MRC Protein Phosphorylation Unit
Centre profile from the MRC Network publication issued Spring 2006.
The MRC Protein Phosphorylation Unit in Dundee leads UK research in the increasingly important field of protein phosphorylation. Network goes north of the border to take a look at the unit’s pioneering work, including its thriving collaboration with the pharmaceutical industry.
A generation ago, the world of science knew very little of protein phosphorylation. The idea that protein kinases and protein phosphatases might be valid drug targets was remote, and the suggestion that this area of science would become a major focus for investment would have been considered laughable. Yet today, it commands 27 per cent of the pharmaceutical industry’s annual R&D spending. Phosphorylation is now recognised as being one of the major ways in which cells coordinate their response to a wide range of agents, such as hormones, growth factors, nutrients, cell damage and even pathogens. Irregularities in phosphorylation can cause, or be the result of, major diseases including cancer, diabetes, heart attack, hypertension and rheumatoid arthritis. This makes protein kinases and phosphatases of enormous importance as potential drug targets.An early success in this field was cyclosporin, a drug launched in the 1980s. It suppresses the immune system by inhibiting a specific protein phosphatase first identified in 1981 in Sir Philip Cohen’s laboratory at the University of Dundee, and is now widely used to prevent tissue rejection after organ transplantation. In response to growing awareness of the importance of phosphorylation, the MRC set up the Protein Phosphorylation Unit (PPU) in Dundee in October 1990, under the directorship of Sir Philip, who is a Royal Society Research Professor. The unit employs over 100 people organised into eight groups, covering a wide range of research into this rapidly developing area.
Watching the signals
For the last three years, Philip’s own research group has been focusing on the study of inflammation. Infection by a pathogen triggers the activation of networks of protein kinases, which leads to the production of pro-inflammatory cytokines (PICs).When released into the blood, PICs stimulate other cells into action, which helps to mount an innate immune response to destroy the invading pathogen. Inflammation, however, is a double-edged sword: an over-reaction in the inflammation response can be as dangerous as no response at all, and this is a major cause of chronic inflammatory diseases such as rheumatoid arthritis and septic shock. Currently, the front line treatments available are drugs which inhibit PICs, but they are expensive, require repeated injections and only about a third of patients respond to them. A more effective treatment that can be taken orally is urgently needed. However, researchers must first understand the signaling pathways that control the production of PICs before they can identify the protein kinases in these pathways that are the best targets for new interventions.
Achieving a balance
Members of Dr Simon Arthur’s group are also exploring the inflammatory process, specifically the role of MAPKs, a particular class of protein kinases. They have recently discovered that the protein kinase MSK plays a critical role in controlling the production of substances called anti-inflammatory proteins. As Simon explains: “Fine-tuning is all important. In the immune response, the balance between inflammatory and anti-inflammatory proteins is crucial. If they are out of balance, diseases such as Crohn’s disease and psoriasis can occur.” This work could eventually lead to the development of drugs which activate specific kinases to trigger an increase in anti-inflammatory proteins, but without increasing inflammatory ones.
Protein kinases that are mutated in inherited diseases
Professor Dario Alessi’s pioneering research has provided “exciting new insights into conditions such as cancer, diabetes and hypertension.” So said the judges when he was awarded the European Molecular Biology Organisation Gold Medal 2005, widely regarded as the most prestigious research prize in Europe for a life scientist under the age of 40.
Helped along by scientific serendipity, one remarkable discovery made by Dario’s group was a breakthrough in understanding how cells manage their energy ‘budget’. The team was investigating an enzyme, LKB1, which is implicated in an inherited cancer syndrome. Their aim was to work out which protein it phosphorylated, in the hope that this would lead to an understanding of how mutations in the gene encoding LKB1 cause cancer. Meanwhile, Professor Grahame Hardie, a colleague working in diabetes research at Dundee University, was investigating a different protein kinase, AMPK. Important in the control of energy levels in cells, AMPK switches off energy-hungry activities and switches on energy generating processes when the supply is low. Grahame was struggling to identify the protein kinase that activates AMPK.
The breakthrough came when one day both teams compared notes and realized that the cancer-linked enzyme LKB1 might be the activator of the energy-controlling kinase AMPK – a theory they went on to verify. The next step for Dario Alessi was to explore whether metformin, a drug used to treat type 2 diabetes by switching on AMPK, might also help to treat cancer because it mimics one of the actions of the tumour-suppressor LKB1.A preliminary epidemiological analysis carried out with clinical colleagues at the University of Dundee Medical School indicated that this idea was likely to be correct. This has encouraged Dario to begin further trials on specific cancers in collaboration with his clinical colleagues. “The connection between AMPK and cell growth could lead to some interesting follow-up work,” he says. “If we can trick a cancer cell into thinking that it doesn’t have enough energy to divide, we may be able to stop it growing.”
Dario has recently made an important discovery about how mutations in the protein kinases WNK1 and WNK4 cause Gordon’s disease, an inherited hypertension syndrome. He has revealed that they switch on two other protein kinases, SPAK and OSR1, that play a critical role in regulating salt balance. These findings suggest that compounds which damp down the activities of WNK, SPAK and OSR1 could lead to new drug treatments for hypertension. Dario and his team are now looking at how mutation of the genes encoding the protein kinases PINK and LRRK1 cause early-onset Parkinson’s disease.
