Congratulations to Ruzica Bago, who is completing her postdoctoral work in Dario Alessi’s lab, on being awarded the University of Dundee School of Life Sciences prestigious Howard Elder Prize for 2016.
Ruzica received the award for her EMBO J publication entitled “The hVps34-SGK3 pathway alleviates sustained PI3K/Akt inhibition by stimulating mTORC1 and tumour growth”.
PI3K and Akt inhibitors are being evaluated in clinical trials for the treatment of human tumours including breast cancer that display driver mutations that inappropriately elevate the PI3K/Akt signalling pathway. Despite initial promise, the majority of these tumours unfortunately rapidly develop resistance the PI3K/Akt pathway therapy. Ruzica Bago, decided to explore the mechanisms by which cancer cells can evolve resistance to PI3K and Akt pathway inhibitors within 2-5 days. Ruzica discovered that prolonged treatment of a panel of breast cancer cells lines for over 2-days with PI3K or Akt inhibitors, led to a marked increase in the expression and activity of a poorly studied protein kinase termed the serum and glucocorticoid-regulated kinase-3 (SGK3), that is closely related to Akt and also activated by the same upstream kinases (PDK1 and mTORC2). Akt kinase possesses a PH domain at its N-terminus that binds to the lipid second messenger PtdIns(3,4, 5)P3 product generated by PI3K, which induces a conformational change promoting phosphorylation and activation of Akt by PDK1 and mTORC2. In contrast, SGK3 instead of a PH domain at its N-terminus possesses another lipid interacting motif termed a PX domain, that binds to PtdIns(3)P-and not PtdIns(3,4, 5)P3. SGK3 is the only known protein kinase to possess a PtdIns(3)P binding domain.
Ruzica then demonstrated that SGK3 was activated in an analogous mechanism to Akt, namely that PtdIns(3)P-binding to the PX domain promoted PDK1 phosphorylation and hence activation of SGK3. This effect is prevented by introducing a mutation within the PX domain that prevents SGK3 from binding to PtdIns(3)P. These findings raised the question as to what was the identity of the lipid kinase in the cell that generates the PtdIns(3)P that stimulates the activation of SGK3 in breast cancer cell lines treated with PI3K or Akt inhibitors. Employing structurally diverse highly selective inhibitors, Ruzica experiments strongly point towards an enzyme termed hVps34, which is one of the major lipid kinase in the cell that generates PtdIns(3)P that is located at endosomes of cells at the same location SGK3 residues.
Given the similarity between Akt and SGK3, Ruzica realised that these kinases could phosphorylate an overlapping set of substrates. Employing an Akt phosphorylation motif antibody-Ruzica found that out of 9 Akt substrates identified in a cell extract 6 of these were likely to be phosphorylated by both Akt and SGK3. Strikingly, Ruzica was able to show that prolonged inhibition of PI3K/Akt enabled SGK3 to fully reactivate the mTORC1 signalling pathway by phosphorylating TSC2. Under these conditions of prolonged treatment with PI3K or Akt inhibitors Ruzica established that mTORC1 activation is now blocked by 14h SGK inhibitor. In collaboration with another postdoc Pau Castel working in the laboratory of José Baselga working at the Memorial Sloan Kettering Cancer Center, they were able to produce decisive data showing that a combination of Akt (MK-2206) and SGK (14h) inhibitors induced marked regression of a breast cancer (BT-474) cell derived tumours in a nude mouse xenograft model, under conditions where either inhibitor administered individually had minimal effects.
Ruzica’s results highlight the importance of the hVps34-SGK3 pathway and suggest it represents a major mechanism, which cells utilise to counteract inhibition of PI3K-Akt signalling. They also provide novel mechanistic insights of how PtdIns(3)P can stimulate activation of SGK3. The characterisation of the 14h SGK inhibitor suggests that it will become a useful research tool to probe biology controlled by SGK isoforms. 14h represents a valuable addition to our growing arsenal of signal transduction inhibitors to dissect functional roles of protein kinases. Finally, and perhaps most importantly findings described in Ruzica’s paper highlight the therapeutic potential of a strategy targeting both the Akt and SGK kinases for the treatment of cancer.
The Howard Elder Prize was endowed by Dr Alison Burt 25 years ago, in memory of her father Dr Howard Elder, who was a former medical graduate of the University of Dundee. The prize is awarded to a postgraduate student or postdoctoral researcher deemed to have published the most significant paper in an area related to cancer research. It was introduced by Dr Elder's daughter who said her father always spoke very highly of his time at Dundee and the opportunities it gave him. The panel, consisting of researchers based in the Division of Cancer Research at the School of Medicine (Professor Kevin Hiom, Professor Albena Dinkova-Kostova and Dr Adrian Saurin), believe that "Dr Bago’s work provides a great example of how detailed understanding of basic biology can be employed for the improvement of human health".
