Regulation of ROCK serine/threonine kinases by Rnd proteins

Lead Research Organisation: University College London
Department Name: Structural Molecular Biology

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


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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 We have shown that the serine/threonine kinases ROCK and PKC phosphorylate Rnd3, and identified the residues phosphorylated by a combination of mutagenesis and mass spectrometry. We have also shown that Rnd1 and Rnd2 can be phosphorylated in cells. We have found that phosphorylated Rnd3 binds to 14-3-3 proteins, and that this binding inhibits Rnd3 activity by translocating it from membranes to the cytosol. This involves phosphorylation of 2 residues on Rnd3 by ROCK and 1 residue by PKC.

When Rnd3 is bound to its target p190RhoGAP, it is not phosphorylated, indicating that it exists in two separate complexes: an active complex with p190RhoGAP, and an inactive complex with 14-3-3.



Rnd3 is also post-translationally modified by addition of a lipid farnesyl group at the C-terminus. We have shown that farnesylation as well as phosphorylation is required for Rnd3 to interact with 14-3-3 proteins. In collaboration with Neil McDonald, we have solved the crystal structure of Rnd3 with 14-3-3 proteins.
Exploitation Route ROCK inhibitors are already in use clinically in Japan. PKC inhibitors are being considered for clinical use. This information on Rnd3, ROCK and PKC, is relevant to the interpretation of clinical results. Rnd3 regulates cell migration and cell contractility and thus methods to incrrease its activity, for example by inhibiting its phosphorylation, could be used to promote cell migration and/or cell contractility. This information on Rnd3 regulation will be relevant to the design and use of ROCK or PKC inhibitors in pre-clinical or clinical settings.

In addition, our novel results on farnesyl involvement in the interaction with 14-3-3 proteins will be useful for the academic community studying a wide variety of other prenylated proteins.
Sectors Healthcare
 
Title Rnd1, Rnd2, Rnd3 mutants 
Description Multiple constructing encoding Rnd1, Rnd2, Rnd3 chimerae, and Rnd1, Rnd2 and Rnd3 point mutations. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2008 
Provided To Others? Yes  
Impact Joint research publications: Madigan et al., Biochemical Journal 2009 Leibig et al., Mol Biol Cell 2009 
 
Description Mass spectrometry analysis of Rnd3 
Organisation University of London
Department Institute of Cancer Research UK (ICR)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
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 of Great Britain & Northern Ireland (UK) 
Sector Public 
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 (CRUK)
Department Cancer Research UK London Research Institute (LRI)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Charity/Non Profit 
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 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