Vosseller K, Stella G, Yee N S, Besmer P
Molecular Biology Program, Sloan Kettering Institute, New York, New York 10021, USA.
Mol Biol Cell. 1997 May;8(5):909-22. 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.
在骨髓来源的肥大细胞（BMMCs）中，Kit受体酪氨酸激酶介导多种反应，包括增殖、存活、趋化性、迁移、分化以及与细胞外基质的黏附。在结缔组织肥大细胞中，Kit在炎症介质分泌中的作用也已得到证实。我们最近证明了磷脂酰肌醇-3'（PI 3）激酶在Kit配体（KL）诱导的BMMCs与纤连蛋白黏附中的作用。在此，我们研究了Kit介导BMMCs中FcεRI介导的脱颗粒增强、细胞骨架重排和黏附的机制。缺乏内源性Kit表达的Wsh/Wsh BMMCs被转导以表达在第719和821位残基处含有Tyr→Phe替代的正常和突变Kit受体。尽管正常的Kit受体完全恢复了Wsh/Wsh BMMCs中KL诱导的反应，但无法结合和激活PI 3激酶的Kitγ719F未能增强脱颗粒，并且在介导膜皱褶和肌动蛋白组装方面受损。用渥曼青霉素或LY294002抑制PI 3激酶也抑制了Kit介导的分泌增强和细胞骨架重排。相反，通过蛋白激酶C（PKC）直接刺激的分泌增强和黏附不需要PI 3激酶。PKC抑制剂钙泊三醇C阻断了Kit介导的与纤连蛋白的黏附、分泌增强、膜皱褶和丝状肌动蛋白组装。尽管细胞松弛素D抑制了Kit介导的丝状肌动蛋白组装和膜皱褶，但分泌增强和与纤连蛋白的黏附不受该药物影响。因此，依赖于肌动蛋白聚合的Kit介导的细胞骨架重排可以与Kit介导的分泌和黏附反应解偶联。我们的结果表明，受体近端PI 3激酶激活和PKC同工型激活参与了Kit介导的分泌增强、黏附和细胞骨架重组。
