Phosphoproteomes are muscle-specific and partially driven by gene expressionWe determined the phosphoproteome of four mouse muscles with diverse anatomical locations and functional properties whose response to aging and long-term CR and RM anti-aging interventions we have previously described9. Of the four muscles, soleus contains predominantly slow-twitch fibers, while tibialis anterior, triceps brachii, and gastrocnemius, all primarily contain fast-twitch fibers9,14 (Fig. 1A). We identified ~7000 non-redundant phosphorylation sites from each muscle (Fig. 1B), with 6780 being detected in all four muscles (Supplementary Data 1). Principal component analysis (PCA) of phosphopeptide signal intensities showed separation of soleus samples from the others along the first PC and a less pronounced separation of tibialis anterior samples along the second PC. Triceps and gastrocnemius samples were largely interspersed with each other (Fig. 1C). To determine whether differences in phosphopeptide abundance between muscles coincided with differences in gene expression, we also obtained mass spectrometry-based measurements of total protein levels in these muscles. Indeed, when comparing either soleus or tibialis anterior muscles with the other muscles, differences in phosphopeptide signal intensities significantly correlated with differences in the protein’s abundance, especially for soleus (Fig. 1D, E). However, the signal intensities of distinct phosphorylation sites within a given protein varied widely, indicating that protein levels do not fully explain phosphopeptide intensities (Supplementary Data 2). Two examples are myomesin 1 (Myom1) in the soleus muscle (Fig. 1F) and actinin alpha 2 (Actn2) in tibialis anterior (Fig. 1G). In both cases, protein level is lower than in other muscles, but the specific phosphorylation site in the protein increases.Fig. 1: Overview of the phosphoproteome data.A Experimental design, mouse image is from Biorender.com. B Venn diagram showing the number of phosphosites detected in each muscle (union of all sites identified in samples from a given muscle, irrespective of age and treatment). C PCA of the entire data set. Each symbol corresponds to a sample, color indicates the muscle type and symbol the condition. D Correlation of changes in the average normalized intensity of 317 phosphopeptides that align with PC1 with changes in corresponding proteins in soleus compared to all other muscles. E Similar for 119 phosphopeptides in tibialis anterior. F Example of the S863 phosphosite on Myom1 with a higher intensity in soleus than other muscles, despite Myom1 protein levels being lowest in this muscle. G S109 phosphorylated Actn2 has a higher intensity in tibialis anterior than other muscles, uncorrelated with the protein level. F, G show boxplots, individual samples being indicated by dots.To identify the biological processes specifically affected in each muscle, we selected phosphopeptides aligning to the PC (absolute z-score ≥ 1.96; correlation ≥ 0.5) that best distinguished each muscle from the others (i.e., PC1 for soleus, PC2 for tibialis anterior), as we have previously described5, and submitted the corresponding genes to Gene Ontology overrepresentation analysis. Soleus-specific phosphopeptides come from proteins involved in cytoskeletal organization, muscle development, and calcium sequestration, while tibialis anterior-specific phosphopeptides come from proteins involved in mRNA processing and localization (Supplementary Fig. 1). Thus, the variation in phosphopeptide intensity is only partially due to the variation in protein abundance between muscles. The soleus and tibialis anterior muscles can be distinguished from the other two muscles based on phosphopeptide signal intensities, despite most peptides being detected in all four muscles.Long-term RM treatment broadly reduces phosphorylation of mTORC1 targetsRM is known to acutely inhibit the activity of mTORC1 towards some, but not all substrates, while mTORC2 substrates are relatively resistant to RM15 (Fig. 2A). To validate our data and better understand the basis of muscle-specific responses to CR and RM9 we calculated log2 fold changes in peptide intensity between 30M_WT and 10M_WT, 30M_CR or 30M_RM samples for individual muscles, thereby capturing AGE, CR and RM effects, respectively. We then interrogated the behavior of direct mTORC1 and Rps6kb substrates, upstream mTORC1 regulators, and mTORC2 substrates (Supplementary Data 3) in our dataset. We tabulated 62 direct mTORC1 substrates, 56 of which came from Battaglioni et al.6, while the other six were E3 ubiquitin-protein ligase parkin (Prkn)16, N-terminal kinase-like protein (Scyl1)17, serine/threonine-protein kinase 11-interacting protein (Stk11iP)18, eukaryotic translation initiation factor 4E-binding protein 2 (Eif4ebp2), Eif4ebp3, and Rps6kb2. The 12 direct Rps6kb substrates were taken from Barilari et al.19.Fig. 2: AGE, CR, and RM effects on mTOR signaling.A Schematic diagram of mTOR signaling, created with Biorender.com. B Phosphosites in direct substrates of mTORC1 and of Rps6kb (see also Supplementary Data 3). Labels on the left indicate that the site has been reported to be RM insensitive (RMI) or sensitive (RMS). Deptor has been shown to be phosphorylated on S287 by casein kinase I isoform alpha (Ck1α)71,72 and by Rps6kb73. We detected 5 phosphosites on Clip1, a protein known to be phosphorylated by mTORC1, at sites so far unmapped74. The 4 phosphosites shown here do not have an associated kinase in the PhosphositePlus database. One site (S311 in mouse) has been assigned to 5’-AMP-activated protein kinase (AMPK) and is not shown. C Phosphorylation sites in upstream mTORC1 regulators. Black arrows on the right indicate the impact of the site’s phosphorylation on mTORC1 (Supplementary Data 3). D Phosphorylation sites in direct substrates of mTORC2. If a site has been captured in multiple distinct peptides, these are numbered and reported individually (e.g., “Flcn_S_62_2”). If the peptides contained multiple phosphosites, all of these are reported (e.g., “Eif4ebp1_S_64, T_69”).We were able to detect 25 sites in 12 mTORC1 substrates and 10 sites in 8 Rps6kb substrates (Fig. 2B). The pattern of aging-associated phosphorylation site changes was muscle-dependent, with some sites in the CAP-Gly domain-containing linker protein 1 (Clip1), La-related protein 1 (Larp1) and 5’-AMP-activated protein kinase catalytic subunit alpha-2 (Prkaa2) proteins showing decreased phosphorylation in the soleus and tibialis anterior muscles, while other sites (Clip1 S313) simultaneously displayed increased phosphorylation in the triceps and gastrocnemius muscles. These results are in line with the initial clustering of the samples based on signal intensities of all phosphosites measured in all muscles (Fig. 1C). Long-term RM treatment reduced the phosphorylation of the majority of RM-sensitive mTORC1 substrates, and most consistently reduced the phosphorylation of Rps6kb substrates (including Rps6 as described in ref. 5). In contrast, CR had more variable effects across muscles than RM, and in the case of Eif4ebp1 T69 phosphorylation, the treatments had opposite effects, with CR increasing and RM decreasing phosphorylation. Thus, RM mitigates aging-associated changes in the phosphorylation mTORC1 substrates, demonstrating that our data accurately capture expected changes. Moreover, as we have previously observed in phenotypic and functional measurements9, the effects of CR and RM on aging processes only partially overlap in mouse muscles.We also checked the 9 sites in 6 upstream mTORC1 regulators (Supplementary Data 3) represented in our data. The most consistent changes in phosphorylation across muscles were in the proline-rich AKT1 substrate 1 (Akt1s1/Pras40), an endogenous inhibitor of mTORC1 that binds regulatory-associated protein of mTOR (Rptor)20,21 (Fig. 2A). Akt1s1 is phosphorylated on different sites (S183/184, S212/213, and S221/S222 in human/mouse numbering) by at least 3 kinases to induce its release from mTORC1, thereby allowing its activation21. In our data, RM (but not CR) consistently reduced the Akt1s1 phosphorylation on S213 (Fig. 2B), a site reported to be phosphorylated in vitro by mTORC1 in a RM-dependent manner, while in vivo the RM sensitivity and the responsible kinase are unclear (reviewed in ref. 22). The S202/S203 and 203/204 (human/mouse) in Akt1s1 were reported to undergo phosphorylation by pyruvate kinase PKM-isoform M2 (Pkm2) in cancer23, leading to Akt1s1 dissociation from Rptor and release of mTORC1 inhibition23. Here we found that the S204 signal was increased by AGE in 3 out of 4 muscles (Fig. 2C), while CR and especially RM mitigated S204 phosphorylation (Fig. 2B). These results are consistent with the previously reported mTORC1 hyperactivity in aged animals, counteracted by the treatments5. CR and RM also increased Rptor phosphorylation on S722, a well known AMPK site24, which is expected to inhibit mTORC1 (Fig. 2C).Finally, we detected 5 phosphosites in 5 of the 30 direct substrates of mTORC2 (Supplementary Data 3). Interestingly, these were all upregulated in AGE (Fig. 2D), indicating that not only mTORC1 but also mTORC2 activity increases in aging muscle. The Akt2 S450 and protein kinase C alpha type (Prkca) T638 sites are known as “turn motif” (TM) sites and are cotranslationally phosphorylated by mTORC2 to stabilize the proteins (reviewed in ref. 6). That CR and RM reduce the phosphorylation of these sites is consistent with previous reports of mTORC2 inhibition upon prolonged RM treatment25,26. Altogether, these observed patterns of