Systematic molecular and cell biological analysis of MST kinase signalling in cell death cell cycle and centrosome biology

Lead Research Organisation: University College London
Department Name: Cancer Biology

Abstract

The human body is composed of diverse cell types with specialised tasks. Much research has been carried out to gain a better understanding of how cells decide where to be and what to do. Recent research has also shown that it is crucial to comprehend how cell number and tissue architecture is controlled under normal physiological conditions such as development or organ regeneration. Normally, to ensure that each tissue is patterned not only to a specific architecture but also to a defined size, multicellular organisms govern the coordination of cell death and multiplication by strict control mechanisms. Therefore, one very important aspect of ongoing research is to define key factors that are essential for this coordination. Interestingly, proteins that modify other proteins by a specific tag (through a molecular mechanism called by specialists phosphorylation) are sometimes central in the coordination of these activities. These modifiers (also termed kinases) represent the field of my interest. Significantly, the tagging of proteins by phosphorylation can change various properties of the tagged protein. For example, enzymatic activities can be increased or decreased, the localisation within the cell might be altered, or binding partners might be lost or gained. Therefore, it is very important that we understand how the activity of kinases is strictly regulated. Equally important, we also must comprehend the importance of (or sometimes also the lack thereof) certain kinase activities. With this study, I will investigate the yet unidentified complexities of an entire family of kinases. Members of this family have already been shown to function as tumour suppressor protein, but I strongly believe that we are yet to unveil important functions of these proteins in healthy normal human cells. By identifying roles of these kinases in the control of how cells die (programmed cell death, also termed apoptosis) and multiplicate (a process termed proliferation), I will decipher the biological significance of the entire kinase family. The studies will involve performing selective manipulation of kinase activities as well as their level of abundance, and the use of additional molecular and cell biological techniques to define which tagging (phosphorylation) events are the most important. Altogether, I strongly feel that the main objectives of this proposal will help to improve our understanding of this essential kinase family. Given that these kinases function in the immune and cardiovascular system, as well as in tumour suppression, discoveries from this proposal could serve as a foundation for improvements regarding lifelong health and wellbeing. On the long-term, understanding these kinases might help to establish how far these proteins can be used as read-outs in the prevention, detection, prognosis and treatment of various human diseases.

Technical Summary

The importance of protein kinases in human disease, and therefore also as therapeutic targets and biomarkers, is well established. In human cells, several hundred kinases catalyse transfer of phosphates (phosphorylation) to serine, threonine, and tyrosine residues, thereby defining protein kinases as one of the largest superfamilies in the human genome. Members of the Germinal Center Kinase (GCK) subfamilies II and III are highly conserved from yeast to man, with functions in the regulation of cell morphology and mitotic exit in yeast, and in the coordination of cell proliferation and death in flies. Recent work has shown that Mammalian serine/threonine STE20-like (MST) kinases play roles in cancer, immune, cardiovascular, and brain function. As a result research is urgently needed to address the complexity of the entire human MST kinase family, composed of MST1/2/3/4 and YSK1. In particular, studies of MST function in normal human cell biology are required. Therefore, we propose the following three main objectives: Objective 1: Systematic testing of loss of function of MST kinases in non-transformed human cells. Studies of cells expressing specific shRNAs in a Tet-on manner together with dominant-negative versions will define which kinases are essential for the regulation of apoptosis, cell cycle progression and centrosome biology in normal healthy human cells. Objective 2: Address the importance of MST kinase activities using chemical genetics. Without affecting total protein expression, 1-NM-PP1 sensitive MST variants will allow us to define how far specific loss of kinase activity actually mimics loss of protein expression. Objective 3: Identify factors functioning downstream of MST kinases in normal human cells. In addition to addressing all currently known substrates, we will screen cell lysates and protein microarrays for novel MST substrates. The aim is to unveil which substrates mediate various MST-driven processes in normal human cells.

