skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Structure of the Shroom-Rho Kinase Complex Reveals a Binding Interface with Monomeric Shroom That Regulates Cell Morphology and Stimulates Kinase Activity

Abstract

Shroom-mediated remodeling of the actomyosin cytoskeleton is a critical driver of cellular shape and tissue morphology that underlies the development of many tissues including the neural tube, eye, intestines, and vasculature. Shroom uses a conserved SD2 domain to direct the subcellular localization of Rho-associated kinase (Rock), which in turn drives changes in the cytoskeleton and cellular morphology through its ability to phosphorylate and activate non-muscle myosin II. Here in this paper, we present the structure of the human Shroom-Rock binding module, revealing an unexpected stoichiometry for Shroom in which two Shroom SD2 domains bind independent surfaces on Rock. Mutation of interfacial residues impaired Shroom-Rock binding in vitro and resulted in altered remodeling of the cytoskeleton and loss of Shroom-mediated changes in cellular morphology. In addition, we provide the first direct evidence that Shroom can function as a Rock activator. These data provide molecular insight into the Shroom-Rock interface and demonstrate that Shroom directly participates in regulating cytoskeletal dynamics, adding to its known role in Rock localization.

Authors:
 [1];  [1];  [1];  [2];  [3];  [1];  [1]
  1. Univ. of Pittsburgh, PA (United States). Dept. of Biological Sciences
  2. Brookhaven National Lab. (BNL), Upton, NY (United States). Dept. of Biology
  3. Univ. of Washington, Seattle, WA (United States). Dept. of Pharmacology
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); National Institutes of Health (NIH)
OSTI Identifier:
1351714
Report Number(s):
BNL-113675-2017-JA
Journal ID: ISSN 0021-9258
Grant/Contract Number:  
SC0012704; GM097204
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 291; Journal Issue: 49; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; Shroom; serine/threonine-protein kinase; coiled-coil; Structural Biology; RhoA; Cell Contractility; cytoskeleton; Rock; X-ray crystallography

Citation Formats

Zalewski, Jenna K., Mo, Joshua H., Heber, Simone, Heroux, Annie, Gardner, Richard G., Hildebrand, Jeffrey D., and VanDemark, Andrew P. Structure of the Shroom-Rho Kinase Complex Reveals a Binding Interface with Monomeric Shroom That Regulates Cell Morphology and Stimulates Kinase Activity. United States: N. p., 2016. Web. doi:10.1074/jbc.M116.738559.
Zalewski, Jenna K., Mo, Joshua H., Heber, Simone, Heroux, Annie, Gardner, Richard G., Hildebrand, Jeffrey D., & VanDemark, Andrew P. Structure of the Shroom-Rho Kinase Complex Reveals a Binding Interface with Monomeric Shroom That Regulates Cell Morphology and Stimulates Kinase Activity. United States. doi:10.1074/jbc.M116.738559.
Zalewski, Jenna K., Mo, Joshua H., Heber, Simone, Heroux, Annie, Gardner, Richard G., Hildebrand, Jeffrey D., and VanDemark, Andrew P. Mon . "Structure of the Shroom-Rho Kinase Complex Reveals a Binding Interface with Monomeric Shroom That Regulates Cell Morphology and Stimulates Kinase Activity". United States. doi:10.1074/jbc.M116.738559. https://www.osti.gov/servlets/purl/1351714.
@article{osti_1351714,
title = {Structure of the Shroom-Rho Kinase Complex Reveals a Binding Interface with Monomeric Shroom That Regulates Cell Morphology and Stimulates Kinase Activity},
author = {Zalewski, Jenna K. and Mo, Joshua H. and Heber, Simone and Heroux, Annie and Gardner, Richard G. and Hildebrand, Jeffrey D. and VanDemark, Andrew P.},
abstractNote = {Shroom-mediated remodeling of the actomyosin cytoskeleton is a critical driver of cellular shape and tissue morphology that underlies the development of many tissues including the neural tube, eye, intestines, and vasculature. Shroom uses a conserved SD2 domain to direct the subcellular localization of Rho-associated kinase (Rock), which in turn drives changes in the cytoskeleton and cellular morphology through its ability to phosphorylate and activate non-muscle myosin II. Here in this paper, we present the structure of the human Shroom-Rock binding module, revealing an unexpected stoichiometry for Shroom in which two Shroom SD2 domains bind independent surfaces on Rock. Mutation of interfacial residues impaired Shroom-Rock binding in vitro and resulted in altered remodeling of the cytoskeleton and loss of Shroom-mediated changes in cellular morphology. In addition, we provide the first direct evidence that Shroom can function as a Rock activator. These data provide molecular insight into the Shroom-Rock interface and demonstrate that Shroom directly participates in regulating cytoskeletal dynamics, adding to its known role in Rock localization.},
doi = {10.1074/jbc.M116.738559},
journal = {Journal of Biological Chemistry},
number = 49,
volume = 291,
place = {United States},
year = {2016},
month = {10}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 4 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Structure of a Highly Conserved Domain of Rock1 Required for Shroom-Mediated Regulation of Cell Morphology
journal, December 2013