protein phosphorylation support the notion that not only mTORC1 but also mTORC2 activities increase during aging, and that CR and especially RM mitigate this increase to maintain muscle functionality9.General changes induced by AGE, CR, and RM in annotated phosphositesThe analysis of mTOR pathway components showed that we recovered expected changes in protein phosphorylation during aging and upon RM treatment, but also revealed substantial variation in the response of individual muscles to the treatments (Supplementary Fig. 2). Between-muscle differences were more pronounced than those induced by AGE, CR or RM (Fig. 1C). PCA of data from individual muscles showed that for soleus and tibialis anterior 10M_WT and 30M_WT samples separated along PC1, for triceps 10M_WT and 30M_WT samples separate along PC2, while for gastrocnemius samples do not clearly separate on any PC (Supplementary Fig. 2). To identify biological processes most generally affected by AGE, CR, and RM, we extracted phosphosites showing common changes in all four muscles. 227, 291, and 172 phosphopeptides were significantly regulated in all muscles in the AGE, CR, and RM conditions, respectively (Supplementary Figs. 3, 4). As previously noted, only a small proportion of these have cognate kinases annotated in the PhosphoSitePlus database27. Therefore, we refer to sites as annotated, unannotated or novel, depending on their presence/absence in PhosphoSitePlus and whether a cognate kinase has been reported.Broadly, AGE-associated changes in the phosphorylation of annotated sites (Fig. 3A, B) occur in many aging-relevant processes, including stress granule formation (Ras GTPase-activating protein-binding protein 1 (G3bp1) S149 phosphorylation28), formation of actin stress fibers (cytoskeleton-associated protein 1, Cap-1 S30729), autophagy (Sequestosome-1 (Sqstm1) T26930), and fatty acid metabolism (acetyl-coA carboxylase, also known as Acc1 or Acaca31). The AMPK catalytic subunit alpha-1 (Ampka1), a cellular sensor of energy levels32 is the kinase associated with most of the AGE-dependent sites. The CR and RM treatments do not consistently mitigate the AGE-associated increase in Ampka1 target site phosphorylation, possibly reflecting the variable activity reported for AMPK in different aging-associated conditions33,34.Fig. 3: General changes induced by individual treatments across all muscles.A Kinase and functionally-annotated phosphorylation sites that are significantly altered by AGE in all muscles (log2 fold changes capped at ±1). Relative changes in phosphopeptide intensity are shown for all muscles and all conditions. The site coordinate within the protein is shown, along with the protein kinase known to phosphorylate the site. If a phosphosite was covered by multiple peptides, the individual peptides are shown, indicated by an additional number in the name. B Biological processes in which the phosphorylation sites from (A) play a role according to the PhosphoSitePlus database. The direction of the phosphorylation signal change is indicated by the red (increase) and blue (decrease) color of the phosphate symbol, and the effect of the phosphorylation on the process is indicated by the color of the arrows leading to the respective process, black for repression and green for activation. If the phosphorylation modifies the activity of the substrate protein, the activity change upon phosphorylation is indicated by the arrow leading to the substrate (black – inhibition, green – activation). C Similar to (B), for CR-altered sites. D Same as in (C), for CR-altered phosphopeptides. E Similar to (B), for RM-altered sites. F Same as in (C), for RM-altered phosphopeptides.The phosphosites that are generally and significantly altered by CR are associated with diverse kinases and impact cell adhesion, motility, pluripotency, and differentiation (Fig. 3C, D). In particular, the CR-reduced S236 phosphorylation in Rps6 may reflect the suppression of mTORC1 activity by dietary restriction. Finally, the general and significant effects of RM are strongly localized to the mTORC1 pathway (Fig. 3E, F), where RM counteracts aging-associated increases in phosphorylation. In addition, RM increases the phosphorylation of some Camk2b substrates, including Camk2b itself, across all muscles, which is intriguing given that Camk2b plays an important role in synaptic transmission35, and neuromuscular junctions (NMJs) are remodeled during aging5.CR and RM mitigate aging-induced changes in unannotated phosphositesNext, we asked whether CR or RM also mitigate aging-induced changes in phosphosites without annotated kinases. There were 204, 255, and 150 kinase-unannotated phosphosites that changed consistently in all muscles with AGE, CR, and RM, respectively (Fig. 4A). The 3 phosphosites that underwent significant changes