Studies on the Regulation of Smooth Muscle Myosin by Light-Chain Phosphorylation

• Project/Area Number: 12680667
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biophysics
• Research Institution: National Cardiovascular Center Research Institute
• Principal Investigator: ONISHI Hirofumi National Cardiovascular Center Research Institute, Structural Analysis, Section chief, 循環器形態部, 室長 (80092542)
• Project Period (FY): 2000 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥3,500,000 (Direct Cost: ¥3,500,000); Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000); Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
• Keywords: Smooth muscle myosin / ATP hydrolysis / Motor function / Calcium regulation / Myosin mutants / Light-chain phosphorylation / 活性中心 / 調節軽鎖リン酸化 / アクチン繊維 / 筋収縮 / 軽鎖成分リン酸化 / 収縮調節 / 分子モーター

Research Abstract

Phosphorylation of the regulatory light chain of myosin is a key reaction for the regulation of smooth muscle contraction. Nevertheless, its working mechanism is not well understood yet. Our purpose in this project is to elucidate which myosin domain is the necessary structure for the regulation. Four major findings are obtained as follows :
(1) From the behavior of recombinant myosin mutants in which Arg-247 and Glu-470 were substituted in several ways, we obtain a conclusion that the force between Arg-247 and the γ-phosphate of ATP may assist in closing the nucleotide-binding cleft and in catalysis, Glu-470 is involved in positioning the lytic water on the γ-phosphorus of the bound ATP.
(2) To investigate which residues in myosin engage in functional interactions with actin, we made myosin mutants in which the putative site inferred from crystallography were replaced with other sequences. Our results suggest that cationic residues Lys-576 and Lys-578 interact with anionic amino acid residues of the so-called "second" actin, and that hydrophobic residues Trp-546, Phe-547, and Pro-548 make connections with the so-called "first" actin.
(3) To investigate the importance of the double-headedness of myosin and of the S2 region for the phosphorylation-dependent regulation, we made three types of recombinant mutants with one intact head and the N-terminally truncated head. Our results suggest that the S2 region has an inhibitory function and that this inhibition is canceled by an interaction between two phosphorylated regulatory light chains.
(4) We isolated reactivatable stress fibers from cultured cells. Using the isolated stress fibers, we showed that in nonmuscle, actomyosin-based contractility was regulated by two kinase systems : the Ca^<2+>-dependent myosin light-chain kinase and Rho-kinase systems.

Research Products

• [Publications] Onishi H, et al.: "Early Stages of Energy Transduction by Myosin : Roles of Arg in Switch I, of Glu in Switch II, and of the Salt-Bridge between Them"Proc.Natl.Acad.Sci.USA. 99. 15339-15344 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Konishi K, et al.: "Two New Modes of Smooth Muscle Myosin Regultation by the Interaction between the Two Regulatory Light Chains, and by the S2 Domain"J.Biochem.(Tokyo). 129. 365-372 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Kojima S, et al.: "Functional Roles of Ionic and Hydrophobic Surface Loops in Smooth Muscle Myosin : Their Interactions with Actin"Biochemistry. 40. 657-664 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Katoh K, et al.: "Rho-Kinase-Mediated Contraction of Isolated Stress Fibers"J.Cell Biol.. 153. 569-583 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Onishi H, et al.: "Molecular and Cellular Aspects of Muscle Contraction"Functional Roles of Ionic and Hydrophobic Surface Loops in Smooth Muscle Myosin : Their Interactions with Actin (in press). (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Onishi, H., Ohki, T., Mochizuki, N and Morales, M. F.: "Early Stages of Energy Transduction in Myosin : Roles of Arg in Switch I, of Glu in Switch II, and of the Salt-Bridge between Them"Proc. Natl. Acad. Sci. U.S.A.. 99. 15339-15344 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Konishi, K., Kojima, S., Katoh, T., Yazawa, M., Katoh, K, Fujiwara, K, and Onishi, H.: "Two New Modes of Smooth Muscle Myosin Regulation by the Interaction between the Two Regulatory Light Chains, and by the S2 Domain"J. Biochem. (Tokyo). 129. 365-372 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Kojima, S., Konishi, K., Katoh, K., Fujiwara, K., Martinez, H. M., Morales, M. F., and Onishi, H.: "Functional Roles of Ionic and Hydrophobic Surface Loops in Smooth Muscle Myosin : Their Interactions with Actin"Biochemistry. 40. 657-664 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Katoh, K, Kano, Y., Amano, M., Onishi, H., Kaibuchi, K., and Fujiwara, K.: "Rho-Kinase Mediated Contraction of Isolated Stress Fibers"J. Cell Biol.. 153. 569-583 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Onishi, H,, Ohki, T., Mochizuki, N., and Morales, M. F., Sugi, H. and Pollack, G. H., eds: "A Hypothesis about Myosin Catalysis, Molecular and Cellular Aspects of Muscle Contraction"Kluwer Academic/Plenum Publishers, New York, in press. (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Onishi H, et al.: "Early stages of energy transduction by myosin : Roles of Arg in Switch I, of Glu in Switch II, and of the salt-bridge between them"Proc. Natl. Acad. Sci. USA 99. 24. 15339-15344 (2002)
• [Publications] Kojima S, et al.: "Functional roles of lonic and hydrophobic surface loops in smooth muscle myosin : Their interactions with actin"Biochemistry. 40. 657-664 (2001)
• [Publications] Konishi K, et al.: "Two new modes of smooth muscle myosin regulation by the interaction between the two regulatory light chains, and by the S2 domain"J. Biochem. 129. 365-372 (2001)
• [Publications] 尾西裕文: "On the trypophan residue of smooth muscle myosin that responds to binding of nucleotide"Proc.Natl.Acad.Sci.USA. 97・21. 11203-11208 (2000)
• [Publications] 孤嶋慎一郎: "Functional roles of ionic and hydrophobic surface loops in smooth muscle myosin : Their interaction with actin"Biochemistry. 40・3. 657-664 (2001)