INDUCTION OF SERINE AND THREONINE PROTEIN-PHOSPHORYLATION BY ENDOTOXIN-ASSOCIATED PROTEIN IN MURINE RESIDENT PERITONEAL-MACROPHAGES

Endotoxin-associated protein (EP) from Salmonella typhi activated muri ne resident peritoneal macrophages to produce prostaglandin E2 (PGE2). Cells from both endotoxin nonresponder (C3H/HeJ) and the endotoxin re sponder (C3H/OuJ) mouse strains were activated by EP. This EP-induced prostaglandin E2 production was blocked by the protein kinase C (PKC) inhibitor H-7 as well as the tyrosine kinase inhibitor genistein, sugg esting the involvement of both serine and threonine phosphorylation an d tyrosine phosphorylation pathways in the activation of resident peri toneal macrophages by EP. Immunoblot analysis using antiphosphoserine and antiphosphothreonine antibodies showed that EP induced the serine and threonine phosphorylation of a 14-kDa protein (p14). This phosphor ylation was not induced by phorbol myristic acid or by lipopolysacchar ide endotoxin. Inhibitors of PKC, PKA, and PKG did not block the phosp horylationof p14. However, the tyrosine kinase inhibitor piceatannol b locked p14 serine and threonine phosphorylation, suggesting that this phosphorylation is dependent upon and preceded by a tyrosine phosphory lation step.