in all muscles and all conditions were located in proteins not known so far to play a role in the context of aging (Fig. 4A). We then turned to phosphosites that showed consistent changes across muscles in at least two conditions, as these could provide novel targets for anti-aging interventions or highlight side-effects of anti-aging treatments. The treatments generally mitigated AGE effects, with signal intensity changes occuring in opposite directions for AGE than CR or RM effects (Fig. 4). Only one site, S1937 in the cytoskeleton-associated ankyrin-3 (Ank3) protein, underwent significant changes in the same direction across all muscles and conditions, with a more pronounced AGE effect than that mediated by RM or CR (Fig. 4B).Fig. 4: Conserved but unannotated protein phosphorylation sites in muscles of aged, treated and untreated mice.A Top: Venn diagram showing the overlap between phosphosites that responded consistently across all four muscles in AGE, CR, and RM treatments. Shown are sites without an annotated kinase. Bottom: log2 fold changes in the intensity of the indicated peptides in each condition and each of the four muscles. Shown are the phosphopeptides that changed significantly and consistently in all muscles in all three conditions. Same as in (A) but conditioned on significant and consistent changes across muscles in AGE and CR (B), AGE and RM (D) and CR and RM (E). C Significantly enriched biological processes (GO terms) in proteins with consistently changing phosphosite in AGE and CR. F Same as (C) but for proteins with consistently changing phosphosite in CR and RM.Phosphopeptides whose aging-associated change was mitigated by CR across muscles (Fig. 4B, C) come from neurofilaments (heavy (Nefh) and medium (Nefm)), proteins associated with the endoplasmic reticulum and related stress responses (UBX domain-containing protein 6 (Ubxn6), protein disulfide-isomerase A6 (Pdia6), endoplasmic reticulum chaperone BiP (Hspa5), nascent polypeptide-associated complex subunit alpha (Naca)), as well as from proteins involved in vesicular trafficking (target of Myb1 membrane trafficking protein (Tom1) and synaptotagmin-2 (Syt2)).RM acts on a different set of targets, mitigating aging-induced phosphorylation changes of proteins involved in glucose metabolism (TBC1 domain family member 4 (Tbc1d4)), mitochondria (sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (Atp2a1)), TAR DNA-binding protein 43 (Tardbp) and muscle structure (e.g., dystrophin (Dmd), Fig. 4D). RM also increased the phosphorylation of a C-terminal serine (S1502) in synemin (Synm), a protein essential for cell adhesion and migration36.A few phosphosites responded significantly across all muscles to anti-aging interventions, but not to AGE itself. CR and RM increased the phosphorylation signal of sites in the muscle contraction-related proteins filamin C (Flnc), leiomodin 2 (Lmod2), and myosin binding protein H (Mybph) as well as the neurofilament protein Nefm. BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3), a protein that mediates mitophagy under stress37, also showed increased treatment-induced phosphorylation. The AGE effect on phosphosites was inconsistent across muscles and it remains to be determined whether these sites are undesired targets of CR and RM or provide unexpected benefits. A few phosphosites displayed lower phosphorylation in response to CR and RM, while displaying higher, but less consistent, phosphorylation during AGE. These proteins are involved in muscle structure (titin, Ttn), contraction (sarco(endo)plasmic reticulum calcium-ATPase 1 (SERCA1) also known as Atp2a1) and glycogen breakdown (skeletal muscle isoform of Phosphorylase B Kinase Regulatory Subunit Alpha, Phka1).Therefore, our data show that CR and RM mitigate the AGE-induced reduction in phosphorylation signal of proteins involved in cell adhesion, muscle contraction, and neuronal function. The two treatments exert largely consistent, but quantitatively distinct effects. Furthermore, for many sites that undergo significant changes in all muscles a cognate kinase has not been reported. Identifying the cognate kinases would further enhance our understanding of skeletal muscle aging.Kinase activity signatures of individual musclesWhile the coverage of phosphosites in any given experiment is still incomplete, changes in individual kinase activity between conditions can be robustly inferred by kinase set enrichment analysis (KSEA)38, based on coherent signal intensity changes in the multiple substrates targeted by a specific kinase (Fig. 5A). Age-related kinase activity patterns varied widely across the four muscles (Fig. 5B), while CR and RM again generally counteracted these changes. The most salient changes were the AGE-associated reduction in Akt2 activity and the increase in Mapk14/p38α