Planned Impact

The deregulation of several kinase pathways is linked to human disease. Significantly, recent research suggests that also members of the MST kinase family might be key regulators of certain processes maintaining human health and wellbeing. Unfortunately, our understanding of this kinase family is limited with respect to normal human cell biology. Therefore, I propose to study the entire human MST kinase family in more detail. Immediate beneficiaries from my proposed research will be various bioscience areas (see section Academic Beneficiaries for details). On the long term, potential outcomes of my research proposal might help to improve human health and wellbeing: The UCL Cancer Institute (UCI) is an excellent hosting institution for this research project. All necessary facilities are on site allowing quick analysis of DNA, protein and cell samples. My laboratory is fully equipped (centrifuges, thermomixers, DNA and SDS-PAGE apparatuses, fridges, freezers, etc.) to process, store and analyse DNA, protein and cell samples appropriately. Significantly, a number of researchers working on experimental therapeutics are based in the UCI, and their experience and expertise is available to my laboratory. Furthermore, the institute has strong links with the University College Hospital (with respect to interactions and proximity), which could serve as excellent environment for exploring clinical avenues. Furthermore, I am already collaborating with Dr. Mark O'Driscoll (University of Sussex, Falmer, Brighton) on the analysis of patient derived cell lines. Altogether, I have access to samples from patients suffering from various diseases/syndromes. Therefore, it might be possible to translate data obtained from my basic research proposal into the clinic on the long term. In summary, my research will have direct impact on various bioscience areas, which on the long term might allow the exploitation of my results in clinical studies. This could ensure a social and economic return to the UK, since health and wellbeing of UK residents would be improved. Noteworthy, one postdoctoral fellow will be trained in the course of this project. The researcher will be trained in generating, reporting, presenting, and publication of data. Furthermore, the fellow will train other lab members in relevant techniques and methods, and has full access to various UCL training resources/courses. Therefore, my research proposal will positively impact the future career path of at least one young scientist. To maximise impact, discoveries made from this research proposal will be communicated to the scientific community through presentations at national and international conferences and through publications in peer-reviewed journals. Moreover, resources will be made available to other researchers upon request. I strongly believe that this data and material sharing enables other researchers, and conveys economic, and social benefits in addition to fostering fruitful collaborations. In this context, it is noteworthy that I am already collaborating with Dr. Jeroen Pasterkamp (University of Utrecht, the Netherlands) on the regulation of MST1 kinase by MICAL-1 (see also attached letter by Dr. Jeroen Pasterkamp). Thus, if this grant would be awarded to me, this international collaboration would strongly benefit, thereby resulting in the UK bioscience community to benefit indirectly from international European funding. Last, but not least, this research grant will help me to establish myself as an internationally competitive researcher. This would enable me to attract international funding (e.g. from the European Research Council, or the European Molecular Biology Organisation) to my UK-based laboratory. Furthermore, this grant would allow me to enhance my creative output, both in my current research and in the conception of future research projects aimed at enhancing quality of life through the application of basic research into fundamental cellular processes

Publications

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Gomez M (2014) The Hippo pathway in disease and therapy: cancer and beyond. in Clinical and translational medicine

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Gomez M (2019) MST1/2 Kinase Assays Using Recombinant Proteins. in Methods in molecular biology (Clifton, N.J.)

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Gomez-Martinez M (2013) Generation of stable human cell lines with Tetracycline-inducible (Tet-on) shRNA or cDNA expression. in Journal of visualized experiments : JoVE

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Hergovich A (2017) YAP needs Nemo to guide a Hippo. in EMBO reports

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Hergovich A (2019) Measuring the Kinase Activities of the LATS/NDR Protein Kinases. in Methods in molecular biology (Clifton, N.J.)

 
Description Thanks to BBSRC funding we studied the roles of the MST kinase family in normal human cell biology. We made four main discoveries, namely:
(1) that one specific MST kinase (termed STK25) is required for cell cycle progression of untransformed normal human cells, while all MST kinases are dispensable for the proliferation of transformed human cell lines.
(2) that MST kinases can phosphorylate MOB3 scallfolding proteins in addition to MOB1, and that several members of the MST kinase family can function as hydrophobic motif kinases upstream of LATS and NDR kinases.
(3) that MST kinases can interact with RASSF scaffolding proteins independent of intact SARAH domains, while interactions with the Salvador scaffolding protein are SARAH domain dependent.
(4) that additional members of the Ste20-like kinase group, besides MST kinases, can function as upstream kinases of LATS and NDR kinases.
In the context of our findings outlined in point 4, we established a very fruiful collaboration with Prof. Kieran Harvey (University of Melbourne, Australia) studying the network of Ste20-like kinases functioning upstream of LATS/NDR-YAP signalling in flies and human cells.
Exploitation Route Collectively, our findings demonstrate that the MST1/2-Hippo tumour suppressor pathway is not a simple linear signalling cascade, but rather represents a complex network that we have only begun to understand. On the one hand, our findings provide an excellent platform for follow up studies from our and other research laboratories. On the other hand, our findings have already started (and will continue) to establish international collaborations that will expand the research horizon of our work.
Considering that the oncogenic transcriptional programmes of YAP/TAZ downstream of Hippo signalling are attractive targets for anti-cancer therapeutics, our work might also be taken forward by clinically interested approaches. However, while YAP/TAZ inhibition should be very beneficial in the treatment of carcinomas in the future, their temporary activation is desirable in regenerative medicine. Therefore, our findings further might help to establish a solid foundation for the development of pharmacological inhibitors and stimulators of YAP/TAZ activities.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology,Other

