The overall objective of the proposed research is to elucidate the biochemical events involved in signal transduction during LPS-triggered tumoricidal activation of macrophages. Based on our initial results, we propose to examine a hypothesis that protein kinase C (PKC), which plays a key role in tumoricidal activation, is activated, following LPS-macrophage interaction, through two different pathways. In both LPS-activatable and nonactivatable macrophage cell lines, LPS activates PIP2-specific phospholipase C, leading to the generation of the second messengers, DAG and Ca2+ released from intracellular storage site by the action of IP3, which then activate PKC. In LPS-activatable cells, LPS may interact with the 30kDa subunit of the Ca2+-dependent neutral protease, calpain, to activate the 80kDa subunit of calpain, which then cleaves PKC at the V3 region to generate the catalytic domain fragment denoted as PKM. Non- activatable cells may lack the second PKC activation mechanism due to either structural alteration of the V3 region of PKC or qualitative and/or quantitative defects in the calpain/calpastatin system. The hypothesis will be tested by pursuing four specific aims: 1) to investigate whether or not LPS treatment of LPS-activatable cells will result in the calpain- catalyzed cleavage of PKC into regulatory and catalytic domain fragments; 2) to determine whether or not PKC in LPS-non-activatable cells id different in structure from that in activatable cells; 3) to characterize the LPS-mediated regulation of calpain in mouse macrophages; and 4) to investigate, in collaboration with Dr. Morrison (project #1 leader), whether or not the 80 kDa LPS-binding protein (an LPS receptor) and the 80 kDa subunit of calpain are related. Our approaches will be: 1) affinity labeling of regulatory and catalytic domains of PKC with the radioactive ligands specific for each domain; 2) production of polyclonal antibodies directed against each domain by using synthetic peptides as antigens; 3) isolation and characterization of PKC and its domains; 4) isolation and sequence analysis of PKCbeta gene of LPC-activatable and non-activatable cells; 5) characterization of calpain and calpastatin; 6) studies of the interaction between LPS and two subunits of calpain isolated from macrophages; and 7) comparison of the LPS-binding protein and the 80kDa subunit of calpain. Many of these studies will be carried out in collaboration with Drs. Morrison and Russell. Some of the biochemical findings should also be applicable to Dr. Parmely's studies of TGF-beta. Results obtained will be important because they are expected to illuminate early signal transduction events in macrophage activation for tumor cell killing, the structure and function of PKC, the nature of the interaction between PKC and calpain/calpastatin, the regulatory mechanism of LPS- induced activation of calpain, and some of the biochemical bases of aberrant macrophage responses.

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