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Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C

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Abstract

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 μM 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.

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Abbreviations

STa:

the heat-stable enterotoxin of E. coli, which has also been called ST-I and STp. The 18 amino acid variant was used throughout

PBS:

phosphate-buffered saline

PDB:

4-β-12, 13-phorbol dibutyrate

ANP:

atrial natriuretic peptide

STaR/GC:

STa receptor/guanylyl cyclase, also called GC-C

PKC:

protein kinase C

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  1. Division of Infectious Diseases, State University of New York at Buffalo, Room 109 Medical Research Bldg., 3435 Main St., 14214, Buffalo, NY, USA

    John K. Crane & Kathryn L. Shanks

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  1. John K. Crane
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Crane, J.K., Shanks, K.L. Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C. Mol Cell Biochem 165, 111–120 (1996). https://doi.org/10.1007/BF00229472

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  • DOI: https://doi.org/10.1007/BF00229472

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