Title: Quantitative analysis of regulatory proteins involved in mast cell FcεRI –mediated signaling
Authors: Diane Jimenez-Stinson1, Mara Steinkamp1, William S. Hlavacek2, and Bridget Wilson1
Affiliations: 1Department of Pathology & Cancer Research & Treatment Center, Albuquerque, NM, 2Los Alamos National Laboratory
Abstract: During an allergic response, mast cells are responsible for the release of pro-inflammatory mediators such as histamine that cause symptoms associated with allergies. Mast cells are activated in a complex cascade of events that starts with an allergen binding to multiple IgE/FcεRI receptor complexes on the mast cell surface. This binding induces receptor clustering and activates regulatory proteins in the FcεRI pathway that will lead to de-granulation. Understanding the regulation of this cellular response may aid in the development of novel treatments for allergic disorders. In order to examine the events that initiate FcεRI signaling, we have turned to in silico modeling that can better probe networks of protein interactions to determine pathway kinetics. In order to accurately predict outcomes, these models must include cellular concentrations of protein kinases that can be determined experimentally. Rat Basophilic Leukemia (RBL) cells serve as our model system, since they have an active FcεRI pathway and can de-granulate in response to allergens. Here, the concentrations of regulatory protein kinases in the FcεRI pathway (Lyn, Syk, Fyn, Csk, and the scaffolding proteins, Pag1 and Lat) were estimated using quantitative western blot analysis of RBL lysates. Protein concentrations are now being incorporated into a model of the FcεRI pathway that will be used to predict the cell's response to allergens under various conditions.