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 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.
Vosseller
K
K
Molecular Biology Program, Sloan Kettering Institute, New York, New York 10021, USA.
Stella
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Yee
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Besmer
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D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology; D015550:Cell Degranulation / Q000502:physiology; D002462:Cell Membrane / Q000648:ultrastructure; D005353:Fibronectins / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D008407:Mast Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010835:Phytohemagglutinins; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases; D012701:Serotonin / Q000378:metabolism; D015398:Signal Transduction; D015854:Up-Regulation
D000199:Actins; D005353:Fibronectins; D007527:Isoenzymes; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D010835:Phytohemagglutinins; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D019009:Proto-Oncogene Proteins c-kit; D019893:Ribosomal Protein S6 Kinases; D012701:Serotonin