Study of SHIP‐binding cell surface proteins suggests c‐kit as a SHIP‐interacting receptor in mast cells

Abstract

AbstractMast cells play a central role in a wide range of immunological and pathological processes, but are most noted for their role in IgE‐dependent allergic responses. Aggregation of the high‐affinity receptor for IgE, FcηRI, stimulates mast cell degranulation, production of lipid mediators, and the synthesis and secretion of cytokines and chemokines. FcηRI‐induced mast cell activation is subject to regulation by inhibitory receptors that transduce intracellular signals via associating phosphatases. The inositol 5‐phosphatase SHIP has been implicated in FcγIIB‐mediated inhibition of FcηRI‐induced mast cell activation. However, SHIP also negatively regulates FcηRI signaling independent of FcγRIIB, suggesting the existence of additional receptors that mediate SHIP recruitment into sites where it mediates its inhibitory function. Here we show that SHIP associates with numerous phosphoproteins from pervanadate‐stimulated mast cells. Based on their sensitivity to PNGase F treatment and cell surface biotinylation, some of these molecules may represent cell surface receptors. A prominent 120−130 kDa SHIP‐binding phosphoprotein was identified in untreated RBL‐2H3 cells and BMMC stimulated with stem cell factor. Based on its molecular weight, sensitivity to PNGase F, and reactivity with an anti‐c‐kit antibody, we conclude that this phosphoprotein is c‐kit. Furthermore, tyrosine phosphorylation of SHIP is enhanced following SCF stimulation. Taken together, these data suggest that SHIP may function as a negative regulator of SCF signaling via direct association with phosphorylated c‐kit.