MRC PPU News
Helen publishes first biochemical analysis of PINK1
24 November, 2011
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Helen Woodroof

Mutations in PINK1 were discovered in 2004 in patients suffering from familial Parkinson’s disease. PINK1 is an unusual protein kinase containing a mitochondrial targeting domain and 3 insertions within its kinase domain. Despite intensive research little was known about the catalytic properties of PINK1 since the human form of the enzyme displayed very little activity. In Drosophila melanogaster, loss of PINK1 function produces a striking phenotype comprising both flight muscle and brain degeneration although the mechanism for this is unknown. This phenotype can be rescued by re-expression of the wild-type but not kinase-inactive version of PINK1 suggesting that the kinase activity of PINK1 is central to its function.

Helen Woodroof, a PhD student in the MRC Protein Phosphorylation Unit, decided to express and test PINK1 from Drosophila melanogaster (dPINK1) and the related insects species, Tribolium castaneum (TcPINK1), and Pediculus humanus corporis (PhcPINK1). She discovered that in contrast to mammalian PINK1, these insect species were active as judged by their ability to phosphorylate the generic substrate myelin basic protein. This was the first time that anyone had been able to measure PINK1 kinase activity directly. Using the most active enzyme (TcPINK1), she undertook a domain analysis and found that the C-terminus was essential for PINK1 activity. In collaboration with Alex Whitworth’s lab in Sheffield, she was also able to confirm that TcPINK1 (and PhcPINK1) were true PINK1 orthologues since they could rescue the Drosophila PINK1 null phenotype.

Next in collaboration with Mike Begley and Lew Cantley at Harvard, an optimal peptide substrate for PINK1, was determined from a positional scanning library screen, which was KKWIpYRRSPRRR and designated PINKtide. Helen studied variants of PINKtide and established that the +1 proline was crucial for PINKtide phosphorylation.

An important and outstanding question in the PINK1 field has been how disease-causing mutations affect PINK1 function. Helen was able to model seventeen conserved missense and truncating human mutations in TcPINK1 including W437X, which was the first mutation, described in a PINK1-affected family (Marsala kindred) and tested their activity against PINKtide. Helen found that nearly all disease-causing mutations of PINK1 confer their effect by inactivating PINK1.

Helen’s findings demonstrate that PINK1 kinase activity is crucial for preventing the onset of Parkinson’s disease. The next major challenge in the field is to identify physiological substrates of PINK1. Helen’s discovery of active PINK1 should aid in this search

Woodroof HI, Pogson JI, Begley M, Cantley LC, Deak M, Campbell DG, van Aalten DMF, Whitworth AJ, Alessi DR, Muqit MMK. Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations. Open Biol 2011 1, 110012




 

Local Diabetes Research Supporters visit MRC-PPU
17 November, 2011
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Kei Sakamoto (front right) and his research team (front row) with the Dundee Doabetes Volunteer’s Group (back row)

Members of the Dundee and District Diabetes UK Volunteers Group visited the MRC Protein Phosphosrylation Unit (PPU) at Dundee on November 8th. The Volunteer Group has raised £20,000 over the past few years and generously donated it to Kei Sakamoto’s research team in the PPU, who are studying the molecular pathways by which nutrients, hormones and exercise regulate blood sugar levels, and how the de-regulation of these systems causes type 2 diabetes. Kei’s team members gave the Volunteers group a tour of the PPU, explained the research projects that they were working on and how the donations have been and are being used. After the tour, members of Volunteer Group and Kei’s team continued their discussions over tea in the Unit’s social room on the 5th floor of the Sir James Black Centre (see picture), which commands great views over the Tay Estuary.

The Volunteer Group will be hosting their next monthly meeting at Ninewells Hospital in Dundee to plan their future fund-raising events. The Group always welcomes new members and anyone who is interested is invited to come!




 

Serge Awarded prize for the best Project
26 October, 2011
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(Serge (left) being presented with his award by William Pralong, the Dean of the Department of Biotechnology at EPFL.

Serge Ducommun, a visiting Master’s student in the Department of Biotechnology at the Ecole Polytechnique Federale de Lausanne (EPFL), Switzerland has been awarded a prize for carrying out the best research project in his year. The project was carried out in the MRC protein Phosphorylation Unit at Dundee and co-supervised by Kei Sakamoto, Carol MacKintosh and Shuai Chen. In this project Serge studied the regulation and physiological roles of AS160 and TBC1D1, two RAB GTPases implicated in regulating the trafficking of the insulin-regulated glucose transporter, GLUT4 in muscle and adipose tissue. Serge’s Master’s degree is coordinated by Professor Johan Auwerx at EPFL.




 

Alex Wins PhD Poster Competition
26 October, 2011
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Alex with his prize winning poster

Alexander von Wilamowitz-Moellendorff in Kei Sakamoto's lab was awarded joint first prize at the poster competition for Ph.D. students based in the College of Life Sciences (CLS) at the University of Dundee who are about to start the third year of their project. 49 students took part in this year’s competition, which was held on October 21st and judged by 10 Principal Investigators in CLS.

Alex's poster presented the work that he has been undertaking to understand how insulin and glucose control hepatic glycogen metabolism and its influence on whole body glucose homeostasis. Alex’s research focuses on role that allosteric activation of glycogen synthase by glucose 6-phosphate plays in controlling the accumulation of liver glycogen. Alex received a cash prize of £125 from QIAGEN who sponsored the competition.




 

Shuai Chen appointed to a Principal Investigator post at Nanjing University
13 October, 2011

Congratulations to Shuai Chen, from Carol MacKintosh’s group, who will soon be taking up a PI position at the Model Animal Research Center (MARC), Medical School of Nanjing University, one of the top universities in China. In the last decade MARC has been taking a leading role in disease research, and it is one of the fifteen institutions in the International Mouse Phenotyping Consortium (IMPC) to systemically generate and phenotype knockout mice. Nanjing, meaning ‘south capital’, was one of the earliest established cities in China and has a history of more than 2, 500 years. The city is on the Yangtze River, about 160 miles away from Shanghai.

Shuai has exciting research plans for the future, and we wish him all the best.




 

New mechanism for preventing autoimmune disease discovered
26 September, 2011
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Dr Kris Clark

Kris Clark, a postdoc in Philip Cohen’s lab has recently discovered a key role for the IκB kinase (IKK)-related protein kinases (IKKε and TBK1) in suppressing the activities of the canonical IKKs (IKKα and IKKβ) when the innate immune system is activated. This regulatory device is needed to prevent the hyper-phosphorylation of the transcription factor NFκB which, if left unchecked, would lead to the overproduction of inflammatory mediators and to the development of chronic inflammatory or autoimmune diseases, depending on cell context (Biochem. J., 2011, 434, 93-104). Now, in a follow up paper (Proc. Natl. Acid Sci USA, 2011) Kris has identified a protein called TANK as a key player in mediating this “crosstalk” between the different IKK family members. He found that the interaction between the canonical IKKs and the IKK-related kinases was abolished (IKKε) or reduced (TBK1) in macrophages from TANK-deficient mice and that, as a consequence, the activation of the IKK-related kinases did not occur (IKKε) or was reduced (TBK1). This resulted in enhanced activity of the canonical IKKs and increased phosphorylation of their substrates. In work that is yet to be published, the laboratory have obtained evidence that the IKK-related kinases may limit the strength of innate immune signalling in additional ways. Taken together, their findings explain why TANK-deficient mice overproduce inflammatory cytokines and develop autoimmune nephritis as they age.




 

Dundee reunion in Barcelona
22 September, 2011
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TOP: Philip beginning his lecture BOTTOM: Philip (centre) with former members of the MRC-PPU (L-R) Jose Lizcano, Ana Cuenda, Antonio Casamayor and Jose Bayascas

On September 6 2011, MRC-PPU Director, Philip Cohen, gave the opening keynote lecture of the Spanish Society for Biochemistry & Molecular Biology (SEBBM), which was sponsored by The Fundacion Ramon Areces Foundation, which was held in Barcelona. Philip was also made an Honorary Member of SEBBM and presented with its Gold Medal on 8 September.

Many scientists from Spain, who have received their Postdoctoral training in the MRC Protein Phosphorylation Unit, are now Principal Investigators in Spain running their own research groups. The pictures below show Philip with four such Spanish scientists who attended the meeting, and the start of his lecture - which was attended by Spanish Minister of Science, Cristina Garmendia Mendizábal, who was originally trained as a Molecular Biologist - entitled 'The interplay between protein phosphorylation and protein ubiquitylation in regulating the innate immune system'.




