/guanylyl cyclase in vitro and in vivo; Ser1029 of the STaR/GC remains a candidate phosphorylation site by PKC.
Crane
J K
JK
Division of Infectious Diseases, State University of New York at Buffalo 14214, USA.
Shanks
K L
KL
D001427:Bacterial Toxins / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D003106:Colon / Q000201:enzymology*; D004768:Enterotoxins / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000378:metabolism*
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D010455:Peptides; D018000:Receptors, Peptide; C034151:heat stable toxin (E coli); D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled