C-KIT RECEPTOR SIGNALING THROUGH ITS PHOSPHATIDYLINOSITIDE-3'-KINASE-BINDING SITE AND PROTEIN-KINASE-C - ROLE IN MAST-CELL ENHANCEMENT OF DEGRANULATION, ADHESION, AND MEMBRANE RUFFLING

In bone marrow-derived mast cells (BMMCs), the Kit receptor tyrosine k inase mediates diverse responses including proliferation, survival, ch emotaxis, migration, differentiation, and adhesion to extracellular ma trix. In connective tissue mast cells, a role for Kit in the secretion of inflammatory mediators has been demonstrated as well. We recently demonstrated a role for phosphatidylinositide-3' (PI 3)-kinase in Kit- ligand (KL)-induced adhesion of BMMCs to fibronectin. Herein, we inves tigated the mechanism by which Kit mediates enhancement of Fc epsilon RI-mediated degranulation, cytoskeletal rearrangements, and adhesion i n BMMCs. W-sh/W-sh BMMCs, lacking endogenous Kit expression, were tran sduced to express normal and mutant Kit receptors containing Tyr --> P he substitutions at residues 719 and 821. Although the normal Kit rece ptor fully restored KL-induced responses in W-sh/W-sh BMMCs, Kit(Y719F ), which fails to bind and activate PI 3-kinase, failed to potentiate degranulation and is impaired in mediating membrane ruffling and actin assembly. Inhibition of PI 3-kinase with wortmannin or LY294002 also inhibited secretory enhancement and cytoskeletal rearrangements mediat ed by Kit. In contrast, secretory enhancement and adhesion stimulated directly through protein kinase C (PKC) do not require PI 3-kinase. Ca lphostin C, an inhibitor of PKC, blocked Kit-mediated adhesion to fibr onectin, secretory enhancement, membrane ruffling, and filamentous act in assembly. Although cytochalasin D inhibited Kit-mediated filamentou s actin assembly and membrane ruffling, secretory enhancement and adhe sion to fibronectin were not affected by this drug. Therefore, Kit-med iated cytoskeletal rearrangements that are dependent on actin polymeri zation can be uncoupled from the Kit-mediated secretory and adhesive r esponses. Our results implicate receptor-proximal PI 3-kinase activati on and activation of a PKC isoform in Kit-mediated secretory enhanceme nt, adhesion, and cytoskeletal reorganization.