| Co-Investigator(Kenkyū-buntansha) |
SASAKAWA Nobuyuki Sophia University, Faculty of Science and Technology, Associate Professor, 理工学部, 助教授 (20187107)
KUMAKURA Konosuke Sophia University, Faculty of Science and Technology, Professor, 理工学部, 教授 (70129790)
NAGAMATSU Shinya Kyorin University School of Medicine, Professor, 医学部, 教授 (80231489)
前川 昌平 神戸大学, 大学院・自然科学研究科, 教授 (40173695)
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| Budget Amount *help |
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2000: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1999: ¥3,200,000 (Direct Cost: ¥3,200,000)
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| Research Abstract |
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
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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
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