細胞質分裂期におけるリン酸化ミオシンIIの役割解明に関する研究

• Project/Area Number: 10213206
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas (B)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Hiroshima University
• Principal Investigator: 細谷 浩史 広島大学, 大学院・理学研究科, 教授 (90183102)
• Co-Investigator(Kenkyū-buntansha): 祐村 恵彦 山口大学, 理学部, 助教授 (70183986)
• Project Period (FY): 1998 – 2001
• Project Status: Completed (Fiscal Year 2001)
• Budget Amount: ¥51,700,000 (Direct Cost: ¥51,700,000); Fiscal Year 2001: ¥12,800,000 (Direct Cost: ¥12,800,000); Fiscal Year 2000: ¥12,900,000 (Direct Cost: ¥12,900,000); Fiscal Year 1999: ¥14,500,000 (Direct Cost: ¥14,500,000); Fiscal Year 1998: ¥11,500,000 (Direct Cost: ¥11,500,000)
• Keywords: ミオシン調節軽鎖 / ミオシン重鎖 / 細胞骨格 / アクチン / ミオシンII / リン酸化 / ストレスファイバー / 収縮環 / ミオシン軽鎖 / 細胞分裂 / 細胞質分裂 / ZIPキナーゼ / MRLC / ミオシン2重リン酸化 / ミオシンリン酸化 / MLCK / Zipキナーゼ / ミオシン軽鎖キナーゼ / MLCIC / ミオシン / HeLu 細胞

Research Abstract

細胞分裂期におけるミオシンリン酸化の役割解明を目的として、ヒーラ細胞を実験材料に使用して以下の成果を得た。
1.19Serがリン酸化されたミオシン調節軽鎖を特異的に認識するモノクローナル抗体を作製した。
2.18Thrおよび19Serの2ケ所が2重リン酸化されたミオシン調節軽鎖を特異的に認識するモノクローナル抗体を作成した。
3.これらの抗体を用いて、間期および分裂期のヒーラ細胞を染色したところ、間期の細胞ではストレスファイバーが染色され、分裂期の細胞では収縮環の領域が染色されることが明らかになった。
4.2重リン酸化ミオシンに対する抗体およびミオシン重鎖の抗体を用いて分裂期の細胞を2重染色したところ、収縮環領域では、前者はdisc状に染色像が観察されたが、後者の染色像はリング状であることが明らかになった。これらの結果から、収縮環でミオシン重鎖と軽鎖が必ずしも共局在しないという事実が明らかにされた。
5.ミオシン調節軽鎖のリン酸化部位に関する変異体を作成しヒーラ細胞への導入を行ったところ、18Thrおよび19Serにアスパラギン酸を導入した疑似2重リン酸化ミオシン調節軽鎖を導入した細胞では、細胞分裂の阻害は特に観察されないが、これらの変異ミオシン調節軽鎖は収縮環領域に局在することが明らかにされた。19Serにアスパラギン酸を導入した疑似1重リン酸化ミオシン調節軽鎖の場合でも同様の結果が観察されたが、収縮環への局在は前者ほど顕著ではなかった。また野生型ミオシン調節軽鎖を導入した場合では、収縮環への局在は顕著に観察されなかった。

