Protein-protein interactions in the regulation of the cardiac sodium pump by phospholemman

Lead Research Organisation: University of Dundee
Department Name: Molecular Medicine

Abstract

Hormones such as adrenaline act on the individual cells of the heart (?myocytes?) to cause the whole heart to change its behaviour according to the needs of the body. This is an exquisitely regulated process that means the heart?s output matches the body?s requirements. Understanding how exposure to hormones causes the whole heart to change its behaviour is crucial in developing an understanding of what can go wrong in heart disease.
Within a myocyte, hormonal signals are transmitted into the cell by groups of proteins (protein ?complexes?). The way these complexes work depends on the proteins involved. We have evidence that a protein complex forms between two proteins, dok-7 and phospholemman. Phospholemman is responsible for transmitting signals from hormones to another protein, the sodium pump, one of the most important enzymes in the heart. The sodium pump is responsible for moving ions across cell membranes, and indirectly it controls many processes that keep the heart alive and allow it to beat. Understanding the relationship between dok-7, phospholemman and the sodium pump will greatly improve our understanding of the way the heart responds to hormones, and possibly why diseased hearts are less able to do so.

Technical Summary

The primary active means of ion transport in the cardiomyocyte sarcolemma is the sodium pump. Acute regulation of the cardiac sodium pump by extracellular stimuli is crucial in allowing the heart to match its output to systemic requirements. Interventions that influence either sodium pump activity, or indirectly the transmembrane sodium gradient, profoundly affect cardiac function. Misregulation of the sodium pump can lead to cardiac insufficiency and heart failure. Phospholemman (PLM) is a novel sodium pump accessory protein that provides the link between kinase activity and modulation of sodium pump function. PLM exerts a tonic inhibition on the sodium pump that is relieved, and activity is in fact stimulated, by phosphorylation of PLM by a variety of kinases. Spatial and temporal control of signalling to PLM is key in controlling sodium pump function. The proteins that direct kinases and phosphatases to the PLM / sodium pump complex are unknown. We have preliminary evidence that the cytoplasmic scaffold protein dok-7, which contains N terminal plekstrin homology and phosphotyrosine binding domains interacts with PLM through its C terminus. Hence dok-7 has the potential to bring PLM together with signalling molecules. This proposal will characterise the functional consequences of this interaction, both in terms of kinase signalling to PLM, and PLM regulation of sodium pump function. Understanding the relationship between dok-7, PLM and the sodium pump will expand our knowledge of basic cardiac physiology and biochemistry, and may identify novel targets for treatment of cardiac ischemia, hypertrophy and heart failure.

Publications

10 25 50
 
Description AHA Council on Basic Cardiovascular Sciences Travel Award/American Heart Association BCVS
Amount £1,000 (GBP)
Organisation American Heart Association (AHA) 
Sector Charity/Non Profit
Country United States
Start 11/2010 
End 12/2010
 
Description BHF PhD studentship
Amount £109,439 (GBP)
Funding ID FS/14/68/30988 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2015 
End 02/2018
 
Description BHF Project Grant
Amount £97,832 (GBP)
Funding ID PG/10/93/28650 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2011 
End 11/2012
 
Description BHF Project Grant
Amount £81,726 (GBP)
Funding ID PG/12/6/29366 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2012 
End 12/2013
 
Description British Heart Foundation PhD studentship
Amount £104,227 (GBP)
Funding ID FS/13/22/30126 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2013 
End 08/2016
 
Description Ninewells Hospital Anonymous Trust
Amount £9,746 (GBP)
Organisation Ninewells Hospital 
Sector Hospitals
Country United Kingdom
Start 06/2012 
End 06/2014
 
Description Ninwells Hospital Anonymous Trust Small Grants Scheme
Amount £10,000 (GBP)
Organisation Ninewells Hospital 
Sector Hospitals
Country United Kingdom
Start 06/2014 
End 06/2015
 
Description Project Grant
Amount £227,111 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2016 
End 12/2018
 
Description SULSA HTS Fund
Amount £18,519 (GBP)
Organisation Scottish Universities Life Sciences Alliance 
Sector Academic/University
Country United Kingdom
Start 06/2012 
End 12/2014
 
Description Small Research Grants Scheme/Ninewells Hospital Anonymous Trust
Amount £9,800 (GBP)
Organisation Ninewells Hospital 
Sector Hospitals
Country United Kingdom
Start 06/2010 
End 06/2011
 
Description Tenovus Scotland Small Research Grants Scheme
Amount £10,000 (GBP)
Organisation Tenovus 
Department Tenovus Scotland
Sector Charity/Non Profit
Country United Kingdom
Start 06/2008 
End 06/2009
 
Title FXYD1 antibody 
Description We have raised an antibody to phospholemman (FXYD1), the principal focus of this research project. 
Type Of Material Antibody 
Year Produced 2011 
Provided To Others? Yes  
Impact There are few if any antibodies available that recognise an extracellular epitope of FXYD1. This is required for this project as the binding of all available antibodies to the intracellular region is influenced by post-translational modifications. 
 
