Award details

DYRK protein kinases regulate p62/SQSTM1 to orchestrate cellular responses to oxidative stress, protein misfolding and nutrient starvation

Reference	BB/P007015/1
Principal Investigator / Supervisor	Dr Simon Cook
Co-Investigators / Co-Supervisors
Institution	Babraham Institute
Department	Signalling
Funding type	Research
Value (£)	327,028
Status	Completed
Type	Research Grant
Start date	18/04/2017
End date	17/04/2020
Duration	36 months

Abstract

p62/SQSTM1 is a multi-functional scaffold protein that acts as a signalling hub. Amongst its several functions p62 can: 1. competitively inhibit the KEAP1-NRF2 interaction, thereby promoting NRF2 nuclear entry and ARE-dependent gene expression; 2. bind Raptor, a subunit of the mTORC1 complex; indeed, p62 is required for mTORC1 activity in response to amino acids; 3. bind to aggregated or damaged proteins at aggresomes and deliver them to autophagosomes for autophagic recycling; 4. translocate into the nucleus in a phosphorylation-dependent fashion to recruit and clear nuclear protein aggregates. We have now found that: 1. The protein kinase DYRK1B can promote expression of canonical NRF2 target genes. 2. DYRK1B and DYRK2 both co-localise with p62 at aggresomes. 3. DYRK2 promotes the assembly p62 foci near the nucleus (typical of aggresomes) and promotes nuclear entry of p62. 4. DYRK1B and DYRK2 can both phosphorylate p62 at sites implicated in its nuclear entry. Our results define p62 as a substrate of DYRK1B/DYRK2 in vitro and in cells and suggest entirely new functions for these kinases in regulating p62 and its functions, including NRF2 activation, mTORC1 signalling and the trafficking of aggregated proteins. These processes are intimately involved in normal age-related declines in cellular fitness; indeed, p62 KO mice exhibit a premature ageing phenotype. We suggest that DYRK signalling promotes the assembly of p62 foci (aggresomes) and the nuclear entry of p62 as part of the cellular response to misfolded or aggregated proteins. This redistribution of p62 impacts on other p62 functions to regulate NRF2 and mTOR signalling. In this way DYRKs and p62 collaborate to orchestrate cellular responses to oxidative stress, protein misfolding and nutrient starvation. This is a fundamental biology study but it may also have important implications for diseases where p62 is implicated, including neurodegeneration and cancer.

Summary

The cells in our body are constantly exposed to chemical and physical 'stress' and accumulate a lifetime of damage to proteins, DNA and various key sub-cellular structures (organelles). It is vital that cells are able to respond appropriately to this damage; failure to do so progressively undermines cellular 'fitness' contributing to age-related declines in cell and tissue function that underpin the normal ageing process and can contribute to age-related diseases such as cancer and dementia. One way that cells respond to stress is to increase the abundance of enzymes that detoxify the cell by removing potentially harmful chemicals. When a cell receives a stress signal the genes that code for detoxifying enzymes are 'read' by 'transcription factors', discrete proteins that bind to DNA and transcribe the DNA information into RNA molecules, which are in turn 'translated' into the relevant proteins. One such transcription factor, called NRF2, coordinates cellular responses to oxidative stress. Another way in which cells deal with cellular damage is through a process called 'autophagy' (self eating) in which damaged proteins are targeted to cellular recycling centres (called autophagosomes), where they are broken down to their raw materials, which can then be re-used. This process of autophagy requires 'cargo receptors', which bind to damaged proteins and transport them to the recycling centre for autophagy. These complex processes are orchestrated by signalling pathways within cells that involve cascades of enzymes called protein kinases. These enzymes 'tag' other proteins with a phosphate group (a process called phosphorylation) and this changes the activity, abundance or localisation of the protein. The tagged protein is referred to as the 'substrate' of the protein kinase enzyme. This project concerns two protein kinases, called DYRK1B and DYRK2, and a specific 'cargo receptor' called p62 or sequestosome-1; here we'll call it p62. Our new results point to a link between the DYRKs and p62 in coordinating how cells respond to stress and damage:- 1. p62 acts as a scaffold to coordinate the activation of the NRF2 transcription factor. We have now discovered that DYRK1B controls the expression of detoxifying enzymes that are known to be targets of NRF2 suggesting that DYRK1B may control activation of NRF2. 2. p62 acts as a cargo receptor for damaged proteins, transporting them to recycling centres for autophagy. It has also been shown that p62 can shuttle in and out of the cell nucleus to collect damaged nuclear proteins for autophagy. This nuclear shuttling requires phosphorylation of p62 but the kinase responsible for this has remained a mystery. We have now discovered for the first time that p62 is phosphorylated by DYRK1B and DYRK2; we propose that this sends p62 into the nucleus to help collect damaged nuclear proteins. 3. p62 coordinates activation of a protein kinase enzyme called mTOR when cells are starved of nutrients. Indeed, mTOR is a critical regulator of lifelong health and controls the lifespan of organisms such as worms, flies, mice and possibly man. Regulation of mTOR by nutrients takes place at specific organelles called lysosomes. However, the phosphorylation of p62 may take it away from lysosomes to the nucleus. Indeed, we find that DYRK2 and p62 co-locate in cells at aggresomes - sites of damaged proteins, consistent with the cargo function of p62. It is not known what effect this re-location of p62 has on nutrient signalling via mTOR at lysosomes. In this study we will define how the DYRKs regulate p62 to coordinate cellular responses to stress and damage. This work is critical to understanding how stress contributes to normal ageing but may also have implications for diseases of old age (dementia, cancer); thus, our results may have wider impacts and we will work with scientists in these areas to progress this.