Research Products

• [Publications] M.A.Okubo: "A monoclonal antibody, mHl, specifically recognizes micrerilli of dividing HeLa cells"Tiss.Cult.Res.Commun.. 18. 113-117 (1999)
• [Publications] G.Totsukawa: "Activation of myosin phosphatase targeting subunit by mitosis-specific phosphorylation"J.Cell Biol.. 144(4). 735-744 (1999)
• [Publications] M.A.Okubo: "Generation and characterization of a monoclonal antibody, mHl, raised against mitotic HeLa cells"Dev.Growth Differ.. 41(4). 381-389 (1999)
• [Publications] N.Nishihara: "Microtubule-dependent movement of symbiotic algae and granules in Paramecium bursaria"Cell Motil.Cytoskeleton.. 43(2). 85-98 (1999)
• [Publications] M.Murata-Hori: "ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells"FEBS Lett.. 451(1). 81-84 (1999)
• [Publications] M.Murata-Hori: "A monoclonal antibody raised against an 86-kD subunit of human DNA helicase II"Zool.Sci.. 17(9). 1255-1259 (2000)
• [Publications] M.Murata-Hori: "Myosin II regulatory light chain as a novel substrate for AIM-l, an Aurora/Ipllp-related kinase from rat"J.Biochem.. 128(6). 903-907 (2000)
• [Publications] B.I.Gerashchenko: "Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria"Cytometry. 41(3). 209-215 (2000)
• [Publications] B.I.Gerashchenko: "Enrichment for late-telophase cell populations using flow cytometry"Cytometry. 41(2). 148-149 (2000)
• [Publications] F.Suizu: "Activation of actin-activated MgATPase activity of myosin II by phosphorylation with MAPK-activated protein kinase-1b (RSK-2)"J.Biochem.. 128(3). 436-440 (2000)
• [Publications] B.I.Gerashchenko: "Myosin regulatory light chain as a critical substrate of cell death : a hypothesis"Med.Hypoth.. 54(5). 850-852 (2000)
• [Publications] M.Hino: "Small heat shock protein 27(HSP27) associates with tubulin/microtubules in HeLa cells"Biochem.Biophys.Res.Commun.. 271(1). 146-169 (2000)
• [Publications] T.Saitoh: "Differential localization of non-muscle myosin II isoforms and phosphorylated regulatory light chains in human MIRC-5 fibroblasts"FEBS Lett.. 509. 365-369 (2001)
• [Publications] M.Murata-Hori: "HeLaZIP kinase induces diphosphorylation of Myosin II regulatory light chain and reorganization of actin filaments in nonmuscle cells"Oncogene. 20. 8175-8183 (2001)
• [Publications] T.Kawano: "A Fungal anxin antagonist, hypaphorine competitively inhibits indole-3-acetic acid-dependent superoxide generation by horseradish peroxidase"Biochem.Biophys.Res.Commun.. 288. 546-551 (2001)
• [Publications] B.I.Gerashchenko: "Model for regulation of non-muscle myosnis"Fiziologichny Zhrbal. 47(2). 100-105 (2001)
• [Publications] S.Komaba: "Identificafion of myosin II kinase from sea urchin eggs as protein kinase CK2"Gene. 275(1). 141-148 (2001)
• [Publications] B.I.Gerashchenko: "Growth kinetics of algal populations exsymbiotic from Paramecium bursaria by flow cytometry measurements"Cytometry. 44(3). 257-263 (2001)
• [Publications] B.I.Gerashchenko: "On regulation of nonmuscle myosin-II"Recent Research Developments in Biophysics and Biochemistry. 1. 1-7 (2001)
• [Publications] B.I.Gerashchenko: "New horizons for endosymbiosis of algae in Paramecium bursaria in light of flow"Recent Research Developments in Biophysics and Biochemistry. 1. 145-160 (2001)
• [Publications] T.Kawano: "Spectroscopic evidence that salicylic acid converts a temporal inactive form of horseradish peroxidase(Compound III) to the irreversibly inactivated verdohemoprotein(P-670)"Biosci Biotechnol.Biochem.. 66(3). 646-650 (2002)
• [Publications] B.I.Gerashchenko: "Phosphorylation at threonine-18 in addition to phosphorylation at Serine-19 on myosin-II regulatory light chain is a mitosis-specific event"Cytometry. 47. 150-157 (2002)
• [Publications] T.Iwasaki: "Diphosphorylated MRLC is required for organization of stress fibers in interphase cells and the contractile ring in dividing cells"Cell struc.Funct.. 26. 677-683 (2001)
• [Publications] T.Kawano: "Spectroscopic evidence in support of horseradish peroxidase Compound II-catalyzed oxidation of salicylic acid but not of phenylethylamine"Biosci.Biotechnol.Biochem.. 66(3). 651-654 (2002)
• [Publications] B.Gerashchen.Ko: "Myosin regulatory light chain as a critical substrate of cell death : a hypothesis."Medical Hypethesis. 54. 850-852 (2000)
• [Publications] M.Hino: "Small heat shack protein 27 (HSP27) associates with tubulin/microtubules in HeLa cells Biochem.Biophys.Res.Commun.,"Biochem.Biophys.Res.Commun. 271. 164-169 (2000)
• [Publications] F.Suizu: "Activation of actin-activated MgATPase activity of myosin II by phosphorylation with MAPK-activated protein kinase-1b (PSK-2)"J.Biochem. 128. 435-440 (2000)
• [Publications] B.Gerashchen Ko: "Strategy for study of host-symbiont cellular interactions : Symbiotic algae in light of flow cytometry."Cytometry. 41. 209-215 (2000)
• [Publications] B.Gerashchen Ko: "Enrichment for late telophase cell populations using flow cytometry."Cytometry. 41. 148-149 (2000)
• [Publications] M.Murata-Hori: "A monoclonal antibody raised against a 86-kD subunit of DNA helicase II."Zool.Sci. 17. 1255-1259 (2000)
• [Publications] M.Murata-Hori: "Myosin II Regulatory Light Chain as a Novel Substrate for AIM-1, an Aurora/Ipl1p-Related Kinase from Rat."J.Biochem. 128. 903-907 (2000)
• [Publications] M-A.Okubo et al: "A monoclonal antibody, mHl, specifically recognizes microvilli of dividing HeLa cells"Tiss.Cult.Res.Comm.. 18. 113-117 (1999)
• [Publications] M-A.Okubo et al: "Generation and characterization of a monoclonal antibody, mHl, raised against mitotic HeLa cells"Develop.Growth.Differ. 41. 381-389 (1999)
• [Publications] G.Totsukawa et al: "Activation of myosin phosphatase targeting subunit by mitosis-specific phosphorylation"J.Cell Biol.. 144. 735-744 (1999)
• [Publications] N.Nishihara et al: "Microtubule-dependent movement of symbiotic algae and small particles in green paramecia Paramecium bursaria"Cell Motil.Cytoskeleton. 43. 85-98 (1999)
• [Publications] M.Murata-Hori et al: "ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells"FEBS lett.. 451. 81-84 (1999)
• [Publications] B.Gerashchenko et al: "Myosin regulatory light chain as a critical substrate of cell death : a hypothesis"Medical Hypothesis. (accepted 20th/July(in press)). (2000)
• [Publications] T,Takahashi: "Regional differentiation of cortical structures and their reorganization during cell division in Paramecium trichium." J,Euk.Microbiol. 45. 369-380 (1998)
• [Publications] M,Murata-Hori: "Singly phosphorylated myosin II regulatory light chain is localized in the cleavage furrow of dividing HeLa cells" Biomed.Res.19. 111-115 (1998)
• [Publications] H-A.Okubo: "A monoclonal antibody,mHl,specifically recognizes microvilli of dividing HeLa cells." Tiss.Cult.Res.Comm.(in press). (1999)
• [Publications] H-A.Okubo: "Generation and characterization of a monoclonal antibody,mHl,raised against mitotic HeLa cells" Develop.Growth Differ.(in press). (1999)
• [Publications] G.Totsukawa: "Activation of myosin phosphatase targeting subunit by mitosis-specific phosphorylation." J,Cell Biol.144. 735-744 (1999)
• [Publications] M.Murata-Hori: "ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells." FEBS lett. (in press). (1999)