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

• Published in: Molecular biology of the cell 1997-05, Vol.8 (5), p.909-922
• Main Authors: Vosseller, K, Stella, G, Yee, N S, Besmer, P
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Animals; Binding Sites; Cell Adhesion - physiology; Cell Degranulation - physiology; Cell Membrane - ultrastructure; Fibronectins - metabolism; Isoenzymes - metabolism; Mast Cells - cytology; Mast Cells - metabolism; Mast Cells - physiology; Mice; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Phytohemagglutinins; Protein Kinase C - metabolism; Protein-Serine-Threonine Kinases - metabolism; Proto-Oncogene Proteins c-kit - metabolism; Ribosomal Protein S6 Kinases; Serotonin - metabolism; Signal Transduction; Up-Regulation
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.8.5.909

In bone marrow-derived mast cells (BMMCs), the Kit receptor tyrosine kinase mediates diverse responses including proliferation, survival, chemotaxis, migration, differentiation, and adhesion to extracellular matrix. 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 investigated the mechanism by which Kit mediates enhancement of Fc epsilon RI-mediated degranulation, cytoskeletal rearrangements, and adhesion in BMMCs. Wsh/Wsh BMMCs lacking endogenous Kit expression, were transduced to express normal and mutant Kit receptors containing Tyr-->Phe substitution at residues 719 and 821. Although the normal Kit receptor fully restored KL-induced responses in Wsh/Wsh BMMCs, Kit gamma 719F, 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 mediated by Kit. In contrast, secretory enhancement and adhesion stimulated directly through protein kinase C (PKC) do not require PI 3-kinase. Calphostin C, an inhibitor of PKC, blocked Kit-mediated adhesion to fibronectin, secretory enhancement, membrane ruffling, and filamentous actin assembly. Although cytochalasin D inhibited Kit-mediated filamentous actin assembly and membrane ruffling, secretory enhancement and adhesion to fibronectin were not affected by this drug. Therefore, Kit-mediated cytoskeletal rearrangements that are dependent on actin polymerization can be uncoupled from the Kit-mediated secretory and adhesive responses. Our results implicate receptor-proximal PI 3-kinase activation and activation of a PKC isoform in Kit-mediated secretory enhancement, adhesion, and cytoskeletal reorganization.