Ruzica is the 6th MRC PPU researcher to be awarded the Howard Elder prize. The previous awardees were Xu Huang 2008, Elton Zeqiraj 2009, Craig MacKay 2010, Kumara Dissanayake 2011 and Marija Maric 2014.
Congratulations to Satpal Virdee for being awarded the prestigious 2016 University of Dundee School of Life Sciences Innovator of the Year Award.
Satpal received his award for the development and patent of the new technology he developed to generate an “E3 ubiquitin ligase activity-based probe” that was recently published in the Nature Chemical Biology manuscript ‘Probes of ubiquitin E3 ligases enable systematic dissection of parkin activation’. The judging panel, consisting of Professor Irwin McLean, Professor Philip Cohen and Dr Fiona Mitchell from Research and Innovation Services, unanimously chose Satpal as the ‘technology is truly innovative in that it represents the creation of a new technology that hitherto did not exist, rather than application of existing technologies.’
The E3 activity probes were generated employing a new approach that Satpal describes as “a fusion of organic synthesis, genetic code expansion technology and protein labelling.” Satpal made the probe by “re-engineering” the ubiquitin charged E2 enzymes that are the co-factors that transfer ubiquitin to E3 ligases. The probe contains a chemical moiety that only reacts with active and not inactive E3 ligases. To demonstrate this system works, Satpal studied whether it could detect activation of endogenous Parkin E3 ligase after treatment of cells with the CCCP agent that induces mitochondrial depolarisation. He obtained striking data showing that the probe only reacted with endogenous Parkin in treated cells. Furthermore, Satpal obtained fibroblasts from Parkinson’s patient who lacked the upstream PINK1 kinase that triggers the activation of Parkin and their unaffected relatives. Using his system Satpal was remarkably able to demonstrate that his probe only reacted with Parkin in CCCP cells derived from unaffected relatives but not in the PINK1 deficient cells. Satpal’s PhD student Kuan-Chuan Pao undertook much of the work for this study.
This technology has significant potential to unbiasedly measure the activity of E3 ligases for the first time that are implicated with a diverse range of diseases. It will also enable to study of how these enzymes are regulated by growth factors, toll-like receptor and DNA damage agonists impact the activity in a wide range of cellular systems.
Dr Pawel Leznicki has been awarded a grant from Tenovus Scotland to broaden our understanding of how deubiquitylating enzymes (DUBs) regulate cancer cell survival. The research will be carried out in the group of Dr Yogesh Kulathu within the Medical Research Council’s Protein Phosphorylation and Ubiquitylation Unit (MRC PPU), University of Dundee.
DUBs reverse and modulate protein ubiquitylation, a versatile post-translational modification that controls virtually all facets of eukaryotic cell biology. Ubiquitylation and deubiquitylation regulate, for example, a controlled programme of cell death (apoptosis) and hence determine cell fate. Deregulation of apoptotic progression has emerged as a key factor that contributes to carcinogenesis. Therefore, better understanding of the mechanisms that control apoptosis is crucial for the development of future therapies targeting cancer.
Pawel’s preliminary results implicate a previously uncharacterised DUB in the regulation of apoptosis and the survival of selected cancer cell lines. The award from Tenovus Scotland will enable Pawel to obtain mechanistic insights into the mode of action of this fascinating enzyme and will open new avenues for future research towards targeting this DUB for anti-cancer therapies.
Congratulations to John Rouse, who has been elected to the Royal Society of Edinburgh, Scotland's National Academy. The Royal Society of Edinburgh each year elects a select number of Fellows who have achieved 'excellence within a wide variety of disciplines, spanning the arts, business, science and technology sectors'. Being elected to the Royal Society of Edinburgh is a very prestigious and a major achievement, illustrating that your research is highly recognised and regarded.
John has been a PI in the Medical Research Council Protein Phosphorylation and Ubiquitylation Unit (MRC PPU) since 2002, and has made an important contribution to our understanding of the molecular mechanisms that allow cells to detect and repair damage to DNA. He has discovered several important factors in our cells that are required for DNA repair and that are vital for preventing human disease such as cancer, kidney disease and Fanconi anaemia. One of John’s biggest achievements was the discovery in 2010 of Fan1, a 'molecular scissors' that cuts off loose DNA ends that occur during DNA repair, in a way that allows DNA repair to go to completion. John recently published papers in the world-leading journals Science, and Genes and Development, revealing that failure of Fan1 to cut these loose DNA ends results in chromosomes becoming unstable, and the result is cancer and chronic kidney diseases. John’s discovery of the SLX4 “molecular toolkit” that also cuts loose DNA ends and removes chromosome tangles ('Holliday junctions') during DNA repair has also gained much international attention, and his discovery of the DVC1 protein that keeps the rate of DNA low is are also regarded as a landmark finding. These finding also pave the way for the development of new anti-cancer therapies.
