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Induction of serine and threonine protein phosphorylation by endotoxin-associated protein in murine resident
1Department of Microbiology and Immunology, State University of New York-Health Science Center at Brooklyn 11203.
Abstract:
Endotoxin-associated protein (EP) from Salmonella typhi activated murine resident peritoneal macrophages to produce prostaglandin E2 (PGE2). Cells from both endotoxin nonresponder (C3H/HeJ) and the endotoxin responder (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, suggesting the involvement of both serine and threonine phosphorylation and tyrosine phosphorylation pathways in the activation of resident peritoneal 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 phosphorylation was not induced by phorbol myristic acid or by lipopolysaccharide endotoxin. Inhibitors of PKC, PKA, and PKG did not block the phosphorylation of p14. However, the tyrosine kinase inhibitor piceatannol blocked p14 serine and threonine phosphorylation, suggesting that this phosphorylation is dependent upon and preceded by a tyrosine phosphorylation step.
Insights
Salmonella typhi endotoxin-associated protein (EP) activates macrophages to produce prostaglandin E2 (PGE2). This process involves both serine/threonine and tyrosine phosphorylation pathways, specifically activating a 14-kDa protein (p14).
Area of Science:
- Immunology
- Cellular Biology
- Biochemistry
Background:
- Salmonella typhi endotoxin-associated protein (EP) is known to interact with host immune cells.
- Macrophages play a crucial role in the inflammatory response, producing mediators like prostaglandin E2 (PGE2).
- The precise signaling pathways activated by EP in macrophages are not fully elucidated.
Purpose of the Study:
- To investigate the signaling mechanisms by which Salmonella typhi EP activates murine resident peritoneal macrophages.
- To identify the specific protein kinases and phosphorylation events involved in EP-induced PGE2 production.
Main Methods:
- Activation of macrophages from endotoxin responder and nonresponder mouse strains with EP.
- Inhibition studies using protein kinase C (PKC) inhibitor H-7 and tyrosine kinase inhibitor genistein.
- Immunoblot analysis using antiphosphoserine and antiphosphothreonine antibodies to detect protein phosphorylation.
- Assessment of the effect of various kinase inhibitors (PKC, PKA, PKG, tyrosine kinase inhibitor piceatannol) on p14 phosphorylation.
Main Results:
- EP activated macrophages from both endotoxin responder and nonresponder mice to produce PGE2.
- EP-induced PGE2 production was inhibited by both PKC and tyrosine kinase inhibitors.
- EP induced serine and threonine phosphorylation of a 14-kDa protein (p14), an event not triggered by phorbol myristic acid or lipopolysaccharide endotoxin.
- Tyrosine kinase inhibitor piceatannol blocked p14 serine and threonine phosphorylation, indicating a preceding tyrosine phosphorylation step.
Conclusions:
- Salmonella typhi EP activates macrophages via signaling pathways involving both serine/threonine and tyrosine kinases.
- EP specifically induces the phosphorylation of a 14-kDa protein (p14), which is dependent on prior tyrosine phosphorylation.
- These findings elucidate novel signaling mechanisms in macrophage activation by bacterial components.