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, trigger ing production of cyclic GMP. Agents which stimulate protein kinase C (PKC), including phorbol esters, synergistically enhance STa effects o n cGMP and secretion. We investigated whether PKC causes phosphorylati on of the STa receptor in vivo and in vitro. Immunoprecipitation of th e STa receptor-guanylyl cyclase was carried out from extracts of T84 c olon cells metabolically labelled with [P-32]-phosphate using polyclon al anti-STa receptor antibody. The STa receptor was phosphorylated in its basal state, and P-32 content in the 150 kDa holoreceptor band inc reased 2-fold in cells exposed to phorbol ester for 1 h. In vitro, imm unopurified STa receptor was readily phosphorylated by purified rat br ain PKC. Phosphorylation was inhibited 40% by 5 mu M of a synthetic pe ptide corresponding to the sequence around Ser(1029) of the STa recept or, 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 vitr o and in vivo; Ser(1029) of the STaR/GC remains a candidate phosphoryl ation site by PKC.