Commenting on the award, John said “I’m delighted to become a Fellow of the Royal Society of Edinburgh. This honour reflects the tremendous efforts of the talented researchers from all over the world I’ve been lucky to have on my research team over the years. I’m looking forward to continuing to help keep Scottish science at the very forefront on the world stage, and to helping the Society with its mission to promote the public understanding of science”.
Dario Alessi, Director of the MRC Unit added "I am delighted that John’s research has reached the level to merit Fellowship of the Royal Society of Edinburgh. The work that John is undertaking on understanding the molecular mechanisms underlying how DNA damage is detected and repaired is amazingly important and is providing fundamentally new understanding of biology. John's research has great potential to help better understand diseases such as cancer and neurodegeneration and is suggesting innovative ideas to better treat these conditions in the future".
Our thirteenth MRC PPU alumni interview is with ex-PPU PhD student Chris Armstrong.
Click the MRC PPU Alumni Interview icon at the top of our website, or here to read the interview.
Over the period 1973-1998, research in Philip Cohen’s lab in the MRC PPU elucidated the signaling pathway by which insulin activates of glycogen synthase and enhances the conversion of blood glucose to tissue glycogen. During the course of this work the Cohen Lab identified the protein kinase glycogen synthase kinase 3 (GSK3) in the late 1970’s which they found had a key role in this process. Because insulin reduces the catalytic power of GSK3, it was initially thought that drugs might be developed that 'switch off' GSK3 activity and so improve the treatment of Type2 diabetes.
However, subsequent research in many laboratories revealed that GSK3 had many other functions in the body, including the attachment of phosphate to a protein in the brain called 'Tau'. When abnormally high levels of phosphate become attached to Tau, they cause it to aggregate and form deposits in the brain called 'tangles', which are one of the hallmarks of Alzheimer’s disease. These findings in turn led to renewed interest in developing drugs that switch off GSK3 in the hope that they would benefit Alzheimer’s patients. A number of pharmaceutical and biotechnology companies took up this challenge and a GSK inhibitor called Tideglusib was developed by the Spanish biotechnology company Noscira and entered clinical trials for the treatment of Alzheimer's and progressive supranuclear palsy, another neurodegenerative disease of the brain. This drug passed Phase I clinical trials indicating that it could be used safely in human patents, and further trials of this drug in larger numbers of patients are now progressing.
Around the same time, it emerged that GSK3 is also switched off when the Wnt signaling pathway is activated. This leads to the accumulation of proteins, such as β-catenin and Axin, which have critical roles in a number of processes, such as the development of the embryo and the repair of tissue damage in adults. For this reason, the same pathway is activated when teeth are damaged. In a remarkable development, Paul Sharpe and his colleagues at King’s College London applied low doses of Tideglusib to biodegradable collagen sponges, which were then inserted into tooth cavities. They found that the sponges gradually degraded over time and were replaced by new dentine, the main supporting structure of the tooth. This could transform the way we treat teeth cavities, making man-made fillings a thing of the past. Since collagen sponges are already available commercially and approved clinically, and Tideglusib has passed safety tests, there is now a real opportunity to get this treatment quickly into dental clinics.
The results of this study were published in Nature Scientific Reports in January 2017 and received worldwide media attention. Paul Sharpe commented, “The simplicity of our approach makes it ideal as a clinical dental product for the natural treatment of large cavities, by providing both pulp protection and restoring dentine.”
One of the fascinations of carrying out fundamental research is that one can never predict what it will eventually lead to and how the discoveries may be used to benefit human health and create wealth. When the Cohen lab discovered GSK3 in the late 1970s the idea that it might revolutionize dentistry or perhaps be beneficial for the treatment of Alzheimer’s would have sounded like science fiction. And who knows, GSK3 inhibitors might yet turn out to be useful for the treatment of diabetes.
This story is also an excellent illustration of how it can take years or even decades before the results of fundamental research reach the stage where it becomes obvious how they can be used to improve health and create wealth. It illustrates once again why sustained long-term funding of basic research by Governments is so important.
The Royal Society open access journal, Open Biology, recently celebrated its 5th anniversary. Amongst the Editor’s picks was the journal’s most cited article by former PhD student, Chandana Kondapalli, which to date has garnered 240 citations since it was published in 2012.
Chandana, who was supervised by Miratul Muqit and Dario Alessi, discovered the first bona fide substrate of the Parkinson’s linked kinase PINK1, namely Parkin, and mapped the phosphorylation site to a highly conserved N-terminal residue Serine65. In the paper Chandana also provided the first evidence that PINK1 was activated by mitochondrial depolarisation.
To read a copy of Chandana’s paper click here.