DOI: 10.25345/c5cc0v46r
title: MassIVE MSV000093798 - Calorie restriction and rapamycin distinctly mitigate aging-associated protein phosphorylation changes in mouse muscles
title: The recognition of aging as a risk factor for chronic, inflammatory and malignant diseases 1 led to increased efforts to identify its underlying molecular mechanisms. The hallmarks of aging, initially defined ten years ago, have been recently expanded to comprise genome instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation and dysbiosis 2. Signaling pathways 3 converging on key transcription factors remodel gene expression and ultimately cellular function in an agedependent manner. Signal transduction relies heavily on protein phosphorylation, the most common posttranslational modification. PhosphoSitePlus, the main repository used in the field, currently contains 320000 nonredundant phosphosites, of which are annotated to a corresponding kinase 12. The catalog continues to grow, due to increasingly sensitive measurement technologies and diverse analysis approaches 13,14. Aging-related changes in protein phosphorylation has so far been studied in the mouse liver 15. Other studies focused on interfering with specific aging hallmarks, for example determining phosphorylation changes induced in the muscle of aged mice by the short term treatment with elamipretide, a drug that reduces the formation of free radicals in mitochondria 16,17. In parallel, numerous studies have started to explore approaches to slow down the aging process and improve lifespan. Ongoing clinical trials involve exercise, intermittent fasting and calorie restriction 18, as well as compounds that target key molecular pathways such as nutrient sensing. The molecular signature of such interventions has been determined at the mRNA and protein level in various tissues 19, including mouse muscles 20,21. However, the remodeling of signaling pathways upon these interventions remains relatively uncharted. In previous work we have demonstrated that individual mouse muscles have distinct functional responses to the longterm treatment with calorie restriction (CR) and rapamycin (RM) and we have determined the underlying mRNA level expression signatures 22. In this study we expand on this work, determining the protein phosphorylation dynamics of four muscles, soleus, tibialis anterior, triceps brachii and gastrocnemius, from adult (10 months 10M), geriatric (30M), and 30 months-old mice that were either calorie restricted (30MCR) or treated with rapamycin (30MRM) from 15 months of age. We robustly detected 6960 phospho sites across samples, 1415 of which are not represented in the PhosphoSitePlus database. We demonstrate that CR and RM have largely consistent, but quantitatively distinct, long-term effects on the phosphoproteome, reverting agerelated changes to different degrees and in muscledependent manners. Our data expands the catalog of protein phosphorylation sites in the mouse, providing important information regarding their tissuespecificity.