 

MRC-PPU Programme Leaders named as highly cited Scientists in Protein Research
15 September, 2011

The September 14th 2011 issue of Lab Times has published citation data for the period 1998-2009 in the field of "Protein Research". MRC-PPU Programme Leaders Philip Cohen and Dario Alessi were named as the 8th and 13th most cited European scientists in the list with 16,244 and 14,431 citations, respectively for papers published between 1998 and 2009. Scotland ranked number one in the world in terms of citations per paper (31.3 citations per paper) with Switzerland 2nd (30.2), England 3rd (29.2) and the USA 15th (20.6)! Remarkably, Philip (134 papers) and Dario (125 papers) published nearly 7% of Scotland's papers in the field of protein research between 1998-2009 (they only co-authored four papers over this period)! Philip's paper published in 2000, which introduced the technology of kinase profiling (Davies et al, Biochem J. 351, 95-105) was the fifth most cited European paper in this field with 2605 citations.




 

From Media Kitchen to Research Laboratory
13 September, 2011

Cameron Spencer, a student at the High School of Dundee, spent 4 weeks between his 5th and 6th years, gaining laboratory experience in the College of Life Sciences Media Kitchen, Division of Signal Transduction Therapy (DSTT) and MRC Protein Phosphorylation Unit (MRC PPU) sponsored by a Nuffield Foundation Science Bursary. In the DSTT, Cameron worked with Lesley Piper and others in the teams who generate highly specialized reagents to accelerate the development of new drug treatments for major diseases. In the PPU, Hilary Smith put Cameron through his paces in an experiment to identify signaling events that are activated when macrophages in the immune system are challenged by bacterial infection. In his own words, Cameron learned that “Although it is the research that has gone on within research laboratories that makes the front pages of newspapers, the structure of a research institute is much more complex, with different layers that form together to build up a working chain that helps sustain the working order within such an institute. From making media in the media kitchen to learning how to use experimental results to draw conclusions, I thoroughly enjoyed my whole experience and from what I have gained during the time I was doing the project, I feel that this is an environment that I could work in.”




 

Juan M. Garcia-Martinez presented with BJ Signal most-cited paper award
16 August, 2011
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Juanma with his award

Juan M. Garcia-Martinez, until recently a postdoc in Dario Alessi's group, has been presented with the Biochemical Journal 'Signal' Paper of the Year 2011 award for the most-cited research article, his paper 'mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1)' having made the biggest contribution to the Biochemical Journal 2011 impact factor – see picture of Juanma with his award.

In this paper, published in December 2008, Juanma demonstrated that the mTOR complex 2 was the key regulator of SGK activity. This study has been cited 112 times.

Juanma is now working in the oncology division of Astrazeneca, developing new anti-cancer drugs.




 

John Rouse is made Professor
9 August, 2011

John Rouse, a Programme Leader in the MRC Protein Phosphorylation Unit (MRC-PPU), has been awarded a Personal Chair in Chromosome Biology by the University of Dundee in recognition of his outstanding contributions to our understanding of the molecular mechanisms involved in repairing damaged DNA. John became a Programme Leader in 2002 when he joined the MRC-PPU after carrying out postdoctoral research with Steve Jackson at the Gurdon Institute, Cambridge. This new accolade is the latest in a series of awards that John has received in recent years, which include the Colworth Medal of the Biochemical Society and the Tenovus Medal. Commenting on the award John said:

"It's a great honour that our work on how cells repair DNA damage has been recognized in this way by the University of Dundee. The research breakthroughs and advances that have emerged from my lab are a reflection of the talents and dedication of the people in my research team, and the stimulating and interactive research environment and outstanding facilities that we have here in the MRC Protein Phosphorylation Unit and College of Life Sciences at Dundee."




 

2011 Tim Hunt prize for Cell Biology awarded to Shuai Chen
9 August, 2011
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Shuai Chen receiving his award from Tim Hunt

Congratulations to Shuai Chen, postdoc in Carol MacKintosh’s group, for winning the 2011 Tim Hunt Prize for Cell Biology for his work on complementary roles of AS160 and TBC1D1 in regulating glucose uptake into muscle in response to insulin and energy stress.
The Tim Hunt prize for Cell Biology is awarded for a significant advance in basic research in any area of cell biology carried out in the College of Life Sciences at the University of Dundee. The award committee commented “We were all very impressed with your use of such a wide range of approaches to develop and test your hypothesis and your discoveries have clearly made a significant contribution to the understanding of the regulation of glucose homeostasis.”
Shuai’s studies used an elegant blend of biochemistry and genetics, the latter in collaboration with Kei Sakamoto, another programme leader in the Unit.
Shuai is the second member of the MRC Protein Phosphorylation Unit to be awarded this prize, following Anna Zagorska last year.




 

New Director for the MRC Protein Phosphorylation Unit Appointed
26 July, 2011

Professor Dario Alessi FRS, FRSE , has been appointed Director of the Medical Research Council’s (MRC) Protein Phosphorylation Unit at the University of Dundee. He will succeed the current Director, Sir Philip Cohen in April 2012.

The MRC Protein Phosphorylation Unit is the only research unit in the world dedicated to the study of protein phosphorylation, a versatile process that regulates almost all aspects of cell life. Abnormalities in protein phosphorylation are a cause of many diseases including cancer, diabetes, high blood pressure, rheumatoid arthritis and Parkinson's disease.

Dario is currently Deputy Director of the MRC Protein Phosphorylation Unit and also Professor of Signal Transduction in the College of Life Sciences at the University of Dundee. His research career spans over 20 years and his appointment as Director is testament to his pioneering research in this field.

In 2005 Dario was awarded the EMBO Gold Medal, Europe’s most prestigious research prize for life scientists under the age of 40. He was also invited to deliver the Francis Crick Prize Lecture of the Royal Society in 2006 and elected a Fellow of the Royal Society in 2008.

Drugs that treat abnormally high levels of phosphorylation are currently having a major impact on the treatment of cancer and over 50 per cent of the pharmaceutical industry's research and development budget is spent on this topic. The MRC Protein Phosphorylation Unit plays a major role in drug discovery and development through a long-standing collaboration with five of the world's major pharmaceutical companies. This partnership, called the Division of Signal Transduction Therapy, has become a model for effective collaboration between academia and industry.

Under Dario Alessi’s direction, the Unit’s future remit will be expanded to study the role of an emerging form of cell biology called ubiquitylation. Early research suggests that ubiquitylation rivals phosphorylation in its global importance for wider scientific understanding. There is great expectation that better knowledge of ubiquitylation and its relationship with phosphorylation could hold the key to determining the causes of many diseases and could lead to the development of new classes of medicine.

Speaking about his appointment, Dario said:

“It is a tremendous privilege to take over the helm of the MRC Protein Phosphorylation Unit from Sir Philip Cohen. Sir Philip will be a hard act to follow, but I am looking forward to the extraordinary opportunities that lie ahead.

“This is an exhilarating time in phosphorylation and ubiquitylation research and my main aim will be to position the unit as a focal point for collaboration between life scientists, pharmaceutical companies and clinicians. It is my strong belief that this is where we can make the most critical contribution to medical research, together reaching a deeper understanding of disease and developing more effective treatments.”

Speaking in support of Dario’s appointment, MRC Chief Executive Sir John Savill said:

"I am delighted that Professor Alessi has been appointed as Director of the MRC Protein Phosphorylation Unit. Professor Alessi is an exceptional scientist having made significant contributions to our understanding of the role of protein phosphorylation in cell regulation and human disease. Under his direction I feel confident that he will build on the unit’s world-leading reputation and excellent track record, ensuring that it remains at the forefront of medical research."

Commenting on Professor Alessi's appointment at the new Director of the MRC Protein Phosphorylation Unit, the current Director, Sir Philip Cohen, said:

"I am really delighted that Dario has been selected to be the next Director of the MRC Protein Phosphorylation, and am confident that he will build on what has been achieved over the last 21 years and bring it to a new level. I should also make it clear that although I am stepping down as the Unit's Director next April, I will not be retiring and indeed have recently signed a new six year contract with the University of Dundee. I am greatly looking forward to pursuing with even greater vigour my research programme aimed at understanding how the innate immune system is regulate, when I will be free from the administrative burden of running the Unit."




 

Developer of Gleevec Awarded Honorary Doctorate by the University of Dundee
20 July, 2011
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Philip Cohen and Nick Lydon

Nick Lydon, who initiated and drove the programme at Novartis, that led to the development of Gleevec, the first drug developed by targeting a specific protein kinase, was awarded an Honorary Doctor of Laws degree by the University of Dundee at the Life Sciences Graduation Ceremony held in the Caird Hall, Dundee on 22 June 2011.

Gleevec, an inhibitor of the Abd tyrosine kinase has transformed a form of leukaemia (CML, chronic myelogenous leukaemia) from a rapidly fatal disease into a manageable condition with survival rates of 90% after five years on the drug. It transformed the perception of protein kinases as drug targets and has become the most important drug marketed by Novartis with sales of US$3 billion in 2010.

Nick Lydon is an alumnus of the University of Dundee, having received a PhD in the Department of Biochemistry in 1982 for research on luteinising hormone in the laboratory of David Stansfield. After the development of Gleevec, Nick Lydon became an entrepreneur in the Biotechnology Industry and has funded four companies since 1988. To read the full laureation address which was delivered by MRC-PPU Director Philip Cohen, click here




 

MRC-PPU students awarded their PhD
4 July, 2011

On the morning of Wednesday June 22nd three of the Unit's students, Laura Pearce, Kirsty Martin and Hilary Smith were awarded their PhD's by Lord Narin Patel, the Chancellor of the University of Dundee, at a graduation ceremony at the Caird Hall in the city centre. Laura (supervisor Dario Alessi), Kirsty (supervisor Simon Arthur) and Hilary (supervisor Philip Cohen) are now carrying out postdoctoral research in Cambridge, Edinburgh and Dundee respectively.




 

Congratulations to Beatrice and Elton
30 June, 2011
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Beatrice and Elton on their wedding day

Two previous members of the MRC-PPU, Beatrice Filippi and Elton Zeqiraj, were married on Saturday 25th June in Toronto, Canada. Beatrice and Elton were members of Dario Alessi’s research group and are presumed to have got together whilst collaborating on a research project aimed at understanding the structure and function of the LKB1 tumour suppressor protein kinase.

Both Beatrice and Elton are now undertaking postdoctoral research at the Samuel Lunenfeld Institute, Mount Sinai Hospital, Toronto. Best wishes from everyone at the MRC-PPU on their marriage.




 

Congratulations to Anna Zagorska for award of prestigious HFSP fellowship
7 June, 2011
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Anna in her new lab in San Diego

Anna Zagorska, an ex-MRC-PPU PhD student who worked in Dario Alessi’s laboratory on the WNK and LKB1 signalling pathways, has been awarded a prestigious Human Frontiers Science Program Fellowship (HFSP)to undertake postdoctoral research in Greg Lemke's lab at the Molecular Neurobiology Laboratory, the Salk Institute, La Jolla, California. Anna is now working on trying to understand the cellular mechanisms that are important for the resolution of the immune system. Human Frontiers Science Program Fellowships are aimed at “supporting top postdoctoral researchers that propose innovative, ground-breaking projects that have the potential to advance the knowledge in their field of study and open a new approach to the research problem”.




 

Laura Pearce awarded prestigious Sir Henry Wellcome postdoctoral Fellowship
1 June, 2011

Laura Pearce who undertook her PhD research project in Dario Alessi's laboratory (October 2007 - December 2010) has been awarded the UK’s most sought-after postdoctoral fellowship to work in the laboratory of Sadaf Farooqi at the University of Cambridge. Laura’s new project will be to make use of the state of the art technologies in this golden era of genetic research to identify mutations in key genes that induce obesity in humans.

During her PhD in the MRC Protein Phosphorylation Unit, Laura identified the Protor-1 and Protor-2 regulatory subunits of the mTOR complex 2. Laura the generated Protor-1 and Protor-2 knock-out mice and found that these subunits are required for normal activation of SGK isoforms in the kidney

The Sir Henry Wellcome Fellowship aims “to provide a unique opportunity for the most promising newly qualified postdoctoral researchers to make an early start in developing their independent research careers, working in the best laboratories in the UK and overseas.” Candidates are required to identify an important biomedical research question and to develop and deliver a personal programme to achieve their research aims. The fellowship is for four years and will provide Laura with an award of £250 000 to undertake her research over this period.

Laura is the second MRC-PPU student to be awarded a Sir Henry Wellcome Fellowship. Elton Zeqiraj who worked jointly between Dario Alessi’s and Daan van Aalten’s laboratories was awarded a Sir Henry Wellcome postdoctoral Fellowship last year to work in Frank Sicheri's lab at the Samuel Lunenfeld Institute in Mount Sinai Hospital (Toronto, Canada) on structural analysis of important signalling and metabolic components.




 

Alban Ordureau is awarded a PhD
24 May, 2011
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Alban enjoying his post-viva celebrations

Alban Ordureau, a graduate student working in Philip Cohen's laboratory, was awarded his PhD on May 23rd for a thesis entitled, "An investigation of the role of E3 ubiquitin ligases in regulating innate immunity".

The examination consisted of a 45 minute lecture by Alban, which was followed by questions from the audience. Following a short break to replenish blood glucose and caffeine levels Alban was then grilled for 3.5 hours about the content of his thesis by external examiner Andrew Bowie from Trinity College, Dublin, Ireland, an expert on innate immunity, and from internal examiner Ron Hay, an expert in the ubiquitin system.




 

MRC-PPU scientists discovery link between polyubiquitylation and autoimmunity
18 May, 2011

Scientists in the MRC Protein Phosphorylation Unit have discovered that a polyubiquitin-binding protein called ABIN1 plays a key role in preventing autoimmune disease and their findings were published on line on 23 May 2011 in the Journal of Experimental Medicine and will appear in the June 6th issue of the journal.

Sambit Nanda, a postdoc working in Philip Cohen's research team, studied a mouse line developed in the Institute in which wild type ABIN1 was replaced by a mutant in which the aspartyl residue at position 485 was converted to asparagine, which prevents ABIN1 from binding to Lys63-linked or linear polyubiquitin chains. The mutant mice progressively developed an autoimmune disease that had all the hallmarks of human lupus. Sambit went on to show that immune cells from the mice produced elevated levels of proinflammatory cytokines in response to ligands that activate Toll-Like receptors (TLRs), and proved that the hyperactivation of TLRs was the cause of the autoimmune disease by showing that autoimmunity could be prevented by crossing to mice that do not express MyD88, an adaptor protein essential for TLR function. The study establishes that the binding of polyubiquitin to ABIN1 is required for this protein to limit the strength of TLR signaling in vivo. More detailed analysis of TLR signaling pathways indicates that ABIN1 exerts its effects between MyD88 and the activation of a protein kinase, termed TAK1. Further studies are underway to elucidate the underlying molecular mechanism.

Over the past couple of years polymorphisms in ABIN1 have been discovered in several human populations which predispose to autoimmune diseases, such as lupus and psoriasis. This suggests that the mouse line developed in this study may be a good model for human autoimmune diseases and be valuable in testing the efficacy of new drugs to treat autoimmunity, which exert their effects by targeting components of TLR signaling pathways.




 

Ben Nevis charity walk
18 May, 2011

The Arthur Research Group are going to walk Ben Nevis (Britain's highest mountain!) on Saturday 2nd July 2011 in aid of Arthritis Research UK. They have chosen this charity as much of their research relates to understanding how inflammation is regulated. This will help them to understand the causes and pathology of inflammatory diseases including arthritis.


As a group of (mostly) novice walkers this is going to be something of a challenge for them so they would REALLY appreciate your support for such a great cause: Just Giving




 

Local fundraising boosts diabetes research within Unit
11 May, 2011
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Fundraising cheque handed to Kei Sakamoto

Diabetes research run by Kei Sakamoto’s group in the Unit has been boosted by the donation of £4,000 from the Dundee and District Diabetes UK Volunteer Group. The group raised money through a number of fundraising activities; hosting a bingo night, organising raffles and as a result of donations. Kei receives the donation during Dundee Diabetes UK’s monthly meeting at Ninewells Hospital on Tuesday, 10th May.

Kei expressed his gratitude to the group, saying, 'I would like to thank all the Dundee Diabetes UK support team members and their family and friends who have raised such a tremendous amount of funds to support my research team in the MRC PPU. It is a huge help for my group to receive such consistently strong support from the people of Dundee and the surrounding area. We will use these funds to carry out important research projects.'
Kei’s team currently studies the molecular signalling and physiological processes by which nutrients, hormones, and exercise control blood sugar levels.




 

Michale and Noor publish MRC Units first spinocerebellar ataxia TTBK2 paper.
10 May, 2011

Recent studies have revealed that mutations within the Tau tubulin Kinase 2 (TTBK2) cause a serious autosomal dominant inherited movement disorder termed spinocerebellar ataxias type 11 (SCA11) [1, 2]. SCA11 is characterised by progressive cerebellar ataxia, pyramidal features, peripheral neuropathy, with age of onset from the early teens to the mid 20s [3]. TTBK2 is closely related to a neuronal specific protein kinase termed TTBK1, which was discovered in 1995 as a protein kinase in bovine brain extract that phosphorylated tau and tublin [4].

TTBK2 consists of 1244 residues and apart from an N-terminal serine/threonine protein kinase domain (residues 20 to 280) possesses no distinctive functional domains or motifs. Thus far, four distinct SCA11 families have been identified, each possessing a mutation leading to premature termination of the TTBK2 protein at around residue 450. This leaves the kinase catalytic domain intact, but eliminates most of the non-catalytic portion of the enzyme.

Michale and Noor decided to investigate some of TTBK2’s properties as nothing is known about TTBK2 substrate specificity and how SCA11 truncating mutations impact on protein expression, kinase activity, stability and localisation.

Noor started by collaborating with Mike Begley and Lew Cantley at Harvard Medical School to analyse TTBK2 substrate specificity. This surprisingly revealed that TTBK2 possesses a conspicuous preference for a phosphotyrosine at the +2 position relative to the phosphorylation site. Noor then exploited this information to develop an optimised peptide substrate (RRKDLHDDEEDEAMSIYpA) to assess TTBK2 catalytic activity, that he termed TTBKtide. Molecular modelling and mutagenesis analysis undertaken by Noor suggest that the TTBK2 catalytic domain may possess a phosphotyrosine binding substrate-docking site within its catalytic domain.

Michale Bouskila next found that all SCA11 truncating mutations markedly enhanced TTBK2 protein expression, but lead to inhibition of TTBK2 kinase activity as well as promoting nuclear localisation. Michale also generated TTBK2 knock-in mice expressing an SCA11 disease causing mutation and found that this resulted in marked inhibition of endogenous protein kinase activity. Michale in collaboration with Emily Fang and Kate Storey found that in homozygosity the SCA11 mutation caused embryonic lethality at E10. The heterozygous SCA11 animals displayed no obvious movement disorder even after 1 year of age.

These results provide initial insights into substrate specificity of TTBK2 and how SCA11 causing mutations impact on kinase activity and localisation.

In future studies it would be important to define the substrates that TTBK2 phosphorylates and investigate whether reduced phosphorylation of these targets contributes to the development of SCA11. In particular, it would be interesting to determine whether physiological TTBK2 substrates were primed with a +2 phosphotyrosine and how regulation of tyrosine phosphorylation was coupled to the control of these TTBK2 targets.

Identifying the key targets of TTBK2 could provide vital new insights into the molecular mechanism underpinning the development of spinocerebellar ataxia and result in new ideas as to how this debilitating disease might be better treated.

To read a copy of Michale and Noor’s paper click here

References
1 Houlden, H., Johnson, J., Gardner-Thorpe, C., Lashley, T., Hernandez, D., Worth, P., Singleton, A. B., Hilton, D. A., Holton, J., Revesz, T., Davis, M. B., Giunti, P. and Wood, N. W. (2007) Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11. Nat Genet. 39, 1434-1436
2 Bauer, P., Stevanin, G., Beetz, C., Synofzik, M., Schmitz-Hubsch, T., Wullner, U., Berthier, E., Ollagnon-Roman, E., Riess, O., Forlani, S., Mundwiller, E., Durr, A., Schols, L. and Brice, A. (2010) Spinocerebellar ataxia type 11 (SCA11) is an uncommon cause of dominant ataxia among French and German kindreds. J Neurol Neurosurg Psychiatry. 81, 1229-1232
3 Houlden, H. (2008) Spinocerebellar Ataxia Type 11. In GeneRevews (Pagon, ed.), University of Washington, Seattle
4 Takahashi, M., Tomizawa, K., Sato, K., Ohtake, A. and Omori, A. (1995) A novel tau-tubulin kinase from bovine brain. FEBS Lett. 372, 59-64




 

Princess Anne visits the MRC Protein Phosphorylation Unit
19 April, 2011
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Princess Anne talks to Dario's PhD students Eva Sommer (centre) and Helen Woodruff (right)

Princess Anne, the Princess Royal, visited the MRC Protein Phosphorylation Unit on April 19 in her role as the President of Tenovus, a Scottish cancer charity. The Princess met John Rouse, one of the Unit's Programme Leaders, who was recently awarded the 2011 Tenovus Medal. Princess Anne also met Deputy Unit Director, Dario Alessi, who has received funding for some of his cancer research from Tenovus. Princess Anne also talked to two of Dario's students, Eva Sommer, who works on the role of the protein kinase SGK in cancer and Helen Woodruff, who is carrying out research to understand how mutations in the protein kinase PINK1 causes Parkinson's Disease.



Dario explaining to Princess Anne what his lab is working on




 

Miratul becomes Hospital Consultant
11 April, 2011

Miratul Muqit, a Wellcome Intermediate Fellow working with Dario Alessi, has been appointed a Consultant Neurologist at Ninewells Hospital in Dundee. Miratul was the first clinician to be appointed to the MRC-PPU and now becomes the first to reach the consultant clinical grade. Since his arrival in 2008, the MRC-PPU has recruited a further four clinicians, the largest number of any Division of the College of Life Sciences.




 

Effects of PI 3-kinase and mTOR inhibitors on treating spontaneous treating B-cell follicular lymphoma revealed
28 March, 2011
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Stephan Wullschleger and Juan M. García-Martínez pictured next to Suzanne Duce's MRI

The PI 3-kinase-mTOR signalling pathway is activated in the majority of human tumours and there is great interest in assessing whether inhibitors of PI 3-kinase or mTOR kinase would have efficacy in treating cancers in which this pathway is over activated.

Stephan Wullschleger and Juan M. García-Martínez, MRC Unit postdocs were interested to answer the question of whether state-of-the-art PI 3-kinase and mTOR inhibitors would be effective at treating tumours that arise spontaneously in mouse model of cancer. These model being more representative of human tumours than Xenograft models frequently employed to assess the effectiveness of potential anti-cancer drugs.

To tackle this question Stephan and Juanma teamed up with Suzanne Duce an MRI expert at the Division of Biological Chemistry & Drug Discovery, College of Life Sciences in Dundee.

Stephan and Juanma decided to make use of a strain of mouse (PTEN+/-LKB1+/hypo) in which the PI 3-kinase/mTOR pathway is activated and spontaneously develop B-cell follicular lymphomas about 6 months of age.

They waited until substantial tumours had formed and then commenced treatment with either a compound called GDC-0941 (a Genentech PI 3-kinase inhibitor) or a compound termed AZD8055 (an AstraZeneca mTOR inhibitor) that are both in clinical trials.

Using MRI methodology, Suzanne was able to demonstrate that administration of either AZD8055 or GDC-0941 induced ~40% reduction in tumour volume within 2 weeks. Signalling experiments showed that this reduction in tumour volume was accompanied by ablation of phosphorylation of PI 3-kinase-mTOR pathway substrates (AKT, S6K and SGK).

The drugs also reduced tumour cell proliferation and promoted apoptosis, which resulted in reduction of the centroblast population within the tumours. Further treatment with either AZD8055 or GDC-0941 beyond two weeks caused a moderate additional decrease in tumour volume, reaching ~50% of the initial volume after 6 weeks of treatment. Upon cessation of drug treatment, tumours grew back at a slightly increased rate compared to aged-matched non-drug treated tumours, and displayed similar high grade and diffuse morphology as the controls.

These results define the effects that newly designed and highly specific mTOR and PI3K inhibitors have on a spontaneous tumour model. These data suggest that AZD8055 and GDC-0941 would offer benefit for the treatment of cancers in which the PI 3-kinase-mTOR pathway is inappropriately activated; however, other combination strategies would need to be tested to induce the complete regression of such tumours.

We propose that the spontaneous cancer model elaborated in this work could be utilised to benchmark the relative effectiveness of PI3K/mTOR inhibitors at suppressing tumour growth. It would be interesting to test whether it was possible to develop signal transduction inhibitors that were more effective than GDC-0941 or AZD8055 at reducing tumour burden as single agents.

To read a copy of Stephan’s, Juanma’s and Suzanne’s paper click here




 

Possible role of mTOR Protor subunits in regulating SGK activation uncovered
23 March, 2011
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Laura Pearce and Eeva Sommer

A few years ago at the beginning of her PhD Laura Pearce identified novel components of the mTOR complex 2 (mTORC2) that she termed Protor-1 and Protor-2. Laura found that Protor isoforms interacted with the mTORC2 component Rictor. Protor-1 and Protor-2 possess significant similarity and are encoded by different genes. Unfortunately, Protor-isoforms do not possess any obvious domains or motifs that yield clues about their physiological roles.

As the role of these subunits was unclear Laura decided to generate knockout mice lacking Protor-1 and/or Protor-2. Laura found that the single and double knockout mice were viable and displayed no obvious phenotype. She also found that loss of Protor-1 and/or Protor-2 did not affect the expression of the other mTORC2 components, nor their ability to assemble into an active complex capable of phosphorylating the hydrophobic motif of Akt (Ser473). Laura next found that Protor knockout mice displayed no defects in the phosphorylation of Akt and PKCα at their hydrophobic or turn motifs.

Just as Laura was giving up hope of finding a phenotype, she observed that Protor-1 knockout mice displayed markedly reduced phosphorylation of SGK1 at its hydrophobic motif a residue that is directly phosphorylated by mTORC2. Moreover, the physiological substrate of SGK1, NDRG1 was also much less phosphorylated in the kidney of Protor deficient mice, indicative of reduced SGK1 activity in these animals. The kidney is the only tissue of the mouse in which it was possible to observe significant basal SGK1 activity and NDRG1 phosphorylation.

This work was greatly aided by Eeva Sommer who helped identify antibody reagents capable of detecting endogenous SGK1 and measuring phosphorylation of the hydrophobic and T-loop residues of SGK1. Eeva also undertook all of the additional experiments that were requested by the Reviewers as Laura had already departed to start her postdoc in Sadaf Farooqi’s laboratory in Cambridge.

These new data suggest that Protor-1 may play a role in enabling mTORC2 to efficiently activate SGK1, at least in the kidney. A key question for future work is what is the mechanism by which Protor isoforms enhance phosphorylation of SGK1 in the kidney? Do Protor subunits act as scaffolding components to aide mTORC2 activating SGK1? Is the dependency of SGK1 activation on Protor subunits specific to kidney tissues?

To read a copy of Laura’s and Eeva’s paper click here




 

Beatrice and Paola discover that MO25 scaffolding protein has roles beyond regulating the LKB1 tumour suppressor
22 March, 2011
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Beatrice Filippi and Paola de los Heros

Beatrice Filippi and Paola de los Heros MRC Unit postdocs working in Dario Alessi’s lab have reevaluated the role of the MO25 scaffolding protein, that Jerome Boudeau - a previous postdoc now running a lab in Montpellier - discovered was a key component required for the activation of the LKB1 tumour suppressor complex [1].

MO25 is required to activate LKB1 and is required for LKB1 to phosphorylate and activate its 14 downstream AMPK family kinase substrates [2-4]. MO25 activates LKB1 through its ability to bind to a catalytically inactive pseudokinase termed STRAD, which is related to STE20 family protein kinases [1, 6, 7]. Elton Zeqiraj, a former PhD student in the MRC Unit (now a Sir Henry Wellcome Fellow working in Toronto), showed that MO25 stabilises STRAD in an active conformation that is capable of binding to and stimulating LKB1.

By analyzing in depth the three-dimensional structure and the interface of MO25-STRAD complex, Beatrice realized that other kinases related to STE20 family of protein kinases posses significant homology with the regions of STRAD that interact with MO25. Beatrice focused on studying five of these STRAD related kinases that play vital biological roles in regulating ion homeostasis and blood pressure (SPAK/OSR1) and cell growth and morphogenesis (MST3/MST4/YSK1).

Beatrice was able to demonstrate that five kinases do indeed interact with MO25 isoforms. Working with Paola they showed that remarkably, MO25 binding stimulated the activity of SPAK and OSR1 over 100-fold. In the case of MST3/MST4/YSK1, binding of MO25 increased the activity of these enzymes ~4-fold.

Beatrice showed that MO25 binds to these STRAD-related STE20 kinases with micromolar affinity, and that mutations that prevent the interaction of MO25 with STRAD also impaired binding of MO25 to SPAK/OSR1 and MST3/MST4/YSK1, as well as activation of these enzymes.

Paola was able to demonstrate that ~70% reduction of MO25α using an siRNA approach in 293 cells inhibited phosphorylation of NKCC1, a physiological substrate of SPAK/OSR1 kinases. Importantly this inhibitory effect was rescued following re-expression of MO25.

Paola also observed that SPAK/OSR1 phosphorylates the three identified substrates of this enzyme (NKCC, NKCC2 and NCC) at a vastly higher rate in the presence of MO25. Significantly, phosphorylation site analysis led to the identification of novel in vitro phosphorylation sites not observed in previous phosphorylation studies undertaken in the absence of MO25. Most of the new reported phosphorylation sites such as Ser71 (mouse sequence of NCC, Ser73 in human sequence) [8] have been identified to be phosphorylated in vivo, following stimulation of cells or tissues with osmotic shock conditions that activate the SPAK/OSR1 pathway. Paola’s new data therefore suggests that SPAK/OSR1 are likely to represent the physiological kinases that phosphorylate these residues rather than another kinase.

There is also great interest in generating inhibitors of SPAK/OSR1 for the treatment of hypertension. The finding that these enzymes can be activated 100-fold by binding to MO25 will be vital for enabling these inhibitor studies. We suggest that screening for SPAK/OSR1 inhibitors are undertaken with the SPAK-MO25 or OSR1-MO25 complex rather than isolated SPAK or OSR1.

Recent findings by Sean Lawler research group at Columbus Ohio (who incidentally was a previous MRC Unit postdoc working with Philip Cohen) revealed that a microRNA (miR-451) that promoted growth of gliomas by inhibiting expression of the MO25α isoform [5]. The effects of miR-451 on cancer cells were interpreted to be all mediated through inhibition of the LKB1 tumour suppressor. However, our new data indicate that the effects of miR-451 are also likely to be contributed to the inhibition of other STE20 kinases that are regulated by MO25.

Our data suggests that MO25 evolved as an ancestral regulator of the active conformation of kinases as well as pseudokinases. It is likely that MO25 may interact with additional kinases that we have not investigated in our study. We also discuss a controversial idea that the original kinase may have evolved from a pseudokinase whose role could have
been to interact with targets in a manner regulated by MO25 and ATP binding.

To read a copy of Beatrice’s and Paola’s paper click here.

Beatrice has recently started working as a postdoc in the MaRS Centre in Toronto.

References
1 Boudeau, J., Baas, A. F., Deak, M., Morrice, N. A., Kieloch, A., Schutkowski, M., Prescott, A. R., Clevers, H. C. and Alessi, D. R. (2003) MO25alpha/beta interact with STRADalpha/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm. EMBO J. 22, 5102-5114
2 Hawley, S. A., Boudeau, J., Reid, J. L., Mustard, K. J., Udd, L., Makela, T. P., Alessi, D. R. and Hardie, D. G. (2003) Complexes between the LKB1 tumor suppressor, STRADalpha/beta and MO25alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol. 2, 28
3 Jaleel, M., McBride, A., Lizcano, J. M., Deak, M., Toth, R., Morrice, N. A. and Alessi, D. R. (2005) Identification of the sucrose non-fermenting related kinase SNRK, as a novel LKB1 substrate. FEBS Lett. 579, 1417-1423
4 Lizcano, J. M., Goransson, O., Toth, R., Deak, M., Morrice, N. A., Boudeau, J., Hawley, S. A., Udd, L., Makela, T. P., Hardie, D. G. and Alessi, D. R. (2004) LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J. 23, 833-843
5 Godlewski, J., Nowicki, M. O., Bronisz, A., Nuovo, G., Palatini, J., De Lay, M., Van Brocklyn, J., Ostrowski, M. C., Chiocca, E. A. and Lawler, S. E. (2010) MicroRNA-451 regulates LKB1/AMPK signaling and allows adaptation to metabolic stress in glioma cells. Mol Cell. 37, 620-632
6 Boudeau, J., Miranda-Saavedra, D., Barton, G. J. and Alessi, D. R. (2006) Emerging roles of pseudokinases. Trends Cell Biol. 16, 443-452
7 Boudeau, J., Scott, J. W., Resta, N., Deak, M., Kieloch, A., Komander, D., Hardie, D. G., Prescott, A. R., van Aalten, D. M. and Alessi, D. R. (2004) Analysis of the LKB1-STRAD-MO25 complex. J Cell Sci. 117, 6365-6375
8 Yang, S. S., Morimoto, T., Rai, T., Chiga, M., Sohara, E., Ohno, M., Uchida, K., Lin, S. H., Moriguchi, T., Shibuya, H., Kondo, Y., Sasaki, S. and Uchida, S. (2007) Molecular pathogenesis of pseudohypoaldosteronism type II: generation and analysis of a Wnk4(D561A/+) knockin mouse model. Cell Metab. 5, 331-344




 

Former MRC-PPU postdoc elected a Fellow of the Royal Society of Edinburgh
15 March, 2011

Hugh Nimmo, the first postdoctoral fellow of Unit Director Philip Cohen, has been elected a Fellow of the Royal Society of Edinburgh. Hugh worked in Philip's laboratory from 1973 to 1976 on a grant funded by the British Diabetic Association (now called Diabetes UK). During this period he discovered that the enzyme glycogen synthase was phosphorylated at more than one site by more than one protein kinase, the first example of such "multisite phosphorylation." He then set up his own laboratory at the University of Glasgow, Scotland, where he has worked ever since, later being promoted to Full Professor and Chairman of the Department of Biochemistry. Hugh has made many important contributions to our understanding of the regulation of plant function by reversible phosphorylation. Most recently, he discovered how phosphorylation enables roots and shoots to operate distinct types of circadian clocks that are synchronized to allow plants to anticipate daily environmental changes. Hugh's wife Gill was also a postdoc in Philip Cohen's lab, while Carol MacKintosh, a Programme Leader in the MRC Protein Phosphorylation Unit, was a Ph.D. student in Hugh's laboratory before coming to Dundee. The Royal Society of Edinburgh, which is Scotland's National Academy of Sciences was founded in 1783 and is one of the world’s oldest scientific academies.