activity in both soleus and tibialis anterior muscles, each of which was counteracted by CR and RM. In gastrocnemius muscle, CR, but not RM, also counteracted the effects of AGE on Akt2 and Mapk14/p38α activity.Fig. 5: Muscle-specific kinase signatures.A Illustration of KSEA38. B Kinases with significant changes in activity between conditions. Each line corresponds to a kinase that has a significant activity change (p < 0.05) in at least one muscle and one condition. The color indicates the class of the kinase. C–E Changes in kinase phosphorylation level in AGE, CR, and RM, respectively. Each ring shows the change in phosphosite (indicated by the label) intensity in the respective condition in one muscle.As kinase activity is typically regulated by phosphorylation, we also examined the complex dynamics of phosphorylation sites located on the kinases themselves (Fig. 5C–E). Among the more consistent changes were the RM-mediated suppression of the age-related increase in stress response-linked protein Ste20/Sps1-related proline-alanine-rich protein kinase (Stk39)39 phosphorylation, the CR-mediated increase in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (Pfkfb2)40 and Glycogen synthase kinase-3 alpha (Gsk3a) phosphorylation, as well as the RM-mediated increase in multiple phosphosites on Camk2b, a kinase with important roles in neuronal plasticity41 and NMJ stability42. RM upregulated the autophosphorylation of Camk2b T287, which makes Camk2b persistently active even when calcium concentrations are low43. Autophosphorylated Camk2b has been shown to phosphorylate histone deacetylase 4 (Hdac4) on S245 to promote its nuclear export44. Indeed, RM also increased Hdac4 S245 phosphorylation across all 4 muscles (Fig. 3E, F). As Hdac4 participates in the muscles response to denervation45, it would be interesting to further investigate a potential role for Camk2b at the NMJ during aging.This analysis reveals that RM and CR counteract age-related changes in kinase activities, but that the effects of individual kinases are quantitatively different between muscles, making it challenging to generalize data obtained in a specific system.Expanding the atlas of protein phosphorylation sitesIn this study we detected 6960 non-redundant phosphosites in at least 2 samples, with 6526 detected in at least 48 of the 95 samples. 5545 of the 6960 sites are already represented in the PhosphoSitePlus database and 485 also have an annotated kinase. However, 1415 are not represented in this database, i.e., they are novel. The known phosphorylation sites come from 597 proteins and the novel sites from 227 proteins, with 208 proteins in the overlap (Fig. 6A). Thus, we have identified 19 novel phosphoproteins in the mouse (Fig. 6A). Both annotated and novel sites are located in proteins involved in muscle development, muscle cell organization, and contraction, as well as energy metabolism (Fig. 6B, Supplementary Data 1). Importantly, absolute intensity of known and novel phosphoproteins were comparable (Fig. 6C), indicating that low protein expression levels cannot account for these novel sites having been overlooked. Furthermore, we did not observe any muscle- or condition-specific expression of the novel phosphosites or phosphorylated proteins (Fig. 6D). However, data from the Human Protein Atlas46 indicate that the expression of genes corresponding to these novel phosphosites is largely restricted to muscle tissue (Fig. 6E–H), which may be underrepresented in proteomics datasets compared to other tissues (e.g., liver or lung). This suggests a more specific context of functionality for the novel sites and underscores the importance of broad surveys that cover multiple organs and multiple interventions.Fig. 6: Expanding the atlas of protein phosphorylation sites.A Venn diagram showing the relationship of proteins with already annotated vs. novel sites. B Gene Ontology overrepresentation analysis of novel Mus musculus phosphoproteins. C Histogram of protein abundances for proteins containing annotated phosphosites (blue) vs. those containing novel sites (red). D Log2 fold change in phosphopeptide signal intensity in the AGE, CR, and RM conditions for phosphosites located in the 19 novel phosphoproteins that are common to all 4 muscles. The order of the muscles in the circular representation from outside to inside is soleus, tibialis anterior, triceps, and gastrocnemius. E–H Single cell RNA-seq data from Human Protein Atlas46 showing the distribution of RNA expression levels for 4 of the novel mouse phosphoproteins (tissue specificity score based on75). Data are represented as dots overlayed over corresponding bars.Our data substantially expand the muscle phosphoproteome and enhance the information about the conditions and specific muscles in which these sites are modified.
