Antigen stimulation to mast cells through high affinity receptors for IgE (FceRI) leads to Ca2+ mobilization and, as a consequence, the release of inflammatory mediators that are responsible for allergic diseases such as asthma. It is generally believed that inositol 1,4,5-trisphoshate (1P3) is responsible for mobilizing Ca2+ from the intracellular endoplasmic reticulum (ER) stores. Dr. Choi?s laboratory has shown that the amount of 1P3 produced by FcERI stimulation is not sufficient to cause Ca2+ mobilization through Fc aboutRl and sphingosine 1-phosphate (SiP), a product of sphingosine kinase (SK) may play a major role in Ca2+ mobilization through FcgRl. Currently the cytosolic form of SK has been cloned and Dr. Choi?s laboratory cloned a putative membrane-associated SK. The goal of this proposal is to test the hypothesis that Syk is upstream of SK activation. The possibility of other signaling molecules to be upstream of SK activation will also be investigated. RBL-2H3 cells will be used as a model system as FcERI-mediated signaling has been extensively studied in this cell line. Strategies will be 1) to determine the involvement of Syk in FcERI-mediated SK activation, 2) to determine the involvement of phospholipase Cy in FcERI-mediated SK activation 3) to determine the involvement of calcineurin in FcERI mediated SK activation 4) to determine the possible cross-talks between the upstream molecules suggested above.