Impact Summary

The primary impact will come from new knowledge of mechanisms of signal transduction, related to the role of DYRKs and p62 in various biological contexts (see Academic beneficiaries). Impacts on other stakeholders: 1. Industry: by enhancing the research capacity and knowledge of businesses and organisations. This proposal developed from a PhD studentship between the Cook lab and AstraZeneca, who had developed a 10nM selective DYRK1B inhibitor. All major pharmaceutical companies remain interested in protein kinases as drug targets for a variety of diseases. Several companies have active DYRK inhibitor programmes whilst others have held back due to the lack of clear substrates to provide context for the known biology or to serve as pathway biomarkers. Our research is therefore relevant to a range of BioPharma companies contributing to UK economic competitiveness. We will work with the Babraham Commercialisation Manager to manage any resultant IP and all interactions with industry. 2. BBSRC: meeting national strategic research priorities. We have defined novel regulators of p62, a protein with a central role in stress responses and cell homeostasis, which is clearly a regulator of normal ageing. Within the BBSRC Strategic Plan 2013/14 refresh this research lies at the heart of Strategic Research Priority 3 - Bioscience For Health - and is relevant to the Societal Grand Challenge of 'maintaining health across the whole lifecourse' and the Key Priority 'Generate new knowledge of the biological mechanisms of development and the maintenance of health across the lifecourse'. In addition, this work 'will provide new insights to potential strategies for health monitoring and intervention, including drug targets and pharmaceuticals' consistent with the aspiration that 'basic bioscience funded by BBSRC underpins the pharmaceutical and healthcare industries'. 3. Healthcare and 3rd sector charities: translation of research into the clinic. The DYRKs are implicated processes that underpin disease and infirmity in old age. DYRK1B is implicated in myogenesis and adipogenesis. Age-related loss of muscle mass significantly impairs quality of life in the elderly. Similarly, adipocytes are critical regulators of metabolism and are involved in a variety of metabolic diseases including obesity. DYRKs are directly implicated in Treg biology in the immune system, cardiac hypertrophy and CNS cell death - all of which have ageing as a key risk factor or drive age-related disease. Furthermore, p62, the protein we have identified as a new target of DYRK1B and DYRK2, is intimately linked to age-related disease as a regulator of NRF2, mTOR and autophagy. Thus, our basic biology will be of interest to a variety of disease charities and healthcare professionals. 4. Training: generating a skilled workforce. This project will provide further training for key researchers (Fortian & Cook) in new scientific skills in growth areas (proteomics, genomics, bioinformatics). Through the links the Cook lab has with industry Fortian will also be exposed to drug discovery research providing training for her future contribution to the academic or commercial sector. 5. Science & Society: influencing and informing policy and increasing public understanding of science. We will continue to contribute to public STEM understanding through our public engagement activities. The PDRA, Fortian, will join other members of the Cook lab in PE activities, communicating their knowledge and enthusiasm to policy makers, the next generation of scientists and interested adults through science exhibitions, science festivals and science visits to schools and local community groups. We will work with the Babraham Public Engagement and Science Communications team to incorporate project specific content into resources for our new 'Science of Ageing' theme for PESC activities 2017-22.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Ageing
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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