 

Characterisation of the first selective inhibitor of the Parkinson’s disease LRRK2 kinase
8 March, 2011

Autosomal dominant missense mutations within the gene encoding for the Leucine-Rich Repeat protein Kinase 2 (LRRK2) predispose humans to develop Parkinson’s disease (PD). Little is understood about how LRRK2 is regulated, or how it functions, or how mutations cause Parkinson’s disease. The most frequent LRRK2 mutation (G2019S) enhances kinase activity suggesting inhibitors may be useful for the treatment of Parkinson’s disease. Whether inhibiting LRRK2 could be used as a strategy to treat Parkinson’s disease is unknown and a key question for research in this field.

Nic Dzamko working in collaboration with Xianming Deng and Nathanael Gray at the Dana-Farber Cancer Institute have characterised a new compound termed LRRK2-IN-1 that potently inhibits LRRK2 (see figure). This compound was generated in the Gray laboratory and inhibits wild type LRRK2 with an IC50 of 13 nM and LRRK2[G2019S] mutant with an IC50 of 6 nM.

LRRK2 specificity was comprehensively evaluated against over 470 protein kinases and found to be remarkably specific, inhibiting only one other kinase namely MAPK7/ERK5 with similar affinity to LRRK2. Through molecular modelling of how LRRK2-IN-1 may bind to LRRK2, we were able to elaborate a LRRK2 mutation (A2016T) which is normally active, but ~400-fold resistant to LRRK2-IN-1.

As no LRRK2 substrates have yet been identified, there was no easy way to evaluate how effectively LRRK2-IN-1 suppressed LRRK2 activity in vivo. However, previous work undertaken by Nicolas Dzamko and a former postdoctoral fellow Jeremy Nichols (now a Principal Investigator, The Parkinson’s Institute, Sunnyvale, California) suggested that phosphorylation of LRRK2 at two residues (Ser910 and Ser935) was controlled by a feedback pathway regulated by LRRK2 kinase activity itself [1, 2].

If this is correct, then LRRK2-IN-1 should induce dephosphorylation of endogenous LRRK2 at Ser910 and Ser935. Nic therefore tested the effect that LRRK2-IN-1 had on Ser910 and Ser935 phosphorylation in various cell types. He found, consistent with our hypothesis, that LRRK2-IN-1 promoted rapid dephosphorylation of Ser910 and Ser935 on endogenous LRRK2. Nic also found that injection of LRRK2-IN-1 into mice induced dephosphorylation of LRRK2 in the kidney and spleen, but not in the brain, indicating that LRRK2-IN-1 poorly penetrates the blood-brain barrier.

Previous work also suggested that Ser910 and Ser935 phosphorylation promoted binding of 14-3-3 and maintained LRRK2 diffusely localised in the cytoplasm of cells [1, 2]. Consistent with this, Nic found that treatment of cells with LRRK2-IN-1 induced dissociation of 14-3-3 binding and promoted the accumulation of LRRK2 within aggregate-like structures in the cytoplasm.

Importantly, LRRK2-IN-1 did not induce dephosphorylation or inhibit 14-3-3 binding or induce aggregation of the drug resistant LRRK2[A2016T] mutant. We would recommend that for all work undertaken with LRRK2-IN1, the drug resistant mutant be employed as a control to ensure effects of LRRK2-IN1 are not mediated inhibition of other targets such as ERK5.

These data confirm that LRRK2-IN1 does indeed effectively inhibit LRRK2 activity in vivo. These results provide further evidence to support the hypothesis that phosphorylation of Ser910 and Ser935 is controlled by LRRK2 kinase activity. We recommend that monitoring phosphorylation of LRRK2 at Ser910 and Ser935 be employed to assess the effectiveness of LRRK2 inhibitors being developed by the pharmaceutical industry. It will also be important to identify the protein kinase that phosphorylates Ser910 and Ser935 and determine whether it is regulated by LRRK2.

The discovery of LRRK2-IN-1 is important, as it will be a very useful tool compound to explore the physiological roles of LRRK2. We hope that LRRK2-IN1 could have a similar impact to the LRRK2 research field as other kinase inhibitors such as PD98059, SB203580 and wortmanin/LY294002 have had on elucidating the roles of the ERK, P38 and PI 3-kinase signalling pathways. We will make LRRK2-IN-1 available with no strings attached to any laboratory working on LRRK2. Hopefully LRRK2-IN-1 will also help with defining whether inhibiting LRRK2 would have utility for the treatment of Parkinson’s disease.

It is also noteworthy that LRRK2-IN-1 was generated in the academic laboratory of Nathanael Gray, who has also developed hugely specific and potent inhibitors to many other important cellular kinases including mTOR, Abl, EGFR, FGFR, Eph receptor, ERK5 and aurora.

There is the perception that pharmaceutical companies can only generate highly specific kinase inhibitors, and that academics are best to stay out of this business. However, the work that Nathanael Gray has undertaken demonstrates that it is indeed possible to elaborate very useful tool compounds with the potential to revolutionise understanding of biological system outside a pharmaceutical environment.

It is likely that many of the future advances in our understanding of biological systems will be made possible by the elaboration of new chemical tools such as LRRK2-IN-1 and therefore medicinal chemistry whether undertaken in company or academic setting deserves great support.

To Read the recent LRRK2-IN-1 paper click here

References
1 Nichols, J., Dzamko, N., Morrice, N. A., Campbell, D. G., Deak, M., Ordureau, A., Macartney, T., Tong, Y., Shen, J., Prescott, A. and Alessi, D. R. (2010) 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease associated mutations and regulates cytoplasmic localisation. Biochem J. 430, 393-404
2 Dzamko, N., Deak, M., Henati, F., Reith, A. D., Prescott, A. R., Alessi, D. R. and Nichols, R. J. (2010) Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser910/Ser935, disruption of 14-3-3 binding and altered cytoplasmic localisation. Biochem J 430, 405-413




 

John Rouse awarded 2011 Tenovus Medal
22 February, 2011

Dr. John Rouse, a Programme Leader at the Medical Research Council (MRC) Protein Phosphorylation Unit in the College of Life Sciences at the University of Dundee has been awarded the 2011 Tenovus Medal.

The Tenovus Medal Lecture has been presented annually at the University of Glasgow since 1992 and is awarded each year to a scientist under the age of 40 with a Scottish link whose work has had a major impact on the field of cancer.

John Rouse was born and educated in Ireland receiving a 1st Class honours degree at Trinity College Dublin. He then moved to Dundee where he carried out research for his PhD degree under the supervision of Sir Philip Cohen in the MRC Protein Phosphorylation Unit. He held a postdoctoral position at the University of Cambridge from 1997-2002 before returning to Dundee to set up his own research laboratory, gaining a Career Appointment from the MRC in 2008.

The focus of John’s research is to understand how cells recognize and repair DNA damage to prevent mutations that lead to cancer. The DNA in every cell of our body is like an instruction manual for the normal functioning of cells. A major problem is that DNA is constantly under attack from agents that cause DNA damage, such as ultraviolet radiation from the sun and toxic substances in the atmosphere, as well as substances that are produced naturally within our cells. This can give rise to changes or “mutations” in DNA. However, DNA damage does not normally cause problems for health because our cells can detect and repair DNA damage. However, if DNA repair fails and mutations gradually accumulate, this can lead to the “re-writing” of the instructions encoded in DNA and it is these undesirable changes that underlie cancer.

