This is a competitive renewal entitled, """"""""Enzymes modulating second messengers in neutrophils"""""""", requesting five more years of finding, for years 10-14. (adapted from the applicant's abstract) The objective of this proposal is to define the exact sequence of molecular events that are involved in the stimulation of phagocytic leukocytes. Knowledge gained from these studies will likely to lead to novel strategies for treating infectious diseases and inflammation. This project will now focus on two distinct but critical reactions in the stimulation of neutrophils: (1) the dephosphorylation/activation of cofilin, and (2) the role of different isozyme of protein kinase C (PKC) in the activation of the NADPH oxidase complex which triggers superoxide generation. The investigator and collaborators have recently reported that cofilin undergoes rapid dephosphorylation/activation in stimulated neutrophils Cofilin is an essential actin depolymerizing agent that is critically involved in regulating the actin cytoskeleton, particularly the functional responses of neutrophils, e.g., chemotaxis, phagosome formation and degranulation. Moreover, the investigator has presented evidence that cofilin is regulated by a novel cycle of phosphorylation and dephosphorylation in neutrophils. The nature of the kinases and phosphatase(s) that participate in this cycle are unknown. Specifically, this project focuses on four unexplored areas (five aims) in the signal transduction pathways of neutrophils. These are (1) identifying and characterizing the protein kinase (aim 1) and phosphatase (aims 2) that catalyze the phosphorylation and dephosphorylation of cofiling in neutrophils, (2) elucidating the regulatory mechanisms that trigger dephosphorylation/activation of cofiling during cell stimulation (aim 3), (3) continue investigating the role of PKC in superoxide production with each of the purified isozymes of PKC that are present in neutrophils and their recombinant substrates, i.e., the oxidase subunits p47-phox and p67- phox (aim 4), and (4) monitoring the assembly of the superoxide generating system by immunofluorescence techniques and characterizing interactions between this complex and the cytoskeleton (aim 5). Co-localization of specific isozymes of PKC with the oxidase subunits will be bought. The goal is to forge a solid line between the regulatory properties of the isolated enzymes and control of the stimulus-response phenomena in phagocytic leukocytes.
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