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
Azzarelli R
(2014)
An antagonistic interaction between PlexinB2 and Rnd3 controls RhoA activity and cortical neuron migration.
in Nature communications
Gottesbühren U
(2013)
Rnd3 induces stress fibres in endothelial cells through RhoB.
in Biology open
Liebig T
(2009)
RhoE Is required for keratinocyte differentiation and stratification.
in Molecular biology of the cell
Madigan JP
(2009)
Regulation of Rnd3 localization and function by protein kinase C alpha-mediated phosphorylation.
in The Biochemical journal
McColl B
(2016)
Rnd3-induced cell rounding requires interaction with Plexin-B2.
in Journal of cell science
Reymond N
(2012)
Cdc42 promotes transendothelial migration of cancer cells through ß1 integrin.
in The Journal of cell biology
Reymond N
(2012)
Rho GTPases and cancer cell transendothelial migration.
in Methods in molecular biology (Clifton, N.J.)
Riou P
(2010)
Rnd proteins: multifunctional regulators of the cytoskeleton and cell cycle progression.
in BioEssays : news and reviews in molecular, cellular and developmental biology
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 | 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 | 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 |