In vivo interaction proteomics reveal a novel role of p38MAPK in controlling proteostasis in ageing Drosophila muscle. Vladimir Belozerov^1,2, Anne-Claude Gingras², Helen McNeill², John McDermott¹. 1) Department of Biology, York University, Toronto, ON, Canada; 2) Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.

   Several recent studies suggest that systemic ageing in metazoans is differentially affected by functional decline in specific tissues, such as skeletal muscle. In Drosophila longevity appears to be tightly linked to the regulation of myoproteostasis, and the formation of misfolded protein aggregates has been shown to be a hallmark of senescence in ageing muscle. Similarly, defective myoproteostasis is described as an important contributor to the pathology of several age-related degenerative muscle diseases in humans, e.g. inclusion body myositis. p38 MAP kinase plays a central role in a conserved signaling pathway activated by a variety of stressful stimuli. In ageing Drosophila muscle p38b/Mef2/MnSOD pathway was shown to control muscle function and longevity by modulating ROS. Concomitant with declining motor functions in ageing p38b mutant flies, we observed enhanced accumulation of detergent-insoluble protein aggregates in flight muscles, suggesting deregulation of myoproteostasis. To define the molecular mechanism of p38MAPK-mediated regulation of protein turnover we used affinity purification and mass spectrometry (AP-MS) to identify proteins interacting with a kinase-dead mutant of p38b (acting as a substrate trap) in ageing flight muscles. One of these substrates, dRack1, is of particular interest as it interacts with the ribosome, and may serve as a link between p38MAPK signaling and translational regulation. Using in vitro kinase assays and mass spectrometry we show that dRack1 is indeed a substrate of p38b. Further genetic and biochemical tests position dRack1 downstream of p38b, and demonstrate that dRack1 inhibits translation in ageing muscle in response to p38b signaling. Finally, we used AP-MS to examine protein interaction network formed by RACK1 in human cells, and identified a novel complex of RACK1 with known translational repressors, providing a likely mechanism of p38MAPK/RACK1-mediated control of proteostasis.