 
Description Our basic research findings have helped to develop a solid foundation for future follow up studies and collaborative projects. Our work might help the development of novel therapeutic approaches to improve healthcare and human wellbeing (see RCUK Key Findings for more details). In addition, by attracting interantiona collaborators the UK ecomony will also benefit (at least indirectly, when not even directly by securing funding from international funding agencies). In summary, our work has the potential for societal and economic impact by supporting different aspects (see RCUK Key Findings for details).
Sector Healthcare,Pharmaceuticals and Medical Biotechnology,Other
Impact Types Societal,Economic

 
Title Development of Tet-on cell systems 
Description The tools and methods have been reported in: Gomez-Martinez, M., Schmitz, D., and Hergovich, A.# Generation of stable human cell lines with tetracycline-inducible (Tet-on) shRNA or cDNA expression. Journal of Visualized Experiments 5(73) (2013). 
Type Of Material Cell line 
Year Produced 2013 
Provided To Others? Yes  
Impact These novel research tools had the following impacts: -resulted in the publication of a manuscript, hence increasing the "visibility" of the lab and new tools/method. -enabled the Hergovich laboratory to study the consequences of overexpression or depletion of our genes of interests in conditions that are normally difficult to maniplulate. -empowered other research laboratories (so far more than 10 different international labs have requested and obtained our novel tools/method) to develop their own Tet-on systems of interest 
URL http://www.jove.com/video/50171/generation-stable-human-cell-lines-with-tetracycline-inducible-tet-o...
 
Description Collaborating between the Hergovich and Camonis (Institut Curie, Paris, France) laboratories on Ste20-like kinases 
Organisation Curie Institute Paris (Institut Curie)
Country France 
Sector Academic/University 
PI Contribution We are studing together the role of additional Ste20-like kinases upstream of NDR kinase signalling. Noteworthy, this has already resulted in the submission of a manuscript: Selimoglu, R., Bettoun, A., Joffre, C., Meunier, B., Fesquet, D., Formstecher, E., Cascone, I., Hergovich, A., and Camonis, J. RalA GTPase and MAP4K4 function through NDR1 activation in stress response and apoptotic signaling. Journal of Cell Biology and Cell Metabolism (in press). While we studied in the UK the specific regulation of NDR kinase by MAP4K4, our French collaborators focused on deciphering the signalling cascade upstream of MAP4K4.
Collaborator Contribution Various research tools (cell lines, plasmids, and antibodies) were exchanged.
Impact Currently the following manuscrip is in press (but not yet available in Pubmed): Selimoglu, R., Bettoun, A., Joffre, C., Meunier, B., Fesquet, D., Formstecher, E., Cascone, I., Hergovich, A., and Camonis, J. RalA GTPase and MAP4K4 function through NDR1 activation in stress response and apoptotic signaling. Journal of Cell Biology and Cell Metabolism (in press). This collaboration was not multi-disciplinary.
Start Year 2013
 
Description Collaborating between the Hergovich and Yao (Morehouse School of Medicine, USA) labs on NDR signalling 
Organisation Morehouse School of Medicine
Country United States 
Sector Academic/University 
PI Contribution We studied in collaboration the role of NDR signalling/phosphorylation in mitosis. We developed relevant tools (e.g. highly specific antibodies) and the Yao laboratory performed the mitotic study.
Collaborator Contribution Highly specific novel antibodies were exchanged.
Impact Currently a manuscript is being assembled to report our novel findings on NDR signalling in mitosis (Yan et al.). Once this manuscript is published we will update the publication output of this BBSRC grant.
Start Year 2013
 
Description Collaboration of the Hergovich and Harvey laboratories with respect to the regulation of MST kinases by TAO1 
Organisation Peter MacCallum Cancer Centre
Country Australia 
Sector Academic/University 
PI Contribution Together with the Cell Growth and Proliferation Laboratory, Peter MacCallum Cancer Centre, Melbourne, Australia, we have started to study the regulation of the entire human MST kinase family by the TAO1 kinase. Particular focus is on the regulation of the non-canonical MST kinases, MST3, MST4 and STK25, since their regulation by TAO1 has not been explored at all. Currently, we are performing a series of biochemical experiments to address this possible crosstalk between MST and TAO1, while the Harvey laboratory examines in Drosophila melanogaster the role of the single MST kinase corresponding to all three human MST kinases - MST3, MST4 and STK25. Significantly, we could already establish that TAO1 can phosphorylate all three MST kinases on the essential T-loop in vitro and in vivo. Combined with the ongoing genetic studies in the Harvey laboratory, our work will be able to elucidate whether this additional non-canonical branch of Hippo signalling can also be regulated by TAO1, as previously reported by the Harvey laboratory for the regulation of Hippo by Tao (Poon et al., Developmental Cell, 2011; doi: 10.1016/j.devcel.2011.09.012)
Start Year 2011