In recent years, John’s laboratory has discovered a range of novel DNA repair proteins that are essential for repairing DNA. These include several enzymes, collectively called the “SLX4 nuclease complex”, which act a “molecular toolkit” for DNA repair. Mutations in SLX4 that adversely affect its function greatly increase susceptibility to cancer. John also identified another enzyme called the “FAN1 structure-specific nuclease”, which acts as a “molecular scissors” for trimming superfluous pieces of DNA during DNA repair, and the “MMS22L–TONSL” complex that initiates the repair of broken DNA.

Commenting on the award John said: “I’m delighted to be awarded the Tenovus Medal, which is a reflection of the talents and efforts of the people who have worked in my lab and the outstanding research environment and facilities that we have here in the MRC Protein Phosphorylation Unit and College of Life Sciences at Dundee”. John will present the Tenovus Medal Lecture, at University of Glasgow on June 8th 2011. He becomes the third researcher from the College of Life Sciences at Dundee to be awarded the Tenovus Medal. The previous recipients were Tom Owen-Hughes in 2007 and Neil Perkins in 2009, who carried out the work for which they received the Tenovus Medal in the Wellcome Trust Centre for Gene Regulation and Expression at Dundee.




 

Human capicúa is a missing link in a cancer cell pathway
26 January, 2011
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Kumara Dissanayake

In the Spotlight in the latest edition of the Biochemical Journal is a paper by Kumara Dissanayake in Carol MacKintosh's group "Erk/p90RSK/14-3-3 signalling impacts on expression of PEA3 Ets transcription factors via capicca". Capicúa, pronounced Cap-ee-Coo-a, is Catalan for head-and-tail, named because mutation of the fruit fly version affects early head and tail development. Now, Kumara reports his discovery that human capicúa is a missing link between the oncogenic Erk signaling pathway and downstream transcription of ETV1 and other genes that are linked to particularly aggressive forms of prostate cancer and melanoma. Specifically, capicúa represses mRNA expression of the PEA3 Ets transcription factors ETV1, ETV4 and ETV5, which may be relieved by multisite controls of capicúa by the protein kinase Erk, p90RSK and 14-3-3 proteins. Experimental depletion of capicúa rendered cancer cells resistant to the therapeutic effects of drugs that block signaling upstream of Erk. As well as its implications for cancer and cancer therapy, the study also suggest capicúa as a nexus that interconnects the signaling networks of growth factor signaling, spinocerebellar ataxias and certain cancers.




 

Craig MacKay wins the Howard Elder Prize
24 January, 2011

Craig MacKay from the MRC Protein Phosphorylation Unit has been awarded the 2010 Howard Elder Prize for his discovery in John Rouse's
lab of the FAN1 nuclease that acts as a “molecular scissors” during DNA repair (MacKay et al, Cell 142, 65-76).

The Fanconi anaemia (FA) proteins are important for DNA repair. Mutations in the proteins of the FA complex result in high levels of chromosomal instability, caused by an inability to repair DNA interstrand crosslinks (ICLs), and hence predisposition to cancer. The central component of the FA pathway is FANCD2 that becomes mono–ubiquitylated at Lys561 in response to ICLs. The mono–ubiquitylation of FANCD2 is essential for ICL repair but despite much work this promotes ICL repair has remained elusive.

During his PhD with John, Craig discovered a novel UBZ domain-containing protein FAN1 that binds to, and is recruited to sites of DNA damage by, the mono–ubiquitylated form of FANCD2. Importantly, the depletion of FAN1 from human cells causes chromosome aberrations and major defects in DNA repair, similar to cells from patients with FA. FAN1 acts at the final stages of ICL repair when homologous recombination (HR) comes into play, and Craig’s data show that FAN1 acts late during HR, at a point that involves the processing or “trimming” of DNA repair intermediates. This led Craig to discover that FAN1 is a 5’ flap endonuclease and his data suggest that FAN1 processes branched recombination intermediates, enabling homologous recombination to go to completion. Craig’s findings at least partly explain the long-standing puzzle of how FANCD2 ubiquitylation promotes DNA repair.

Craig’s FAN1 paper is has been featured in several recent News articles: (e.g. Mol. Cell 39, 167; Nat. Struct. Mol. Biol. 17, 926; Nat. Rev. Mol. Cell. Biol. 11, 603; Cell Cycle 9, 4259; Cell Cycle 9, 4261). To read a copy of Craig's paper click here

Following the completion of his Ph.D. Craig has taken up a postdoctoral position with Arno Alpi in the Scottish Institute for Cell Signaling (scills.ac.uk) at the University of Dundee, where he is working on different aspects of the Fanconi anaemia pathway.

The Howard Elder Prize was endowed 25 years ago by Dr Alison Burt in memory of her father (Dr Howard Elder, a former medical graduate of the University). The prize is awarded each year to a PhD student or postdoctoral researcher in the College of Life Sciences at the University of Dundee deemed to have published the most significant paper in an area related to cancer research. The award is accompanied by a cash prize of £500.

This is the third year in a row that an MRC Unit researcher has been awarded the Howard Elder Prize. In 2009, Elton Zeqiraj won it for his work on the structure of the LKB1 tumour supressor (to read the relevant news item click here), and in 2008 Xu Huang got the prize for his work on the role of LKB1 and AMPK in regulating tumorigenesis (to read the relevant news item click here).




 

The MacKintosh and Sakamoto teams define a key step in insulin control of blood sugar
21 January, 2011
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Shuai Chen

Glucose sugar surges into our bloodstream after a meal, and is directed to enter muscle and fat cells under control of the hormone insulin. Understanding how insulin controls glucose distribution is of enormous practical significance because an increasing worldwide epidemic of people are developing sluggish responses to insulin -- a condition called 'insulin resistance' that is a hallmark of type 2 diabetes.

A few years ago, Shuai Chen and Kathryn Geraghty in Carol MacKintosh’s group as well as David James’ group at the Garvan Institute in Sydney, found that insulin triggers a protein named AS160 (Akt substrate 160kDa) to be captured by C-shaped proteins named 14-3-3s inside muscle and fat cells [PMID: 17617058]. Suspecting AS160 to be involved in insulin control of glucose metabolism, Carol and Shuai teamed up with Kei Sakamoto, a Programme Leader in the Unit who has set up a state-of-the-art Molecular Physiology Laboratory. Together the team discovered that mice whose AS160 is genetically mutated so that it cannot bind to 14-3-3s are markedly impaired in clearing their blood glucose into muscle in response to insulin.

The AS160-mutant mice have abnormally high amounts of a specialised glucose transporter called GLUT4 that assists glucose delivery into muscle and fat cells. But, despite having more GLUT4, the mutants do not move the GLUT4 efficiently into the correct position to let glucose into the muscle. These findings establish that AS160 plays a key role in glucose control by correctly navigating GLUT4 in response to insulin. Recent genetic studies have also identified a few people with a rare form of diabetes who have faulty AS160, and it would be very informative to characterise GLUT4 function in these people.

Just as you need to understand how engines work before you can fix a car, understanding the internal machinery of glucose control in muscle should provide new ways to improve blood glucose management in people. One idea is that a drug could be developed to mimic the function of AS160 in controlling the movement of GLUT4 inside muscles, and thereby lower blood glucose levels.

Drugs are not the only answer however. Exercise has a powerful effect in regulating blood glucose, even in people with insulin resistance. How? Here too, the team have clues. TBC1D1, which is a sister protein to AS160, also binds to 14-3-3s – but this time during exercise, and also in response to drugs that mimic some effects of exercise [PMID: 17995453]. AS160 and TBC1D1 therefore seem to be the complementary Yin and Yang of glucose control – with insulin regulating glucose levels via AS160, and exercise regulating glucose via TBC1D1. Lots of experiments are under way to test these hypotheses!

The researchers thank the Medical Research Council, Diabetes UK and Dundee and District Diabetes UK Voluntary Group for their enthusiastic support and funding. Also, David Wasserman of Vanderbilt University who contributed critical experiments to the new study, which is published in the journal 'Cell Metabolism'. [PMID: 21195350] and highlighted in the Diabetes UK website.




 

Craig MacKay wins the Howard Elder Prize
19 January, 2011
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Craig MacKay

Craig MacKay has been awarded the 2010 Howard Elder Prize for his discovery in John Rouse’s lab of the FAN1 nuclease that acts as a “molecular scissors” during DNA repair (MacKay et al, Cell 142, 65-76).

The Fanconi anaemia (FA) proteins are important for DNA repair. Mutations in the proteins of the FA complex result in high levels of chromosomal instability, caused by an inability to repair DNA interstrand crosslinks (ICLs), and hence predisposition to cancer. The central component of the FA pathway is FANCD2 that becomes mono–ubiquitylated at Lys561 in response to ICLs. The mono–ubiquitylation of FANCD2 is essential for ICL repair but despite much work this promotes ICL repair has remained elusive.

