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Solution NMR structure of the myosin phosphatase inhibitor protein CPI-17 shows phosphorylation-induced conformational changes responsible for activation.

Ohki S, Eto M, Kariya E, Hayano T, Hayashi Y, Yazawa M, Brautigan D, Kainosho M
J Mol Biol 314 839-49 (2001)
Cited: 25 times
EuropePMC logo PMID: 11734001

Abstract

Contractility of vascular smooth muscle depends on phosphorylation of myosin light chains, and is modulated by hormonal control of myosin phosphatase activity. Signaling pathways activate kinases such as PKC or Rho-dependent kinases that phosphorylate the myosin phosphatase inhibitor protein called CPI-17. Phosphorylation of CPI-17 at Thr38 enhances its inhibitory potency 1000-fold, creating a molecular on/off switch for regulating contraction. We report the solution NMR structure of the CPI-17 inhibitory domain (residues 35-120), which retains the signature biological properties of the full-length protein. The final ensemble of 20 sets of NMR coordinates overlaid onto their mean structure with r.m.s.d. values of 0.84(+/-0.22) A for the backbone atoms. The protein forms a novel four-helix, V-shaped bundle comprised of a central anti-parallel helix pair (B/C helices) flanked by two large spiral loops formed by the N and C termini that are held together by another anti-parallel helix pair (A/D helices) stabilized by intercalated aromatic and aliphatic side-chains. Chemical shift perturbations indicated that phosphorylation of Thr38 induces a conformational change involving displacement of helix A, without significant movement of the other three helices. This conformational change seems to flex one arm of the molecule, thereby exposing new surfaces of the helix A and the nearby phosphorylation loop to form specific interactions with the catalytic site of the phosphatase. This phosphorylation-dependent conformational change offers new structural insights toward understanding the specificity of CPI-17 for myosin phosphatase and its function as a molecular switch.

Reviews citing this publication (8)

  1. Functional diversity of protein phosphatase-1, a cellular economizer and reset button. Ceulemans H, Bollen M. Physiol Rev 84 1-39 (2004)
  2. Role of protein phosphatase type 1 in contractile functions: myosin phosphatase. Hartshorne DJ, Ito M, Erdödi F. J Biol Chem 279 37211-37214 (2004)
  3. Mass spectrometric approaches using electrospray ionization charge states and hydrogen-deuterium exchange for determining protein structures and their conformational changes. Yan X, Watson J, Ho PS, Deinzer ML. Mol Cell Proteomics 3 10-23 (2004)
  4. SAIL--stereo-array isotope labeling. Kainosho M, Güntert P. Q Rev Biophys 42 247-300 (2009)
  5. Diversity and plasticity in signaling pathways that regulate smooth muscle responsiveness: Paradigms and paradoxes for the myosin phosphatase, the master regulator of smooth muscle contraction. Eto M, Kitazawa T. J Smooth Muscle Res 53 1-19 (2017)
  6. Characterizing Post-Translational Modifications and Their Effects on Protein Conformation Using NMR Spectroscopy. Kumar A, Narayanan V, Sekhar A. Biochemistry 59 57-73 (2020)
  7. Phosphorylation Sites in Protein Kinases and Phosphatases Regulated by Formyl Peptide Receptor 2 Signaling. Annunziata MC, Parisi M, Esposito G, Fabbrocini G, Ammendola R, Cattaneo F. Int J Mol Sci 21 (2020)
  8. Regulation of myosin light chain phosphatase and pulmonary arterial relaxation. Dakshinamurti S. Can J Physiol Pharmacol 83 893-898 (2005)

Articles citing this publication (17)