Protein phosphatases in diabetes and cancer
Professor Tricia Cohen’s group is focusing on protein phosphatases, which remove rather than add phosphate from proteins. This is still a relatively unexplored area of signal transduction compared to protein kinases. They use a multidisciplinary approach, including gene disruption in mammals and RNA interference techniques in fruit flies. By expanding their investigation of the roles of protein phosphatase complexes that are critical for human health, Tricia and her team hope to identify targets for new drug treatments for cancer and diabetes. In diabetes, they recently showed that the disruption of a mouse gene encoding a glycogen-targeted form of a specific protein phosphatase can result in obesity and insulin resistance in later life. As Tricia said: “Understanding the regulation of the phosphatase which is targeted to glycogen is very important. A drug capable of altering its regulation may have potential for the treatment of diabetes, a disorderthat affects 150 million people worldwide.”
Cellular secrets of flora and fauna
Professor Carol MacKintosh and her group study plant and animal cells side by side, as they have much in common at a molecular level. For example, the team found that C-shaped proteins called 14-3-3s prevent plants from suffering metabolic collapse in the dark. “I called it ‘cuddle and squeeze’ because that’s what the C-shaped proteins seemed to be doing. They bind to enzymes of sugar metabolism inside leaf cells when the sun goes down, and through these actions help the plant to survive the night,” Carol explained. Following clues from their plant studies, the group looked at 14-3-3s in human cells and discovered that these help drive cancer cells to scavenge for nutrients and regulate how glucose is used to generate cellular energy and growth. 14-3-3s have also given an unexpected insight into how viruses, for example HIV, hijack the internal machinery of a cell. The next step will be to develop drugs that antagonise or enhance the functions of 14-3-3s inside cells.
The 3-D structure of PDK1, a protein kinase discovered by Dario Alessi, which has become an important target for developing anti-cancer drugs. Diagram: Daan van Aalten and Dario Alessi, PPU.
Protein kinases and DNA damage
The PPU’s most recent arrival is Dr John Rouse, who came from the Gurdon Insitute at Cambridge three years ago. His group is looking at how cells deal with damage to DNA. Genetic material is unstable and chemically reactive. If DNA damage is left unchecked, the sequence and the structure of the genome changes and part of the instruction set is lost. This type of genome instability is seen in a large number of tumours. To prevent it, healthy cells are able to recognise and repair DNA damage. “Cells can launch a series of events to ensure rapid DNA repair, and to ensure that damaged chromosomes aren’t replicated or segregated until they’re repaired,” John explained. “And here’s the crunch – these responses to DNA damage are regulated by protein kinases.”
The protein kinases in question bind to the damaged DNA and this activates other kinases which, in turn, phosphorylate proteins that protect genome stability and control repair. The challenge lies in pinpointing exactly how this control mechanism works. If the group can unravel it, they may be able to develop an understanding of the mechanisms that are deregulated in cancer cells.
Working with industry
In addition to excellent science, another of the PPU’s great successes is its collaboration with several of the world’s leading pharmaceutical companies, including AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Merck & Co Inc, Merck KGaA and Pfizer. Set up in 1998, this is probably the largest partnership ever undertaken between the pharmaceutical industry and a UK research institution. It allows the companies to share the unit’s unpublished information, reagents, technology and know-how and to have the first right to licence the intellectual property generated.
Over the past five years, income from the venture totalling £15 million has been ploughed back into MRC research at the unit. But the collaboration doesn’t steer the research – it is in fact the other way round. And the benefits aren’t just financial, as Philip Cohen explains: “For example, we gain access to company compounds that are valuable reagents for our own research and our post-doctoral researchers and PhD students get a great insight into the pharmaceutical industry; many more go into industry as a result,” he explained. “The collaboration happened almost by chance, but now it’s established I can see what a good idea it is and well worth expanding further.” The unit also has interactions with other companies. For example, its long-standing collaboration with USA company Upstate Inc led to the setting up of a new division of Upstate in Dundee in 1999, which now employs over 100 people.
All for one and one for all
The PPU aims for a unified approach across its activities. It has a shared services division – with teams for DNA cloning, DNA sequencing, antibody and protein production and cell culture – and pools its funding from all sources so that the money can be used strategically to yield maximum benefit for the unit as a whole. For example, it has invested a large sum of money in state-of-the-art mass spectrometers to identify phosphorylated sites in proteins – an area in which Dr Nick Morrice of the PPU is a world expert – and in mouse genetics. “The unit is a pioneer in its field, and has developed a very efficient way of operating,” Philip Cohen says. “Central budgets will never be able to fund everything we would like to do, and collaboration with industry helps to provide the additional funding needed. As a result, we’re working hard for both the health and wealth of the nation.”
Future challenges
Thanks to the work of the PPU and its colleagues worldwide, the importance of phosphorylation to the body’s processes is now widely recognised. But this fascinating area of medical science will continue to challenge scientists for many years to come, as Philip is quick to point out: “The identification of the major substrates of each protein kinase and phosphatase is a major undertaking – and one that will take several decades to solve – but it’s at the heart of what we need to know in the twenty-first century.”