Regulation of ROCK serine/threonine kinases by Rnd proteins

Lead Research Organisation: King's College London
Department Name: Unlisted

Abstract

In mammals, cells need to move during development, wound healing and in response to infection. Cell movement is driven by the cytoskeleton, principally by filaments made of actin molecules. Part of cell movement involves contractile forces, which are generated by myosin filaments moving along actin filaments. Similar interactions between myosin and actin filaments generate muscle contraction. The ability of myosin filaments to move along actin filaments is increased by a protein called ROCK-I. Several human diseases could be treated by chemicals that inhibit ROCK-I, including heart disease, Alzheimers' disease and some cancers. We have identified a protein in cells, called RhoE, that binds to and inhibits ROCK-I, and thus reduces the interaction of myosin filaments with actin filaments. We want to understand more about how RhoE inhibits ROCK-I, and predict that this will help in the design of new chemicals to inhibit ROCK-I.

Technical Summary

ROCK-I and ROCK-II are serine/threonine kinases that phosphorylate a number of proteins involved in regulating the actin cytoskeleton and actomyosin-based contractility. They thereby contribute to muscle contraction, cell migration, phagocytosis, and cell-cell junction integrity. RhoE is an atypical Rho family GTP-binding protein that binds to and inhibits ROCK-I but not ROCK-II. ROCK-I in turn phosphorylates RhoE and thereby induces its translocation from intracellular membrane to the cytosol and increases its stability. We propose to investigate the molecular basis for RhoE inhibition of ROCK-I, by mapping the regions of RhoE required for ROCK-I inhibition. In addition, we will determine where RhoE interacts with and inhibits ROCK-I in a variety of mammalian cell types, and how this interaction is regulated by extracellular stimuli. We will also investigate how phosphorylation alters RhoE localization and leads to increased stability, and whether RhoE interaction with other proteins affects its ability to inhibit ROCK-I. Furthermore, we will investigate whether there are other post-translational modifications on RhoE apart from phosphorylation, and if so how they affect its function. Finally, we will compare the responses of mammalian cells to RhoE with those to its closest relatives, Rnd1 and Rnd2, which do not bind to or inhibit ROCK-I/II.

Publications

10 25 50

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/E004083/1 01/09/2006 31/03/2007 £280,880
BB/E004083/2 Transfer BB/E004083/1 01/04/2007 31/08/2009 £225,362
 
Description In mammals, cells need to move during development, wound healing and in response to infection. Cell movement is driven by the cytoskeleton, principally by filaments made of actin molecules. Part of cell movement involves contractile forces, which are generated by myosin filaments moving along actin filaments. Similar interactions between myosin and
actin filaments generate muscle contraction. The ability of myosin filaments to move along actin filaments is increased by a protein called ROCK1. We have identified a protein in cells, called Rnd3, that is regulated by ROCK1, and reduces the interaction of myosin filaments with actin filaments. We have identified how ROCK1 affects the activity of Rnd3 by changing where it goes inside cells.
Exploitation Route Inhibitors of ROCK1 and PKC will affect the activity of Rnd3 and hence cell motility, relevant to healthy development and human diseases involving cell migration.
Sectors Healthcare

 
Description AMS Team Science Working Group
Geographic Reach Asia 
Policy Influence Type Participation in a advisory committee
URL http://www.acmedsci.ac.uk/policy/policy-projects/team-science/
 
Description Co-Chair AMS Careers Committee
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Scientific Advisory Board CNB
Geographic Reach Asia 
Policy Influence Type Participation in advisory committee
 
Description Scientific Advisory Board SBI
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Participation in a advisory committee
 
Description Analysis of RhoE and 4EBP1 
Organisation University of the Balearic Islands
Department Research Institute of Health Sciences
Country Spain 
Sector Academic/University 
PI Contribution Some of the experiments were carried out in our laboratory. We also provided RhoE plasmids and RhoE-inducible cell lines generated in our laboratory.
Collaborator Contribution The partners carried out experiments leading to publication of a joint paper.
Impact The output is the following publication: Villalonga, P., Fernandez de Mattos, S., Ridley, A.J. (2009) RhoE inhibits 4E-BP1 phosphorylation and eIF4E function impairing cap-dependent translation. J. Biol. Chem. 284, 35287-35296.
 
