The major current goal of project is to study the biochemistry and cell biology of Macrophage Stimulating Protein (MSP) and its receptor protein tyrosine kinase (RON). Investigations to date suggest that this ligand/ receptor pair may affect a number of cells that participate in the response to tissue injury, including skin cells, macrophages and capillary endothelium. Our major findings this year are summarized below. 1. We have made progress in purifying the wound fluid enzyme that cleaves pro-MSP to active MSP. After multiple purification steps, SDS-PAGE of the product reveals about 8 bands by silver stain. We can detect enzymatic activity in one spot in the gel after eluting out the SDS, by cleavage of a model substrate incorporated in a membrane applied to the gel. Since the cleaved substrate becomes fluorescent, its location can be ascertained. Our hope is to scale up the procedure to the point where the correctly selected segment of the gel can be cut out for protein identification by mass spectroscopy. 2. RON, the receptor for MSP, can be activated without specific ligand when epithelial cells are plated onto an extracellular matrix (ECM) like collagen. Adhesion of RON-expressing epithelial cells to ECM caused phosphorylation of RON, which depended on kinase activity of both RON itself and c-Src. This conclusion is based on the following observations. [1] ECM-induced RON phosphorylation was inhibited in cells expressing kinase-inactive c-Src. [2] Active c-Src could phosphorylate immunoprecipitated RON from ECM-stimulated cells, but not unstimulated cells. [3] ECM did not cause RON phosphorylation in cells expressing kinase-dead RON, nor could active c-Src phosphorylate RON immunoprecipitated from these cells. The data fit a pathway in which ECM-induced integrin aggregation causes both c-Src activation and RON oligomerization followed by RON kinase-dependent autophosphorylation. This results in RON becoming a target for activated c-Src, which phosphorylates additional tyrosines on RON. Integrin-induced epidermal growth factor receptor (EGFR) phosphorylation also depended on both EGFR and c-Src kinase activities. Taken together our results suggest that this sequence appears to be a general pathway for integrin-dependent growth factor receptor activation. 3. Beta-catenin is an oncogenic protein involved in regulation of cell-cell adhesion and gene expression. Accumulation of cellular beta-catenin occurs in many types of human cancers. Four mechanisms are known to cause increases in beta-catenin: mutations of beta-catenin, adenomatous polyposis coli (APC) or axin genes and activation of Wnt signaling. We have identified a new cause of beta-catenin accumulation involving oncogenic mutants of RON, a member of the MET receptor tyrosine kinase (RTK) family. Cells transfected with oncogenic RON were characterized by beta-catenin tyrosine phosphorylation, beta-catenin protection from proteasomal degradation through inhibition of complex formation with axin, and increased beta-catenin potential targets