  1. Functional sites in protein families uncovered via an objective and automated graph theoretic approach. Wangikar PP, Tendulkar AV, Ramya S, Mali DN, Sarawagi S. J Mol Biol 326 955-978 (2003)
  2. Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits. Eto M, Kitazawa T, Brautigan DL. Proc Natl Acad Sci U S A 101 8888-8893 (2004)
  3. Cerebellar long-term synaptic depression requires PKC-mediated activation of CPI-17, a myosin/moesin phosphatase inhibitor. Eto M, Bock R, Brautigan DL, Linden DJ. Neuron 36 1145-1158 (2002)
  4. Detailed structural characterization of unbound protein phosphatase 1 inhibitors. Dancheck B, Nairn AC, Peti W. Biochemistry 47 12346-12356 (2008)
  5. Direct activation of human phospholipase C by its well known inhibitor u73122. Klein RR, Bourdon DM, Costales CL, Wagner CD, White WL, Williams JD, Hicks SN, Sondek J, Thakker DR. J Biol Chem 286 12407-12416 (2011)
  6. Phosphorylation-induced conformational switching of CPI-17 produces a potent myosin phosphatase inhibitor. Eto M, Kitazawa T, Matsuzawa F, Aikawa S, Kirkbride JA, Isozumi N, Nishimura Y, Brautigan DL, Ohki SY. Structure 15 1591-1602 (2007)
  7. Involvement of Cpi-17 and zipper-interacting protein kinase in the regulation of protein kinase C-alpha, protein kinase C-epsilon on vascular calcium sensitivity after hemorrhagic shock. Xu J, Yang G, Li T, Ming J, Liu L. Shock 33 49-55 (2010)
  8. Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA. Smet-Nocca C, Launay H, Wieruszeski JM, Lippens G, Landrieu I. J Biomol NMR 55 323-337 (2013)
  9. Novel in vitro and in vivo phosphorylation sites on protein phosphatase 1 inhibitor CPI-17. Dubois T, Howell S, Zemlickova E, Learmonth M, Cronshaw A, Aitken A. Biochem Biophys Res Commun 302 186-192 (2003)
  10. A versatile mass spectrometry-based method to both identify kinase client-relationships and characterize signaling network topology. Ahsan N, Huang Y, Tovar-Mendez A, Swatek KN, Zhang J, Miernyk JA, Xu D, Thelen JJ. J Proteome Res 12 937-948 (2013)
  11. Beneficial effects of activation of PKC on hemorrhagic shock in rats. Fang Y, Li T, Fan X, Zhu Y, Liu L. J Trauma 68 865-873 (2010)
  12. Distinctive solution conformation of phosphatase inhibitor CPI-17 substituted with aspartate at the phosphorylation-site threonine residue. Ohki Sy, Eto M, Shimizu M, Takada R, Brautigan DL, Kainosho M. J Mol Biol 326 1539-1547 (2003)
  13. Protein kinase C activation decreases peripheral actin network density and increases central nonmuscle myosin II contractility in neuronal growth cones. Yang Q, Zhang XF, Van Goor D, Dunn AP, Hyland C, Medeiros N, Forscher P. Mol Biol Cell 24 3097-3114 (2013)
  14. Structure determination and conformational change induced by tyrosine phosphorylation of the N-terminal domain of the alpha-chain of pig gastric H+/K+-ATPase. Fujitani N, Kanagawa M, Aizawa T, Ohkubo T, Kaya S, Demura M, Kawano K, Nishimura S, Taniguchi K, Nitta K. Biochem Biophys Res Commun 300 223-229 (2003)
  15. Phospho-pivot modeling predicts specific interactions of protein phosphatase-1 with a phospho-inhibitor protein CPI-17. Matsuzawa F, Aikawa SI, Ohki SY, Eto M. J Biochem 137 633-641 (2005)
  16. Computational design of molecular motors as nanocircuits in Leishmaniasis. Kosey D, Singh S. F1000Res 6 94 (2017)
  17. Possible roles of N- and C-terminal unstructured tails of CPI-17 in regulating Ca2+ sensitization force of smooth muscle. Eto M, Katsuki S, Ohashi M, Miyagawa Y, Tanaka Y, Takeya K, Kitazawa T. J Smooth Muscle Res 58 22-33 (2022)


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