During his PhD with John, Craig discovered a novel UBZ domain-containing protein FAN1 that binds to, and is recruited to sites of DNA damage by, the mono–ubiquitylated form of FANCD2. Importantly, the depletion of FAN1 from human cells causes chromosome aberrations and major defects in DNA repair, similar to cells from patients with FA. FAN1 acts at the final stages of ICL repair when homologous recombination (HR) comes into play, and Craig’s data show that FAN1 acts late during HR, at a point that involves the processing or “trimming” of DNA repair intermediates. This led Craig to discover that FAN1 is a 5’ flap endonuclease and his data suggest that FAN1 processes branched recombination intermediates, enabling homologous recombination to go to completion. Craig’s findings at least partly explain the long-standing puzzle of how FANCD2 ubiquitylation promotes DNA repair.

Craig’s FAN1 paper is has been featured in several recent News articles: (e.g. Mol. Cell 39, 167; Nat. Struct. Mol. Biol. 17, 926; Nat. Rev. Mol. Cell. Biol. 11, 603; Cell Cycle 9, 4259; Cell Cycle 9, 4261). To read a copy of Craig's paper click here. For a previous news item announcing the discovery of FAN1 click here.

Following the completion of his Ph.D. Craig has taken up a postdoctoral position with Arno Alpi in the Scottish Institute for Cell Signaling (SCILLS) at the University of Dundee, where he is working on different aspects of the Fanconi anaemia pathway.

The Howard Elder Prize was endowed 25 years ago by Dr Alison Burt in memory of her father (Dr Howard Elder, a former medical graduate of the University). The prize is awarded each year to a PhD student or postdoctoral researcher in the College of Life Sciences at the University of Dundee deemed to have published the most significant paper in an area related to cancer research. The award is accompanied by a cash prize of £500.




 

Roles of the TAPP adaptor proteins defined by Stephan Wullschleger
7 January, 2011

Pleckstrin homology (PH) domains play vital roles in enabling proteins to recognise phosphoinositides. Simon Dowler (Dario Alessi’s 2nd PhD Student) identified two proteins over 10 years ago now termed the Tandem PH containing Protein-1 (TAPP1) and TAPP2. These are related adaptor proteins consisting of two sequential PH domains in which the C-terminal PH domain binds with high affinity to PtdIns(3,4)P2¬ [1]. Despite intensive analysis of many PH domain containing proteins, TAPP1 and TAPP2 to our knowledge are the only proteins known to specifically interact with PtdIns(3,4)P2 with high affinity.

Christine Milburn (Dario Alessi’s 3rd PhD Student, jointly supervised by Daan van Aalten) solved the structure of the C-terminal PH domain of TAPP1 revealing the mechanism by which it recognised PtdIns(3,4)P2 specifically [2]. Christine demonstrated that mutation of residues, Arg211 on TAPP1 or the equivalent Arg218 residue in TAPP2, that interacted with PtdIns(3,4)P2, completely prevented interaction of these proteins with PtdIns(3,4)P2 [2]. Interestingly, Christine structural data also suggested that binding of TAPP1 to PtdIns(3,4,5)P3 was inhibited by steric hindrance of an Ala residue located close to the position in which the 5’-phosphate would be expected to reside. The equivalent residue in PtdIns(3,4,5)P3-binding PH domains is frequently a Gly. Mutation of the Ala residue to Gly in TAPP1 resulted in it being capable of interacting with PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with similar affinity [2].

Wendy Kimber (Dario Alessi’s 6th PhD Student) next provided the first evidence suggesting that TAPP1 binds PtdIns(3,4)P2 selectively in vivo, by showing that TAPP1 relocated from the cytosol to the plasma membrane of cells, following stimulation with agonists that induced PtdIns(3,4)P2, but not with those that induced mainly PtdIns(3,4,5)P3 [3]. Aaron Marshall’s group in Winnipeg also demonstrated in B cells, both TAPP1 and TAPP2 translocated to the plasma membrane in response to antigen stimulation and this correlated with the formation of PtdIns(3,4)P2 rather than production of PtdIns(3,4,5)P3 [4].

Despite this work the biological functions of TAPP1 and TAPP2 remained elusive. Apart from the PH domains the only other known functional region is a C-terminal PDZ-binding motif that interacts with several PDZ binding proteins including the tyrosine-phosphatase-13 (PTPN13, previously known PTPL1 or FAP-1) [5] as well as the scaffolding proteins MUPP1 [3] and syntrophin [6].

One hypothesis that we had was TAPP1 and TAPP2 by specifically recognising PtdIns(3,4)P2 could function to recruit signalling molecules or complexes to the plasma membrane that down-regulate the PI 3-kinase signalling pathways [5]. Arguably, it makes sense to employ PtdIns(3,4)P2 as a signal to down-regulate the PI 3-kinase pathway, as the levels of this 3-phosphoinositide peaks later than PtdIns(3,4,5)P3. PtdIns(3,4)P2 would serve to down-regulate PI 3-kinase and signal the need for decreased formation of PtdIns(3,4,5)P3 production.

To test this idea Stephan Wullschleger generated knock-in mice expressing point mutants of TAPP1 and TAPP2 unable to interact with PtdIns(3,4)P2. The resulting TAPP1R211L/R211L TAPP2R218L/R218L double knock-in mice are viable and exhibited significantly enhanced activation of the Akt kinase a key downstream mediator of insulin signalling. By collaborating with David H. Wasserman at the Vanderbilt-NIH Mouse Metabolic Phenotyping Center, we were able to establish that double TAPP1/TAPP2 knock-in mice displayed significantly enhanced whole body insulin sensitivity in a hyperinsulinemic-euglycemic clamp study.

Stephan also demonstrated that TAPP1R211L/R211L TAPP2R218L/R218L double knock-in fibroblast cells that he derived displayed enhanced PtdIns(3,4,5)P3 levels and Akt activation in response to insulin. Overall Stephan’s data suggests that enhanced insulin sensitivity is mediated by increased Akt kinase activation thereby stimulating glucose uptake in heart and skeletal muscle.

These results are important as the TAPP1R211L/R211L TAPP2R218L/R218L knock-in mice represent the first mouse model for these proteins and support the notion that TAPP1/TAPP2 operate as negative regulators of the PI 3-kinase signalling pathway. In future it will be exciting to work out what proteins TAPP1 and TAPP2 are interacting with and recruiting to the plasma membrane to dampen down the PI 3-kinase pathway. One idea we had was that it might be mediated by binding to PTPN13/PTPL1 but Stephan found that knock-in mice expressing catalytically inactive PTPN13C2374A/C2374A did not display enhanced insulin sensitivity.

To read a copy of Stephan’s TAPP paper click (http://www.biochemj.org/bj/imps/abs/BJ20102012.htm)

References

1 Dowler, S., Currie, R. A., Campbell, D. G., Deak, M., Kular, G., Downes, C. P. and Alessi, D. R. (2000) Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities. Biochem J. 351, 19-31

2 Thomas, C. C., Dowler, S., Deak, M., Alessi, D. R. and van Aalten, D. M. (2001) Crystal structure of the phosphatidylinositol 3,4-bisphosphate-binding pleckstrin homology (PH) domain of tandem PH-domain-containing protein 1 (TAPP1): molecular basis of lipid specificity. Biochem J. 358, 287-294

3 Kimber, W. A., Trinkle-Mulcahy, L., Cheung, P. C., Deak, M., Marsden, L. J., Kieloch, A., Watt, S., Javier, R. T., Gray, A., Downes, C. P., Lucocq, J. M. and Alessi, D. R. (2002) Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo. Biochem J. 361, 525-536

4 Marshall, A. J., Krahn, A. K., Ma, K., Duronio, V. and Hou, S. (2002) TAPP1 and TAPP2 are targets of phosphatidylinositol 3-kinase signaling in B cells: sustained plasma membrane recruitment triggered by the B-cell antigen receptor. Mol Cell Biol. 22, 5479-5491

5 Kimber, W. A., Deak, M., Prescott, A. R. and Alessi, D. R. (2003) Interaction of the protein tyrosine phosphatase PTPL1 with the PtdIns(3,4)P2-binding adaptor protein TAPP1. Biochem J. 376, 525-535

6 Hogan, A., Yakubchyk, Y., Chabot, J., Obagi, C., Daher, E., Maekawa, K. and Gee, S. H. (2004) The phosphoinositol 3,4-bisphosphate-binding protein TAPP1 interacts with syntrophins and regulates actin cytoskeletal organization. J Biol Chem. 279, 53717-53724