Role of activated protein kinase C in the mechanism for MgATP-dependent Priming of exocytosis in adrenal chromaffin cells

以前，我们认为蛋白激酶C（PKC）在调节牛肾上腺嗜铬细胞胞吐过程中MgATP需要期，而不影响晚期MgATP非依赖性Ca^2+触发的膜融合步骤。RACK 1是一种活化PKC的受体，已通过筛选大鼠脑表达文库中结合活化PKC的蛋白质来鉴定。为了研究RACK 1可能参与PKC介导的MgATP需要阶段的胞吐调节，我们研究了RACK 1和PKC亚型在肾上腺嗜铬细胞的免疫细胞化学亚细胞定位。抗RACK 1抗体的免疫印迹测试表明RACK 1富集在细胞的Triton不溶部分中。用RACK 1抗体进行的免疫荧光实验表明，RACK 1蛋白主要定位于质膜下区，也存在于嗜铬细胞的核周区和胞浆中。质膜下区域的RACK 1与皮质共定位 ...更多信息 称为丝状肌动蛋白（F肌动蛋白）。嗜铬细胞内含有PKC α、β、ε和ε亚型，这些亚型分布于对照细胞的胞浆中。在12-0-tetradecanoylphorbol-13 acetate（TPA）刺激的细胞中，只有传统的PKCs α和β易位。PKCα与RACK 1共定位于质膜下区，PKCβ与RACK 1共定位于质膜下区、核周区和胞浆中。用肌动蛋白解聚剂mycalcohol B（MLB）将F-肌动蛋白解聚为G-肌动蛋白，处理细胞后，质膜下区皮质F-肌动蛋白和RACK 1的免疫反应性以剂量依赖的方式消失。MLB可抑制TPA刺激的PKCα和β向质膜下的转位。细胞松弛素D（cytochalasin D，CD）可使F-肌动蛋白断裂成短纤维，破坏FITC-鬼笔环肽皮质荧光环，使质膜下RACK 1的免疫荧光强度略有下降。CD不能阻止TPA刺激的PKCα和β向质膜下区域的移位。免疫印迹分析表明，MLB促进肌动蛋白和RACK 1从细胞的Triton不溶性部分的释放，表明RACK 1在细胞中与F肌动蛋白相互作用。抗RACK 1抗体免疫沉淀RACK 1和肌动蛋白从对照细胞的提取物。RACK 1与TPA处理的细胞提取物中的肌动蛋白、PKCα和PKC β在抗RACK 1抗体的作用下发生共沉淀。这些结果表明RACK 1与质膜下的F-actin紧密结合，并且在嗜铬细胞中活化的PKCα和β通过与RACK 1结合而与F-actin相互作用。TPA选择性增强MgATP依赖性从毛地黄皂苷透性嗜铬细胞释放。这种增强作用可被PKCα和β的选择性抑制剂抑制，并被常规PKC的选择性激活剂激活。MLB以剂量依赖性方式抑制TPA对释放的增强作用。相反，CD对增强作用没有影响。这些结果表明，激活的PKCα和β通过与RACK 1结合与F actin相互作用，是PKC激活MgATP需要胞吐途径启动阶段所必需的，可能在制备渗出机制中起重要作用。少

Previously, we suggested that protein kinase C (PKC) regulates MgATP-requiring stage without affecting the late MgATP-independent Ca^<2+>-triggered step immediately prior to membrane fusion in exocytosis in bovine adrenal chromaffin cells. RACK1, a receptor for activated PKC, has been identified by screening a rat brain expression library for proteins that binds activated PKC. To study a possible involvement of RACK1 in the PKC mediated regulation of the MgATP-requiring stage for exocytosis, we examined the subcellular localization of RACK1 and isoforms of PKC in adrenal chromaffin cells by immunocytochemistry. Immunoblotting tests with anti-RACK1 antibody indicated that RACK1 is enriched in Triton-insoluble fraction of the cells. Immunofluorescence with the anti-RACK1 antibody demonstrated that this protein is primarily localized in the subplasmalemmal region, and also in perinuclear region and cytosol of chromaffin cells. RACK1 in the subplasmalemmal region is co-localized with corti … More cal filamentous actin (F actin). Chromaffin cells contained PKC α, β, ε and ξ, and these PKC isoforms were distributed in the cytosol of control cells. In 12-0-tetradecanoylphorbol-13acetate (TPA) stimulated cells, only the conventional PKCs, α and β were translocated. After the translocation, PKCα was colocalized with RACK1 in subplasmalemmal region, and PKCβ was colocalized with RACK1 in subplasmalemmal region, perinuclear region and cytosol. Treatment of cells with an actin depolymerizing agent, mycalolide B (MLB) which severs F-actin to G-actin, produced a disappearance of the immunoreactivity of cortical F-actin and RACK1 in subplasmalemmal region in a dose-dependent manner. MLB prevented the translocation of PKCα and β to subplasmalemmal region by TPA stimulation. Treatment of cells with cytochalasin D (CD) which severs F-actin into short filaments, produced a disruption of FITC-phalloidin cortical fluorescent ring and the immunofluorescence intensity of RACK1 in subplasmalemmal region was slight decreased by CD. CD did not prevent the translocation of PKCα and β to subplasmalemmal region by TPA stimulation. Immunoblot analysis demonstrated that MLB facilitates the release of both actin and RACK1 from Triton-insoluble fraction of the cells, suggesting that RACK1 interacts with F actin in the cells. Anti-RACK1 antibody immunoprecipitated both RACK1 and actin from the extract of control cells. In addition, RACK1 was coimmunoprecipitated with actin, PKCα and β from the extract of TPA-treated cells by anti-RACK1 antibody. These results suggest a tight binding of RACK1 with subplasmalemmal F-actin, and that activated PKCα and β interact with F-actin through the binding to RACK1 in chromaffin cells. TPA selectively potentiated MgATP-dependent release from digitonin-permeabilized chromaffin cells. The potentiation was inhibited by a selective inhibitor of PKCα and β, and was activated by a selective activator of conventional PKCs. MLB inhibited the potentiation of release by TPA in a dose dependent manner. In contrast, CD had no effect on the potentiation. These results suggest that the interaction of activated PKCα and β with F actin through the binding to RACK1 is essential for the activation by PKC of MgATP requiring priming stage of exocytotic pathway, possibly in the preparation effusion machinery. Less