Title Tet-inducible cell lines 
Description We have generated a panel of cell lines expressing wild type and palmitoylation point-mutants of FXYD1 under the control of a tetracycline sensitive promoter. These have been used to define the functional effect of palmitoylation of FXYD1 on the sodium pump, and the functional crosstalk between phosphorylation and palmitoylation of FXYD1 
Type Of Material Cell line 
Year Produced 2013 
Provided To Others? Yes  
Impact Publication PubMed id 21868384 
URL http://europepmc.org/abstract/MED/21868384
 
Description Aberdeen 
Organisation University of Aberdeen
Department Scottish Biologics Facility
Country United Kingdom 
Sector Academic/University 
PI Contribution Evaluate monoclonal antibodies as sodium pump activators in assays in my lab
Collaborator Contribution Produce monoclonal antibodies by screening phage display library
Impact therapeutic monoclonal antibodies
Start Year 2012
 
Description COST 
Organisation European Cooperation in Science and Technology (COST)
Country European Union (EU) 
Sector Public 
PI Contribution Secondary Proposer (COST Action Proposal oc-2013-2-17379, European Interdisciplinary Network on Na, K-ATPase in disease)
Collaborator Contribution Primary Proposer Dr Tsvetana Schyns-Liharska
Impact none - too early
Start Year 2013
 
Description Duisberg Essen 
Organisation University Duisburg-Essen
Country Germany 
Sector Academic/University 
PI Contribution Provided reagents
Collaborator Contribution none
Impact None
Start Year 2011
 
Description Edinburgh 
Organisation University of Edinburgh
Department Centre for Integrative Physiology
Country United Kingdom 
Sector Academic/University 
PI Contribution Shared reagents and ideas
Collaborator Contribution Shared reagents
Impact ongoing
Start Year 2011
 
Description Harvard 
Organisation Harvard University
Department Harvard Medical School
Country United States 
Sector Academic/University 
PI Contribution Provided antibodies
Collaborator Contribution Evaluated antibodies
Impact none
Start Year 2010
 
Description Karolinska 
Organisation Karolinska Institute
Department Department of Molecular Medicine and Surgery
Country Sweden 
Sector Academic/University 
PI Contribution Provided antibody reagents
Collaborator Contribution Evaluated antibody reagents
Impact PubMed ID 21653224,
Start Year 2009
 
Description King's College London 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution This collaboration aims to define the physiological role of FXYD1. Physiological measurements are made at King's College London, and biochemical experiments at my laboratory in Dundee. Physiological data from London inform experimental design in my lab, and biochemical data from Dundee inform experimental approaches in London. Through investigating FXYD1 from a molecular to a whole animal level, we aim to define the physiological role of the biochemical relationships in which it participates. This relationship has led to one publication to which my laboratory was the principal contributor: FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C. Fuller W, Howie J, McLatchie LM, Weber RJ, Hastie CJ, Burness K, Pavlovic D, Shattock MJ. Am J Physiol Cell Physiol. 2009 Jun;296(6):C1346-55. PMID 19339511
Collaborator Contribution Data sharing to inform each other's experimental design. Regular meetings to review experimental data and plan future investigations
Impact Publications PMID 18718984, 18065526, 19339511, 20543084, 21849407, 21868384, 22955490
 
Description Norway 
Organisation University of Oslo
Department Institute for Experimental Medical Research
Country Norway 
Sector Academic/University 
PI Contribution Provided reagents
Collaborator Contribution none
Impact Conference presentations, publications under preparation
Start Year 2013
 
Description UT South Western 
Organisation University of Texas Southwestern Medical Center
Country United States 
Sector Academic/University 
PI Contribution Biochemical information and expertise on the palmitoylation of the cardiac sodium pump catalytic and regulatory subunits. Research materials as they become available
Collaborator Contribution Functional information and expertise on the regulation of the cardiac sodium pump by palmitoylation.
Impact A PhD student from my lab will travel to Dallas
Start Year 2012
 
Description University of Leeds 
Organisation University of Leeds
Department Institute of Membrane and Systems Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution We have shared data and experience with the lab of Dr Sarah Calaghan in Leeds. She has provided advice on protocols for our experiments, and we have offered experience and and advice with the experiments ongoing in her laboratory.
Collaborator Contribution Provided experimental protocols
Impact Joint project grant application to the British Heart Foundation funded and now in progress
Start Year 2010
 
Description Weizmann 
Organisation Weizmann Institute of Science
Country Israel 
Sector Academic/University 
PI Contribution Provided reagents
Collaborator Contribution Provided reagents
Impact Publications under preparation
Start Year 2012
 
Description Tour of Department 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact As part of of our engagement with the local community, groups that have contributed or are considering contributing to the local Tayside Institute for Cardiovascular Research charity are invited on tours of the department. The tour involves several demonstrations of the different types of research that are undertaken in the department, and a short seminar detailing our research achievements.

The local Tayside Institute for Cardiovascular Research continues to fundraise successfully, and has recently received a substantial bequest in the will of an individual who was part of the 2007 department tour.
Year(s) Of Engagement Activity 2007,2009,2010,2011