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

我们身体中的细胞不断暴露于化学和物理“压力”，并积累了对蛋白质，DNA和各种关键亚细胞结构（细胞器）的终身损伤。细胞能够对这种损伤做出适当的反应是至关重要的;如果不能做到这一点，细胞的“健康”就会逐渐受损，导致细胞和组织功能与年龄相关的下降，而细胞和组织功能是正常衰老过程的基础，也可能导致与年龄相关的疾病，如癌症和痴呆症。细胞应对压力的一种方式是增加酶的丰度，通过清除潜在的有害化学物质来解毒细胞。当细胞接收到压力信号时，编码解毒酶的基因被“转录因子”“读取”，这些转录因子是与DNA结合并将DNA信息转录成RNA分子的离散蛋白质，这些RNA分子又被“翻译”成相关蛋白质。其中一种转录因子NRF 2负责协调细胞对氧化应激的反应。细胞处理细胞损伤的另一种方式是通过一种称为“自噬”（自噬）的过程，在该过程中，受损的蛋白质被靶向细胞回收中心（称为自噬体），在那里它们被分解为原材料，然后可以重新使用。这种自噬过程需要“货物受体”，它与受损的蛋白质结合，并将它们运送到自噬的回收中心。这些复杂的过程是由细胞内的信号通路精心策划的，其中涉及称为蛋白激酶的级联酶。这些酶用磷酸基团（称为磷酸化的过程）“标记”其他蛋白质，这会改变蛋白质的活性，丰度或定位。标记的蛋白质被称为蛋白激酶的“底物”。该项目涉及两种蛋白激酶，称为DYRK 1B和DYRK 2，以及一种称为p62或螯合体-1的特定“货物受体”;在这里我们称之为p62。我们的新结果指出了DYRKs和p62在协调细胞如何应对压力和损伤方面的联系：p62作为一个支架来协调NRF 2转录因子的激活。我们现在已经发现DYRK 1B控制已知是NRF 2靶点的解毒酶的表达，这表明DYRK 1B可能控制NRF2.2的激活。p62作为受损蛋白质的货物受体，将它们运送到回收中心进行自噬。研究还表明，p62可以穿梭进出细胞核，收集受损的核蛋白进行自噬。这种核穿梭需要p62的磷酸化，但负责这一点的激酶仍然是一个谜。我们现在首次发现p62被DYRK 1B和DYRK 2磷酸化;我们认为这将p62送入细胞核以帮助收集受损的核蛋白。当细胞缺乏营养时，p62协调一种称为mTOR的蛋白激酶的激活。事实上，mTOR是终身健康的关键调节剂，并控制生物体如蠕虫、苍蝇、小鼠和可能的人的寿命。营养素对mTOR的调节发生在称为溶酶体的特定细胞器中。然而，p62的磷酸化可能将其从溶酶体带到细胞核。事实上，我们发现DYRK 2和p62在细胞中共定位于受损蛋白的侵袭体位点，与p62的货物功能一致。目前尚不清楚p62的这种重新定位对通过mTOR在溶酶体的营养信号传导有什么影响。在这项研究中，我们将确定DYRKs如何调节p62以协调细胞对应激和损伤的反应。这项工作对于理解压力如何促进正常衰老至关重要，但也可能对老年疾病（痴呆症，癌症）产生影响;因此，我们的结果可能会产生更广泛的影响，我们将与这些领域的科学家合作，以推动这一点。

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.