H2O2 produced by the respiratory burst of alveolar macrophages participates in autocrine signaling for activation of the protein kinases, ERKI and ERK2, and the transcription factor, AP-1. The goal of this proposal is to identify the key redox sensitive proteins in the ERK and AP-1 signaling pathways and determine the chemical modifications produced by H2O2 that modulate their activities. Preliminary studies and recent literature support the hypothesis that H2O2 participates in both ERK and AP-1 activation through reversible oxidation of critical cysteine residues in key signaling proteins. Stimulation of ERK activation by zymosan activated serum (ZAS) depends upon H2O2 produced by the respiratory burst. We hypothesize that H2O2 inhibits a protein tyrosine phosphatase (PTP) with substrate specificity for X, a component of a signaling pathway triggered by ZAS whose phosphorylation is essential for activation of the ERK pathway. We propose that H2O2 inhibits this PTP through reversible formation of a sulfenic acid intermediate. We also propose that H2O2 generated by the respiratory burst activates AP-1 through the JNK pathway by reversibly oxidizing the thioredoxin (Trx) bound to ASK1, resulting in the release and activation of ASK1, an upstream activator of the JNK module. Aim 1 is to determine the steps in the ERK pathway stimulated by ZAS that are dependent or independent on the generation of H2O2. Aim 2 is to determine the chemical mechanisms through which H2O2 acts on the ERK pathway. Aim 3 is to determine the pathways and chemical mechanisms through which H2O2 activates AP-1. The experimental plan is designed to test these hypotheses in a sequential manner. State-of-the-art and novel modifications of immunological and biochemical assays for signal transduction and measurement of oxidizing species, transfection of signaling proteins and antioxidant enzymes, and MALDI-TOF mass spectrometry will be used. Alveolar macrophages play a critical role in pulmonary inflammation through the production of inflammatory mediators that is mediated, in part, by the pathways to be investigated.