Description Mass spectrometry analysis of Rnd3 
Organisation Institute of Cancer Research UK
Country United Kingdom 
Sector Academic/University 
PI Contribution Methods to identify farnesylated C-terminal peptides of Rnd3 were developed
Collaborator Contribution We provided cell samples for analysis
Impact Publication: Cell. 2013 Apr 25;153(3):640-53. doi: 10.1016/j.cell.2013.03.044. 14-3-3 proteins interact with a hybrid prenyl-phosphorylation motif to inhibit G proteins. Riou P1, Kjær S, Garg R, Purkiss A, George R, Cain RJ, Bineva G, Reymond N, McColl B, Thompson AJ, O'Reilly N, McDonald NQ, Parker PJ, Ridley AJ.
Start Year 2012
 
Description Proneural transcription factors regulate different steps of cortical neuron migration through Rnd-mediated inhibition of RhoA signaling. 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution A new collaboration between the laboratories of Dr Francois Guillemot at MRC NIMR and Prof Anne Ridley resulted in two publications. The Ridley laboratory contributed Rnd plasmids and expertise on Rnd/Rho/ROCK signalling.
Collaborator Contribution The partners contributed the research identifying Rnd proteins as targets for transcription factors in the neuronal cortex and in vivo studies of their functions during cortical development.
Impact Publications in journals: Neuron. 2011 Mar 24;69(6):1069-84. doi: 10.1016/j.neuron.2011.02.018. Proneural transcription factors regulate different steps of cortical neuron migration through Rnd-mediated inhibition of RhoA signaling. Pacary E1, Heng J, Azzarelli R, Riou P, Castro D, Lebel-Potter M, Parras C, Bell DM, Ridley AJ, Parsons M, Guillemot F. Nat Commun. 2014 Feb 27;5:3405. doi: 10.1038/ncomms4405. An antagonistic interaction between PlexinB2 and Rnd3 controls RhoA activity and cortical neuron migration. Azzarelli R1, Pacary E1, Garg R2, Garcez P3, van den Berg D3, Riou P4, Ridley AJ2, Friedel RH5, Parsons M2, Guillemot F3.
Start Year 2009
 
Description Rnd3 interaction with 14-3-3 proteins 
Organisation Cancer Research UK
Department Cancer Research UK London Research Institute (LRI)
Country United Kingdom 
Sector Academic/University 
PI Contribution The collaboration involves biophysical analysis of Rnd3 interaction with 14-3-3 proteins
Collaborator Contribution They synthesized peptides, carried out biophysical analysis of the interactions of the peptides with 14-3-3 proteins, and solved the crystal structure of the peptides with 14-3-3.
Impact Publication:Cell. 2013 Apr 25;153(3):640-53. doi: 10.1016/j.cell.2013.03.044. 14-3-3 proteins interact with a hybrid prenyl-phosphorylation motif to inhibit G proteins. Riou P1, Kjær S, Garg R, Purkiss A, George R, Cain RJ, Bineva G, Reymond N, McColl B, Thompson AJ, O'Reilly N, McDonald NQ, Parker PJ, Ridley AJ.
Start Year 2008
 
Description Academy of Medical Science Team Science 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Academy of Medical Sciences Working Group published 'Team Science' report, after holding a series of workshops with different sectors. I was chair of the Working Group.
Year(s) Of Engagement Activity 2016
URL https://acmedsci.ac.uk/policy/policy-projects/team-science
 
Description National Science Week Workshop - London 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact This was a regular event hosted by the Ludwig Institute for Cancer Research, UCL. We presented information to A-level students and teachers on cancer research and movies about 'life in a cancer research laboratory' and then had a question and answer session.

School feedback about the event was very positive.
Year(s) Of Engagement Activity 2006
 
Description Open days - King's College London 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach National
Primary Audience Schools
Results and Impact A-level students and their parents visit King's College London to find out about courses and research. We take groups on guided tours of our laboratories and describe our research to them.

Increased awareness that universities carry out research as well as teach undergraduates
Year(s) Of Engagement Activity 2007,2008,2012,2013,2014,2015,2016
 
Description School Visit (North London) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact Approximately 60 students and 5 teachers attended my talk on cancer and cancer metastasis. They asked lots of questions at the end, which showed a clear interest in the area.

My visit led to one student visiting my laboratory for work experience.
Year(s) Of Engagement Activity 2013
 
Description Work experience - A-level students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Schools
Results and Impact One or two biology A-level students carried out work experience in my laboratory for a week. They spent time with each member of the laboratory and with me. We demonstrated experimental techniques and discussed the work we do. Some of them also carried out mini-projects analysing movies of cells.

As a consequence of the work experience, the students started undergraduate degrees in biomedical sciences, medicine or veterinary medicine.
Year(s) Of Engagement Activity 2009,2012,2013,2014,2015