Shape Complementarity at Protein/Protein Interfaces
journal, December 1993

  • Lawrence, Michael C.; Colman, Peter M.
  • Journal of Molecular Biology, Vol. 234, Issue 4
  • DOI: 10.1006/jmbi.1993.1648

Linking Crystallographic Model and Data Quality
journal, May 2012


Shroom4 (Kiaa1202) is an actin-associated protein implicated in cytoskeletal organization
journal, January 2006

  • Yoder, Michael; Hildebrand, Jeffrey D.
  • Cell Motility and the Cytoskeleton, Vol. 64, Issue 1
  • DOI: 10.1002/cm.20167

Regulation of Myosin Phosphatase by Rho and Rho-Associated Kinase (Rho-Kinase)
journal, July 1996


Rho–Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells
journal, January 2001


Structure of Shroom domain 2 reveals a three-segmented coiled-coil required for dimerization, Rock binding, and apical constriction
journal, June 2012

  • Mohan, Swarna; Rizaldy, Ryan; Das, Debamitra
  • Molecular Biology of the Cell, Vol. 23, Issue 11
  • DOI: 10.1091/mbc.E11-11-0937

hShroom1 links a membrane bound protein to the actin cytoskeleton
journal, January 2009


Direct cleavage of ROCK II by granzyme B induces target cell membrane blebbing in a caspase-independent manner
journal, February 2005

  • Sebbagh, Michael; Hamelin, Jocelyne; Bertoglio, Jacques
  • The Journal of Experimental Medicine, Vol. 201, Issue 3
  • DOI: 10.1084/jem.20031877

Crystal Structure of a Coiled-Coil Domain from Human ROCK I
journal, March 2011


Cell dynamics in fetal intestinal epithelium: implications for intestinal growth and morphogenesis
journal, August 2011

  • Grosse, A. S.; Pressprich, M. F.; Curley, L. B.
  • Development, Vol. 138, Issue 20
  • DOI: 10.1242/dev.065789

Shroom, a PDZ Domain–Containing Actin-Binding Protein, Is Required for Neural Tube Morphogenesis in Mice
journal, November 1999


Phosphorylation of Myosin-Binding Subunit (Mbs) of Myosin Phosphatase by Rho-Kinase in Vivo
journal, November 1999

  • Kawano, Yoji; Fukata, Yuko; Oshiro, Noriko
  • The Journal of Cell Biology, Vol. 147, Issue 5
  • DOI: 10.1083/jcb.147.5.1023

The COOH Terminus of Rho-kinase Negatively Regulates Rho-kinase Activity
journal, November 1999

  • Amano, Mutsuki; Chihara, Kazuyasu; Nakamura, Nao
  • Journal of Biological Chemistry, Vol. 274, Issue 45
  • DOI: 10.1074/jbc.274.45.32418

MolProbity: all-atom contacts and structure validation for proteins and nucleic acids
journal, May 2007

  • Davis, I. W.; Leaver-Fay, A.; Chen, V. B.
  • Nucleic Acids Research, Vol. 35, Issue Web Server
  • DOI: 10.1093/nar/gkm216

Actin Out: Regulation of the Synaptic Cytoskeleton
journal, October 2015

  • Spence, Erin F.; Soderling, Scott H.
  • Journal of Biological Chemistry, Vol. 290, Issue 48
  • DOI: 10.1074/jbc.R115.655118

Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing
journal, March 2001

  • Sebbagh, Michaël; Renvoizé, Claire; Hamelin, Jocelyne
  • Nature Cell Biology, Vol. 3, Issue 4
  • DOI: 10.1038/35070019

Congenital myopathies: diseases of the actin cytoskeleton: Congenital myopathies
journal, October 2004

  • Clarkson, Emilie; Costa, Celine F.; Machesky, Laura M.
  • The Journal of Pathology, Vol. 204, Issue 4
  • DOI: 10.1002/path.1648

Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains
journal, November 1993

  • Moussavi, Robabeh S.; Kelley, Christine A.; Adelstein, Robert S.
  • Molecular and Cellular Biochemistry, Vol. 127-128, Issue 1
  • DOI: 10.1007/BF01076773

Improving FRET dynamic range with bright green and red fluorescent proteins
journal, September 2012

  • Lam, Amy J.; St-Pierre, François; Gong, Yiyang
  • Nature Methods, Vol. 9, Issue 10
  • DOI: 10.1038/nmeth.2171

Structural Insights into the Interaction of ROCKI with the Switch Regions of RhoA
journal, December 2003

  • Dvorsky, Radovan; Blumenstein, Lars; Vetter, Ingrid R.
  • Journal of Biological Chemistry, Vol. 279, Issue 8
  • DOI: 10.1074/jbc.M311911200

The interaction between Shroom3 and Rho-kinase is required for neural tube morphogenesis in mice
journal, August 2014


Arachidonic acid-induced Ca2+ sensitization of smooth muscle contraction through activation of Rho-kinase
journal, November 2000

  • Araki, Shigemasa; Ito, Masaaki; Kureishi, Yasuko
  • Pflügers Archiv, Vol. 441, Issue 5
  • DOI: 10.1007/s004240000462

Targeted inhibition of the Shroom3–Rho kinase protein–protein interaction circumvents Nogo66 to promote axon outgrowth
journal, June 2015


Shroom2 regulates contractility to control endothelial morphogenesis
journal, March 2011

  • Farber, Matthew J.; Rizaldy, Ryan; Hildebrand, Jeffrey D.
  • Molecular Biology of the Cell, Vol. 22, Issue 6
  • DOI: 10.1091/mbc.E10-06-0505

Empirical Statistical Model To Estimate the Accuracy of Peptide Identifications Made by MS/MS and Database Search
journal, October 2002

  • Keller, Andrew; Nesvizhskii, Alexey I.; Kolker, Eugene
  • Analytical Chemistry, Vol. 74, Issue 20
  • DOI: 10.1021/ac025747h

Associations between the Rho kinase-1 catalytic and PH domain regulatory unit
journal, November 2013

  • Craft, John W.; Zhang, Hua; Charendoff, Marc N.
  • Journal of Molecular Graphics and Modelling, Vol. 46
  • DOI: 10.1016/j.jmgm.2013.09.009

NIH Image to ImageJ: 25 years of image analysis
journal, June 2012

  • Schneider, Caroline A.; Rasband, Wayne S.; Eliceiri, Kevin W.
  • Nature Methods, Vol. 9, Issue 7
  • DOI: 10.1038/nmeth.2089

Pax6-dependent Shroom3 expression regulates apical constriction during lens placode invagination
journal, January 2010

  • Plageman, T. F.; Chung, M. -I; Lou, M.
  • Development, Vol. 137, Issue 3
  • DOI: 10.1242/dev.045369

Toward the structural genomics of complexes: Crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis
journal, May 2006

  • Strong, M.; Sawaya, M. R.; Wang, S.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 21
  • DOI: 10.1073/pnas.0602606103

Phosphorylation and Activation of Myosin by Rho-associated Kinase (Rho-kinase)
journal, August 1996

  • Amano, Mutsuki; Ito, Masaaki; Kimura, Kazushi
  • Journal of Biological Chemistry, Vol. 271, Issue 34
  • DOI: 10.1074/jbc.271.34.20246

Shroom Induces Apical Constriction and Is Required for Hingepoint Formation during Neural Tube Closure
journal, December 2003

  • Haigo, Saori L.; Hildebrand, Jeffrey D.; Harland, Richard M.
  • Current Biology, Vol. 13, Issue 24
  • DOI: 10.1016/j.cub.2003.11.054

Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I
journal, March 2001

  • Coleman, Mathew L.; Sahai, Erik A.; Yeo, Margaret
  • Nature Cell Biology, Vol. 3, Issue 4
  • DOI: 10.1038/35070009

Kinase-interacting substrate screening is a novel method to identify kinase substrates
journal, June 2015

  • Amano, Mutsuki; Hamaguchi, Tomonari; Shohag, Md. Hasanuzzaman
  • The Journal of Cell Biology, Vol. 209, Issue 6
  • DOI: 10.1083/jcb.201412008

The cytoskeleton and cancer
journal, January 2009


Rho-Kinase Inhibitor Retards Migration and in Vivo Dissemination of Human Prostate Cancer Cells
journal, March 2000

  • Somlyo, Avril V.; Bradshaw, Dawn; Ramos, Susan
  • Biochemical and Biophysical Research Communications, Vol. 269, Issue 3
  • DOI: 10.1006/bbrc.2000.2343

Coot model-building tools for molecular graphics
journal, November 2004

  • Emsley, Paul; Cowtan, Kevin
  • Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
  • DOI: 10.1107/S0907444904019158

Shroom3 and a Pitx2-N-cadherin pathway function cooperatively to generate asymmetric cell shape changes during gut morphogenesis
journal, September 2011

  • Plageman, Timothy F.; Zacharias, Amanda L.; Gage, Phillip J.
  • Developmental Biology, Vol. 357, Issue 1
  • DOI: 10.1016/j.ydbio.2011.06.027

Cytoskeletal mechanisms of neuronal degeneration
journal, February 2001


Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

Protein production by auto-induction in high-density shaking cultures
journal, May 2005


Ca 2+ Sensitivity of Smooth Muscle and Nonmuscle Myosin II: Modulated by G Proteins, Kinases, and Myosin Phosphatase
journal, October 2003


Sonic hedgehog induces angiogenesis via Rho kinase-dependent signaling in endothelial cells
journal, September 2010

  • Renault, Marie-Ange; Roncalli, Jérôme; Tongers, Jörn
  • Journal of Molecular and Cellular Cardiology, Vol. 49, Issue 3
  • DOI: 10.1016/j.yjmcc.2010.05.003

PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton.
journal, October 1996

  • Leung, T.; Chen, X. Q.; Manser, E.
  • Molecular and Cellular Biology, Vol. 16, Issue 10
  • DOI: 10.1128/MCB.16.10.5313

Differential Actin-dependent Localization Modulates the Evolutionarily Conserved Activity of Shroom Family Proteins
journal, May 2006

  • Dietz, Megan L.; Bernaciak, Teresa M.; Vendetti, Frank
  • Journal of Biological Chemistry, Vol. 281, Issue 29
  • DOI: 10.1074/jbc.M512463200

NMRPipe: A multidimensional spectral processing system based on UNIX pipes
journal, November 1995

  • Delaglio, Frank; Grzesiek, Stephan; Vuister, GeertenW.
  • Journal of Biomolecular NMR, Vol. 6, Issue 3
  • DOI: 10.1007/BF00197809

Specific isoforms of drosophila shroom define spatial requirements for the induction of apical constriction
journal, May 2010

  • Bolinger, Cory; Zasadil, Lauren; Rizaldy, Ryan
  • Developmental Dynamics, Vol. 239, Issue 7
  • DOI: 10.1002/dvdy.22326

Shroom2 (APXL) regulates melanosome biogenesis and localization in the retinal pigment epithelium
journal, September 2006


A molecular ruler regulates cytoskeletal remodelling by the Rho kinases
journal, December 2015

  • Truebestein, Linda; Elsner, Daniel J.; Fuchs, Elisabeth
  • Nature Communications, Vol. 6, Issue 1
  • DOI: 10.1038/ncomms10029

Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network
journal, October 2005

  • Hildebrand, J. D.
  • Journal of Cell Science, Vol. 118, Issue 22
  • DOI: 10.1242/jcs.02626

Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension
journal, October 1997

  • Uehata, Masayoshi; Ishizaki, Toshimasa; Satoh, Hiroyuki
  • Nature, Vol. 389, Issue 6654
  • DOI: 10.1038/40187

WASp-dependent actin cytoskeleton stability at the dendritic cell immunological synapse is required for extensive, functional T cell contacts
journal, November 2015

  • Malinova, Dessislava; Fritzsche, Marco; Nowosad, Carla R.
  • Journal of Leukocyte Biology, Vol. 99, Issue 5
  • DOI: 10.1189/jlb.2A0215-050RR

Competing sorting signals guide endolyn along a novel route to lysosomes in MDCK cells
journal, November 2001


Rho-associated Kinase of Chicken Gizzard Smooth Muscle
journal, February 1999

  • Feng, Jianhua; Ito, Masaaki; Kureishi, Yasuko
  • Journal of Biological Chemistry, Vol. 274, Issue 6
  • DOI: 10.1074/jbc.274.6.3744

Characterization of RhoA-binding Kinase ROKα Implication of the Pleckstrin Homology Domain in ROKα Function Using Region-specific Antibodies
journal, January 2002

  • Chen, Xiang-qun; Tan, Ivan; Ng, Chong Han
  • Journal of Biological Chemistry, Vol. 277, Issue 15
  • DOI: 10.1074/jbc.M109839200

Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.
journal, May 1996


    Works referencing / citing this record:

    Scoria: a Python module for manipulating 3D molecular data
    journal, September 2017

    • Ropp, Patrick; Friedman, Aaron; Durrant, Jacob D.
    • Journal of Cheminformatics, Vol. 9, Issue 1
    • DOI: 10.1186/s13321-017-0237-8

    Scoria: a Python module for manipulating 3D molecular data
    journal, September 2017

    • Ropp, Patrick; Friedman, Aaron; Durrant, Jacob D.
    • Journal of Cheminformatics, Vol. 9, Issue 1
    • DOI: 10.1186/s13321-017-0237-8