The long range objective of this proposal is to gain an understanding of the intracellular signalling pathway(s) used by bacterial endotoxin (LPS), a well known primary initiator of endotoxin shock. It is well established that LPS, when complexed with a plasma protein, LPS binding protein, binds to CDl4 on the surface of macrophages. While consequence of LPS binding to cells is clearly cell activation, intracellular signalling activated by LPS is ill-defined. Recently we identified and purified p38, a novel substrate of LPS induced protein tyrosine phosphorylation, which is homologous to. several nonmammalian protein kinases, and which may provide new insights to LPS signalling. We propose that protein tyrosine phosphorylation in general is a downstream event occurring after LPS engages CD14 on the cell surface and that tyrosine phosphorylation of p38 in particular transduces signal in LPS-induced cell activation. To evaluate this hypothesis we will use the following approaches. We will isolate and characterize a full-length cDNA clone of p38, then express recombinant p38 for use in anti-p38 antibody production and characterization of the biochemical and enzymatic properties of p38. Inhibition or overexpression of p38 in in vitro cell system will be performed to evaluate the. importance of p38 in LPS-induced cell activation. Effort will also be given to identify upstream or downstream molecules that interact with p38. The proposed work in this application will contribute to the eventual definition of the LPS signalling path way. Understanding the mechanism for LPS induced cell responses will help to develop therapeutic procedures for the treatment of endotoxin shock.
