The cross-regulation of Gi-protein by cholera toxin involves a phosphorylation by protein kinase A.
Pretreatment of alveolar macrophages with cholera toxin inhibits the release of arachidonic acid induced by the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine. The results presented here show that cholera toxin might exert its inhibitory effect through the phosphorylation of Gi alpha by protein kinase A (PKA). (1) Gi-proteins from cells pretreated with cholera toxin showed parallel increases in their sensitivity to ADP-ribosylation by toxins in vitro and in Gi alpha phosphorylation. By contrast, the Gi alpha concentration was unchanged. (2) Cholera toxin pretreatment also decreased the functional activity of Gi, as assessed by the inhibition (80%) of agonist-induced binding of guanosine-5'-[gamma-thio]triphosphate (GTP[gamma S]). (3) These effects of cholera toxin were blocked by a specific PKA inhibitor, N-(2-[methyl-amino]ethyl)-3-isoquinolinesulphonamide dihydrochloride (H8) and mimicked by a cyclic AMP (cAMP) analogue and a phosphatase inhibitor. (4) Gi alpha was also phosphorylated in vitro by the catalytic subunit of PKA. In contrast with other cell systems, the stimulation of protein kinase C seems to have no effect on the sensitivity of Gi to ADP-ribosylation or on its phosphorylation. Therefore, the phosphorylation of Gi-proteins by PKA seems to be the actual target of the negative control of arachidonic acid release via the cAMP-mediated pathway.
Levistre
R
R
URA CNRS 1283, Laboratoire de Biochimie, CHU Saint-Antoine, Paris, France.
Berguerand
M
M
Bereziat
G
G
Masliah
J
J
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006168:Guinea Pigs; D016676:Macrophages, Alveolar / Q000378:metabolism; D010766:Phosphorylation; D015290:Second Messenger Systems; D015398:Signal Transduction
D002772:Cholera Toxin; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins