Abstract
Background
Skeletal muscle is central to locomotion and metabolic homeostasis. The laboratory worm Caenorhabditis elegans has been developed into a genomic model for assessing the genes and signals that regulate muscle development and protein degradation. Past work has identified a receptor tyrosine kinase signalling network that combinatorially controls autophagy, nerve signal to muscle to oppose proteasome‐based degradation, and extracellular matrix‐based signals that control calpain and caspase activation. The last two discoveries were enabled by following up results from a functional genomic screen of known regulators of muscle. Recently, a screen of the kinome requirement for muscle homeostasis identified roughly 40% of kinases as required for C. elegans muscle health; 80 have identified human orthologues and 53 are known to be expressed in skeletal muscle. To complement this kinome screen, here, we screen most of the phosphatases in C. elegans .
Methods
RNA interference was used to knockdown phosphatase‐encoding genes. Knockdown was first conducted during development with positive results also knocked down only in fully developed adult muscle. Protein homeostasis, mitochondrial structure, and sarcomere structure were assessed using transgenic reporter proteins. Genes identified as being required to prevent protein degradation were also knocked down in conditions that blocked proteasome or autophagic degradation. Genes identified as being required to prevent autophagic degradation were also assessed for autophagic vesicle accumulation using another transgenic reporter. Lastly, bioinformatics were used to look for overlap between kinases and phosphatases required for muscle homeostasis, and the prediction that one phosphatase was required to prevent mitogen‐activated protein kinase activation was assessed by western blot.
Results
A little over half of all phosphatases are each required to prevent abnormal development or maintenance of muscle. Eighty‐six of these phosphatases have known human orthologues, 57 of which are known to be expressed in human skeletal muscle. Of the phosphatases required to prevent abnormal muscle protein degradation, roughly half are required to prevent increased autophagy.
Conclusions
A significant portion of both the kinome and phosphatome are required for establishing and maintaining C. elegans muscle health. Autophagy appears to be the most commonly triggered form of protein degradation in response to disruption of phosphorylation‐based signalling. The results from these screens provide measurable phenotypes for analysing the combined contribution of kinases and phosphatases in a multi‐cellular organism and suggest new potential regulators of human skeletal muscle for further analysis.
Keywords: Phosphatase, C. elegans, Sarcomere, Proteostasis, Protein degradation, Muscle
Introduction
Skeletal muscle is required for locomotion and maintaining posture and gait. These roles are facilitated by the actin/myosin‐based contractile units. Frequently, the clinical focus on loss of muscle function is on the loss of locomotor function, for example, with trauma or age or in the muscular dystrophies. In the USA, the costs associated with such musculoskeletal conditions were estimated at 5.73% of the GDP in 2011, up from 3.43% in 1998, and expected to continue to rise as the population continues to age.1 However, the establishment, maintenance, and operation of the contractile units require substantial metabolic input. This explains why a muscle is a major contributor to overall metabolic homeostasis both as the major site of glucose storage and disposal and as the main protein/nitrogen reserve. Disruption of muscle glucose disposal likely contributes to the larger public health crisis of type II diabetes,2 and the loss of muscle protein seen in various clinical conditions such as burns, sepsis, and cancer can be the proximal cause of death.3 Thus, muscle has multiple functions of important clinical relevance.
Like many clinical problems, the establishment and maintenance of muscle homeostasis are studied not only in human subjects but also in laboratory animals. The worm Caenorhabditis elegans is one such animal. Its small size, transparency, and rapid development coupled with the genetic and genomic tools available make it an ideal model for foundational studies.4 The worm has been used to study muscle development,5 muscular dystrophy,6 fat metabolism,7 sarcopenia,8 spaceflight‐induced changes in muscle,9 and muscle protein degradation (Figure 1A);10, 11, 12, 13, 14, 15, 16, 17, 18, 19 in each instance, the uncovered genes, signals, and/or underlying concepts of control mechanism(s) have been found to have direct relevance to the same processes and/or conditions in humans.
Three recent kinome‐wide RNAi screens performed in C. elegans to identify the kinome requirement for normal muscle development and homeostasis20 identified roughly 40% of the kinome as being important for establishing and/or maintaining proteostasis, mitochondrial structure, or sarcomere structure in muscle. Of these kinases identified in C. elegans, 80 have identified human orthologues and 53 are known to be expressed in skeletal muscle. To complement this data set and to study phosphatases on a genome‐wide scale, we undertook a systematic analysis of phosphatases required for establishing or maintaining muscle cell health in C. elegans. For this study, we employed RNAi to systematically knockdown most individual phosphatases in the C. elegans genome. RNAi was utilized because of both the lack of specificity of available protein phosphatase inhibitors as well as the lack of inhibitors for most of the phosphatome.
Methods
Nematode handling and RNA interference screening
Nematode handling, strains utilized, RNAi screening, epistasis testing of identified genes against known protein degradation pathways, and assessment of autophagic vesicles via transgenic reporter protein were all as previously described and diagrammed for the RNAi screen of the C. elegans kinome requirement for a muscle.20
A screening list of phosphatase‐encoding genes was constructed from a C. elegans RNAi phosphatase list of 167 genes supplied by Source BioScience LifeSciences Ltd. (Nottingham, UK) and a list of 207 genes supplied by Plowman et al.,21 the latter of which was based on a genome‐wide HMM search for phosphatase motifs in the C. elegans genome. Comparison of the lists yielded 106 genes that were represented in both lists. The remaining genes unique to one of the two lists were further examined for phosphatase annotation in www.wormbase.org.22 Thereupon, a further 67 genes from the Plowman list and a further 25 genes from the Source BioScience LifeSciences Ltd. list were found. Thus, a total of 198 phosphatase‐encoding genes (106 matches +25 Source Bioscience Ltd. +67 Plowman) were collated from both. Where possible, sequence verified RNAi clones against each individual phosphatase were obtained from either of two previously constructed genome‐wide RNAi bacterial feeding clones.23, 24 These clones were obtained from Source BioScience (Nottingham, UK). After sequence verifying all positive results from our screen, we identified that previously utilized, sequence verified, RNAi constructs were available for 183 putative phosphatase‐encoding genes (see Supporting Information Data S1).
Quality control of our RNAi screens was as previously described and diagrammed for the RNAi screen of the C. elegans kinome requirement for a muscle.20 By comparing the developmental phenotypes, such as growth or uncoordinated movement observed in this study to developmental phenotypes observed in RNAi experiments by other investigators using the same RNAi bacteria clone, a potential discrepancy of RNAi results for 17% of total genes screened was identified. This is in concordance with published RNAi screens17, 20 and half of these potential discrepancies are cases in which we identified a developmental phenotype in response to RNAi but for which a wild‐type phenotype was observed in RNAi experiments by others, indicating that either the RNAi was more effective in this study and therefore these results may be new findings, or these results are false positives. This is again consistent with published RNAi screens17, 20 and most likely represents our method producing a first discovery of function rate that is higher than past studies. Technical details, including why our false positive rates are lower and first discovery rates are higher than past studies, can be found elsewhere.25
Network analysis
Data from meta‐analyses of physical and functional interactions between the genes identified during the chronic and acute RNAi screen were extracted manually from the following databases: WormBase,22 GeneMANIA,26 and PhosphoPOINT.27 Only interactions between the genes identified to potentially regulate a specific process were searched to construct process‐specific network models. To use PhosphoPOINT data, a human orthologue for the gene identified was searched. The assignment of orthology was taken from a recent meta‐analysis28 and review29; orthologies used are in Data S1 for phosphatases and in Lehmann et al. 20 for kinases. PhosphoPOINT data for the human orthologues were then converted back to the C. elegans orthologues. Some of the genes identified had the same human orthologue and therefore appear as one node in the networks (see Supporting Information Data S2 and S4); these genes are egl‐4 and pkg‐2; kin‐14 and frk‐1. All extracted interactions were visualized using CytoScape.30 All extracted data are available for use and similar visualization (see Supporting Information [Link], [Link]); data are divided by individual networks. Data for physical networks are from C. elegans genome‐wide known physical interactions and predicted physical interactions based upon known physical interactions of orthologues in a different species (human, rodent, fly, yeast) both which were retrieved from WormBase and GeneMANIA, as well as on kinome‐wide biochemical data for directly interacting human orthologues, which were retrieved from PhosphoPOINT. Data for functional networks are mainly based on kinome‐wide biochemical data of shared substrates and/or interacting phosphoproteins for the human orthologue derived from PhosphoPOINT. These networks also contain C. elegans known gene product interactions and predicted gene product based upon known gene product interactions for the orthologue in a different species, both which were retrieved from WormBase and GeneMANIA.
Western blot
For western blot analysis of MEK phosphorylation, 30 worms were picked into 20 μl sterile ddH2O and immediately frozen in liquid nitrogen and stored at −20°C. Later the same week, 8 μL of 3× Laemmli buffer was added to each sample and heated for 5 min at 95°C in a hot block, whereupon they were vortexed for 30 s and centrifuged for 1 min and placed on ice. The entirety of each sample was then loaded into a 12% Bis‐Tris SDS PAGE gel (Bio‐Rad, Hemel Hempstead, UK) for electrophoresis for 1 h at 200 V. Separated proteins were transferred onto a PVDF membrane (Bio‐Rad) for 45 min at 100 v, then placed in 3% bovine serum albumin (BSA) in Tris‐buffered saline and 0.1% Tween‐20 (TBST) for 1 h at room temperature. Membranes were washed 3× for 5 min in TBST then incubated at 4°C overnight in primary antibody solution. Anti‐P‐MEK 1/2Ser 217/221 (no. 9121) (Cell Signalling Technology, Beverly, MA, USA) was diluted 1:1000 in TBST. Afterwards, the membrane was washed 3× for 5 min in TBST before incubation in the secondary antibody solution of 3% BSA in TBST containing HRP conjugated anti‐rabbit secondary antibody (Cell Signalling Technology), 1:2000 for 1 h at room temperature. The membrane was then washed 3× in TBST, before incubation for 5 min in enhanced chemiluminescence reagent (Millipore, Watford, UK) and visualized using a Chemidoc XRS system. Band volumes were quantified using ImageJ (NIH).
Results
Phosphatases required for establishing or maintaining muscle health
To establish the role of each phosphatase‐encoding gene in the genome of C. elegans in establishing and/or maintaining muscle homeostasis, we obtained a set of RNAi constructs against phosphatases from Source BioScience LifeSciences Ltd. and also RNAi constructs against phosphatases identified using a hidden Markov model (HMM) search for phosphatase motifs in the C. elegans genome.21 This lead us to identify 198 putative phosphatase‐encoding genes of which 106 were identified by both sources, 25 were unique to Source Bioscience, and 67 were unique to the HMM search; sequence verified RNAi constructs were available for 183 of these genes. Utilizing these 183 RNAi constructs, we repeated the RNAi screening protocol used to identify kinases required for normal muscle proteostasis, protein degradation, mitochondrial structure, and sarcomere structure (diagrammed in Lehmann et al.20). Briefly, worms were treated with RNAi against a single gene throughout development, and adults were scored at multiple time points during adulthood for normal reporter protein levels, mitochondrial structure, and sarcomere structure. RNAi treatments that produced lethality or abnormal protein levels or structure were then applied to previously untreated, normal, adults to determine if the knockdown produced a defect solely due to a requirement of the gene during development or if the gene was also required for continued maintenance of fully developed muscle. Additionally, a key feature of the protein degradation screen was that RNAi treatments were not only identified as inducing altered proteostasis and increased protein degradation but they were also examined for the requirement of UNC‐51/ATG1 in producing the increased protein degradation and, if UNC‐51 was required, if increased autophagic vesicles were observed. This autophagy screen is graphically displayed in Figure 1B.
As shown in Figures 2, 3, and 4, RNAi against 97 of 183 putative phosphatases produced a subcellular defect in a muscle. This suggests that roughly half of all phosphatases are required for normal development and/or maintenance of muscle. This percentage requirement is slightly higher than the roughly 40% of kinases that are required for normal development or maintenance of muscle and likely reflects the fact that because there are fewer phosphatases than kinases, there is less redundancy. Again, like the kinase requirement for muscle, more phosphatases are required for normal proteostasis than for mitochondrial structural homeostasis, and the least phosphatases are required for normal sarcomere homeostasis. This suggests that there are more signals impinging upon muscle metabolism than upon muscle sarcomere structure. Similarly to the kinase requirement for muscle, most phosphatases identified as required for normal development of muscle are also required for maintenance of adult muscle.
Included in the results are the identification of genes that were already known to regulate a muscle, such as a negative regulator of fibroblast growth factor receptor (FGFR), clr‐1,14, 31 and myosin phosphatase, mel‐11, which is known to be involved in elongation during development.32 These screens also identified embryonic lethality as expected for let‐92 and cdc‐25.1. Although the identification of these genes appears to validate the RNAi results, not many of the other genes identified have been studied in detail or are known to regulate any of the processes examined. This was confirmed by gene ontology analysis using the online software DAVID,33 which failed to recognize a third of the genes we identified as having previously been assigned a biological function. This suggests that the approach taken in this study may be an important first step forward understanding the functions of previously unstudied phosphatase‐encoding genes. Interestingly, a little over half of the genes identified in these screens have homologues expressed in human skeletal muscle (see Supporting Information Data S1), suggesting that these genes may be candidates for further study of the regulation of muscle protein degradation, mitochondrial fission, and sarcomere maintenance in humans.
Epistasis testing of potential degradation‐regulating phosphatases versus known signals
To further identify how the RNAi knockdowns were producing cytosolic protein degradation, we functionally clustered the genes identified as required to prevent induction of protein degradation into those appearing to be required to prevent autophagy or proteasome‐mediated degradation. This was accomplished by treating unc‐51 (ATG1) mutants or proteasome inhibitor‐treated animals with each RNAi treatment that induced protein degradation. Additionally, we used mpk‐1 and daf‐18 loss of function mutations to cluster these genes into FGFR‐mediated and IGFR‐mediated pathways, respectively.13 Half of the phosphatase‐encoding genes appear to be potential regulators of autophagy‐mediated protein degradation (Figure 5A), which is similar to the finding when the kinase‐encoding genes were previously knocked down. To confirm that autophagy was indeed induced in response to these RNAi treatments, we examined if GFP::LGG‐1 autophagic vesicles increased in muscle in response to treatment, which they did (Figure 5B). These findings suggest that when protein phosphorylation is perturbed either by increasing phosphorylation, in phosphatase RNAi knockdowns, or decreasing phosphorylation, in kinase knockdowns, that autophagy is triggered. In other words, autophagy appears to be sensitive to the global balance of numerous signals in muscle. Interestingly, most of the kinases and phosphatases that were identified to potentially regulate protein degradation required MPK‐1 (mammalian extracellular signal‐regulated kinase (ERK)). This suggests that MPK‐1 and other MAPKs may play a central role in the regulation of overall protein degradation within a cell. Given that ERK is known to be expressed and active in human skeletal muscle,34 perhaps a similar metabolic role for ERK in human skeletal muscle exists.
Identification of let‐92 as a putative central node for protein degradation
To examine if the identified phosphatases and recently identified kinases that may regulate subcellular processes within muscle might act within a network regulating muscle homeostasis, we used past C. elegans genome‐wide known and predicted gene product physical interaction maps from published meta‐analyses,35, 36, 37 as well human kinome‐wide known gene product physical interaction data from a published meta‐analysis,27 to construct potential physical networks for the kinases identified in each screen. We also used past C. elegans genome‐wide known and predicted gene product functional interactions from published meta‐analyses,35, 36, 37 as well human kinome‐wide known gene product functional interaction data from a published meta‐analysis, to construct potential functional networks for the kinases identified in each screen. The physical networks are based upon binding data (e.g. yeast two hybrid, co‐immunoprecipitation) for the C. elegans kinase and/or data for the yeast, fly, rodent, and/or human orthologue35, 36, 37 while the functional networks are based upon limited genetic interactions for the C. elegans kinase and/or data for the yeast, fly, rodent, and/or human orthologue35, 36, 37 and a large amount of biochemical data for shared interacting phospho‐proteins for the human orthologue.27 Visualization of these predicted interactions using cytoscape did indeed reveal some potential interaction networks (see Supporting Information [Link], [Link]). Of note, there were not many known or predicted interactions between the phosphatases identified here. However, the combination of data on identified kinases and phosphatases resulted in a more integrated network than kinase or phosphatase‐specific networks alone. Also, within these potential networks emerged a phosphatase, let‐92, and kinase, abl‐1, that appeared to be central nodes as indicated by the number of connections to other identified genes (Figure 6A). The identification of such central nodes suggests one strategy in prioritizing phosphatases and kinases for further study.
Knockdown of protein phosphatase 2A catalytic or regulatory subunit‐encoding genes results in increased MEK phosphorylation
Because LET‐92 appeared to be a central node and because PP2A is known to interact with Akt,38 a kinase known to control mammalian muscle size via both well‐appreciated39 and recently demonstrated mechanisms,40 we decided to further investigate the role of LET‐92 as a regulator of muscle protein degradation. The data presented in Figure 5 suggest that let‐92 knockdown induces MAPK‐dependent autophagy. This is consistent with early reports of protein phosphatase 2A (PP2A) being a negative regulator of MAPK both in vitro 41 and in cultured cells42 and is also consistent with past reports of constitutive, autocrine, FGFR activation of Ras‐MAPK in C. elegans muscle being subject to negative regulation.13 Therefore, we tested if knockdown of PP2A catalytic and regulatory subunits resulted in increased phosphorylation of MEK, which should increase activation of MAPK. Western blots (Figure 6B) confirmed increased phosphorylation of MEK in response to knockdown of let‐92, paa‐1, and C06G1.5 as well as the clr‐1 positive control. These results, coupled with those shown in Figure 5, suggest that PP2A is required to prevent excessive activation of autophagy in C. elegans muscle by modulating the activity of Ras‐MAPK signalling, which appears to act upstream of UNC‐51/ATG1.13
Discussion
Functional analysis of the phosphatome of Caenorhabditis elegans
Post‐translational modifications are a widely appreciated mechanism of modulating protein function. Phosphorylation is arguably one of the best studied such modifications, and the ability to modulate phosphorylation status of key proteins is clinically desirable.43, 44, 45 Much progress has been made on understanding the role that protein kinases play in phosphorylating their targets and in understanding the specificity of compounds against the kinome.46, 47 In contrast, the progress on understanding the role the protein phosphatases play in dephosphorylating their targets has lagged behind. Here, we have conducted three near full genome RNAi screen to identify phosphatases that when knocked down result in abnormal development and/or maintenance of muscle. Using this approach, we have found that roughly half of the phosphatome is required for normal development or maintenance of muscle. These data provide the first potential functional importance of more than a third of the C. elegans phosphatome and a preliminary picture of how many phosphatases are important for the proper development and maintenance of muscle. Further work is needed to determine if these phosphatases are required within muscle or other tissues for normal muscle health and to understand why and how these phosphatases are important. Given that putative human homologues of roughly half of the identified phosphatases are already known to be expressed in muscle (Supporting Information Data S1), it is likely that a good portion of the identified phosphatases act within muscle to modulate development and/or maintenance. While it is perhaps surprising that so many phosphatases appear to be required for normal development and/or maintenance of a muscle, the requirement is roughly similar to the kinome requirement for a muscle.20 The combined C. elegans phosphatome and kinome requirement for muscle provides a platform for future mechanistic studies of individual phosphatases and kinases, further unravelling of the complexity of the regulation of muscle, and a starting point for further therapeutic modulation of human muscle health.
Disruption of phosphorylation events frequently triggers autophagy
Here, we have found that autophagic protein degradation is triggered in roughly half of individual phosphatase knockdowns that induce degradation. This result is intriguing for two reasons. First, as there are four major proteolytic systems in a muscle,10 this implies that a phosphatase is more likely to be important to prevent autophagy than to prevent proteasome‐meditated, caspase‐meditated, or calpain‐meditated degradation. Second, as knockdown of individual kinase‐encoding genes most frequently triggered autophagy,20 this implies that both increased and decreased phosphorylation events are likely to trigger autophagy. This finding from the combined work on the kinome and phosphatome suggests that autophagy is controlled by a balance of positive and negative signals and is consistent with past suggestions that in C. elegans, muscle autophagy is controlled by counterbalanced, constitutive pro‐degradation signalling from FGFR, and anti‐degradation signalling from insulin‐like growth factor receptor (IGFR).13 While the current observation is consistent with the past findings, what is surprising is the large extent to which both individual kinases and phosphatases appear to be required to prevent autophagy. One possible explanation for the more extensive requirement for kinases and phosphatases to prevent autophagy is that autophagy might be a default state that is subject to negative regulation in the presence of multiple signals that indicate favourable growth conditions. Such a notion is consistent with the previous suggestion that mTor is an integrator of multiple favourable growth conditions to modulate both protein synthesis and degradation.48, 49 This also raises the question of the relative importance of autophagic‐mediated as opposed to proteasome‐mediated protein degradation for maintaining human muscle homeostasis.
Mitogen‐activated protein kinase as a central regulator of protein degradation
In addition to finding that autophagic protein degradation is the type of protein degradation most commonly triggered in response to knockdown of any individual kinase or phosphatase, we have found that functional MPK‐1 is very frequently required for the protein degradation that is triggered in response to knockdown of any individual kinase20 or phosphatase (Figures 3 and 4). Thus, analysis of both the kinome and phosphatome suggests a central role of MPK‐1 in modulating muscle protein degradation in response to phosphorylation events. This observation, like the observation of both increased and decreased phosphorylation events being associated with increased autophagy, suggests that perhaps a central integrator of multiple favourable growth conditions exists. Our connectivity analysis of the kinome and phosphatome with respect to protein degradation suggests that LET‐92 is a central node and that it appears to be a modulator of muscle protein degradation with knockdown producing mpk‐1‐dependent autophagic degradation. These results, coupled with the fact that ERK is known to be expressed and active in human skeletal muscle,34 raise the question of if Raf‐MAPK is a central modulator of autophagic degradation, with a significant number of kinases and phosphatases providing modulatory signals for this central pathway. This also raises the question of if Raf‐MAPK is not just a central player in controlling protein synthesis but also of autophagy, perhaps acting to either modulate or complement a similar role of mTor. Thus, our results from C. elegans open the door to further mechanistic studies of the regulation of human muscle metabolism.
Potential implications for human health and disease
We have identified phosphatases that are required for normal muscle health in a worm. Eighty of these phosphatases have human counterparts and 53 are already known to be expressed in human muscle. If they control human muscle health like they do worm muscle health, then these phosphatases are important for normal muscle health and may contribute to human muscle disease; translational work that remains to be completed. This has several implications for the clinic. First, these phosphatases, like the previously uncovered kinases,20 are potential druggable targets for therapeutic intervention in muscle health. For example, as has recently been reported for mouse muscle, stimulation of protein kinase A results in increased proteasome‐mediated protein degradation, whereas treatment with protein phosphatase 1 decreases proteasome‐mediated protein degradation.50 Thus, with further work, it is highly probable that protein kinase and phosphatase inhibitors can be used to modulate protein degradation levels in either direction, work that will no doubt be accelerated by the cancer field's push to identify effective protein kinase and phosphatase inhibitors that are safe for human use.51, 52 Inhibition/activation of kinases and phosphatases may also prove useful in other respects. For example, the phosphatase PTPH1 is known to regulate p97,53 which has recently been suggested to extract proteins from the highly organized, protein dense sarcomeres.54 Therefore, clinical modulation of multiple molecular processes within human muscle is likely to be achievable just by targeting these two classes of druggable proteins. Second, drugs that are used to target protein phosphatases or kinases in other diseases, for example cancer, may produce myopathy as a side effect due to the normal role of the phosphatase or kinase in muscle health. For example, inhibition of the protein kinase MEK produces rhabdomyolysis55 and is known to be important for worm muscle health.14 Third, mutations in protein phosphatases or kinases may account for some rare as yet molecularly uncharacterized muscular dystrophies. For example, mutations in the phosphatase myotubularin 1 are known to cause X‐linked myotubular myopathy56 and a mutation in the phosphatase myotubularin‐releated protein 14 has been shown to cause centronuclear myopathy.57 Fourth, declines in expression of phosphatases or kinases with age may contribute to the onset and/or progression of sarcopenia. For example, myotubularin‐releated protein 14 displays reduced expression with age in mice and its loss accelerates sarcopenia.58 Lastly, alterations in expression of phosphatases or kinases with activity may contribute to individual differences in muscular adaptation to exercise. For example, the kinase MARCKS and phosphatase PTEN display increased expression following a programme of resistance exercise training.59 Given that inactivity is one of the top non‐communicable diseases in the world,60 this suggests a substantive new avenue of research into combating the negative muscular consequences of inactivity, the impact of phosphatase or kinase modulators on muscular adaptation to activity.
Conflict of interest
Susann Lehmann, Joseph J Bass, Thomas F Barratt, Mohammed Z Ali, and Nathaniel J Szewczyk declare that they have no conflict of interest.
Supporting information
Acknowledgements
Our thanks to L.A. Jacobson (University of Pittsburgh) for useful discussions and the Gieseler laboratory (Université Claude Bernard Lyon 1) for making and providing strain KAG146 prior to publication. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors of this manuscript certify that they comply with the ethical guidelines for authorship and publishing in the Journal of Cachexia, Sarcopenia, and Muscle.61
The manuscript does not contain clinical studies or patient data. The use of invertebrate models of human disease is fully compliant with the replacement, reduction, and refinement of animal models and is therefore ethically preferred.
This work was supported by the US NIH‐NIAMS [grant number AR‐054342], the Medical Research Council [grant numbers J500495, K00414X]; and Arthritis Research UK [grant number 19891].
Lehmann, S. , Bass, J. J. , Barratt, T. F. , Ali, M. Z. , and Szewczyk, N. J. (2017) Functional phosphatome requirement for protein homeostasis, networked mitochondria, and sarcomere structure in C. elegans muscle. Journal of Cachexia, Sarcopenia and Muscle, 8: 660–672. doi: 10.1002/jcsm.12196.
References
- 1. United States Bone and Joint Initiative . The Burden of Musculoskeletal Diseases in the United States (BMUS), 3rd ed, Rosemont, IL; 2014. Available at http://www.boneandjointburden.org.
- 2. Petersen KF, Morino K, Alves TC, Kibbey RG, Dufour S, Sono S, et al. Effect of aging on muscle mitochondrial substrate utilization in humans. Proc Natl Acad Sci U S A 2015;112:11330–11334. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Cohen S, Nathan JA, Goldberg AL. Muscle wasting in disease: molecular mechanisms and promising therapies. Nat Rev Drug Discov 2015;14:58–74. [DOI] [PubMed] [Google Scholar]
- 4. Corsi AK, Wightman B, Chalfie M. A Transparent window into biology: a primer on Caenorhabditis elegans . WormBook 2015;1–31, https://doi.org/10.1895/wormbook.1.177.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Moerman DG, Williams BD. Sarcomere assembly in C. elegans muscle. WormBook 2006;1–16, https://doi.org/10.1895/wormbook.1.81.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Brouilly N, Lecroisey C, Martin E, Pierson L, Mariol MC, Qadota H, et al. Ultra‐structural time‐course study in the C. elegans model for Duchenne muscular dystrophy highlights a crucial role for sarcomere‐anchoring structures and sarcolemma integrity in the earliest steps of the muscle degeneration process. Hum Mol Genet 2015;24:6428–6445. [DOI] [PubMed] [Google Scholar]
- 7. Ashrafi K. Obesity and the regulation of fat metabolism. WormBook 2007;1–20, https://doi.org/10.1895/wormbook.1.130.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Herndon LA, Schmeissner PJ, Dudaronek JM, Brown PA, Listner KM, Sakano Y, et al. Stochastic and genetic factors influence tissue‐specific decline in ageing C. elegans . Nature 2002;419:808–814. [DOI] [PubMed] [Google Scholar]
- 9. Higashibata A, Hashizume T, Nemoto K, Higashitani N, Etheridge T, Mori C, et al. Microgravity elicits reproducible alterations in cytoskeletal and metabolic gene and protein expression in space‐flown Caenorhabditis elegans . NPJ Microgravity 2016;2: https://doi.org/10.1038/npjmgrav.2015.22.15022 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Lehmann S, Shephard F, Jacobson LA, Szewczyk NJ. Integrated control of protein degradation in C. elegans muscle. Worm 2012;1:141–150. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Zdinak LA, Greenberg IB, Szewczyk NJ, Barmada SJ, Cardamone‐Rayner M, Hartman JJ, et al. Transgene‐coded chimeric proteins as reporters of intracellular proteolysis: starvation‐induced catabolism of a lacZ fusion protein in muscle cells of Caenorhabditis elegans . J Cell Biochem 1997;67:143–153. [PubMed] [Google Scholar]
- 12. Szewczyk NJ, Peterson BK, Jacobson LA. Activation of Ras and the mitogen‐activated protein kinase pathway promotes protein degradation in muscle cells of Caenorhabditis elegans . Mol Cell Biol 2002;22:4181–4188. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Szewczyk NJ, Peterson BK, Barmada SJ, Parkinson LP, Jacobson LA. Opposed growth factor signals control protein degradation in muscles of Caenorhabditis elegans . EMBO J 2007;26:935–943. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Szewczyk NJ, Jacobson LA. Activated EGL‐15 FGF receptor promotes protein degradation in muscles of Caenorhabditis elegans . EMBO J 2003;22:5058–5067. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Szewczyk NJ, Hartman JJ, Barmada SJ, Jacobson LA. Genetic defects in acetylcholine signalling promote protein degradation in muscle cells of Caenorhabditis elegans . J Cell Sci 2000;113:2003–2010. [DOI] [PubMed] [Google Scholar]
- 16. Etheridge T, Rahman M, Gaffney CJ, Shaw D, Shephard F, Magudia J, et al. The integrin‐adhesome is required to maintain muscle structure, mitochondrial ATP production, and movement forces in Caenorhabditis elegans . FASEB J 2015;29:1235–1246. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Shephard F, Adenle AA, Jacobson LA, Szewczyk NJ. Identification and functional clustering of genes regulating muscle protein degradation from amongst the known C. elegans muscle mutants. PLoS One 2011;6: https://doi.org/10.1371/journal.pone.0024686.e24686 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Etheridge T, Oczypok EA, Lehmann S, Fields BD, Shephard F, Jacobson LA, et al. Calpains mediate integrin attachment complex maintenance of adult muscle in Caenorhabditis elegans . PLoS Genet 2012;8: https://doi.org/10.1371/journal.pgen.1002471.e1002471 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Gaffney CJ, Shephard F, Chu J, Baillie DL, Rose A, Constantin‐Teodosiu D, et al. Degenerin channel activation causes caspase‐mediated protein degradation and mitochondrial dysfunction in adult C. elegans muscle. J Cachexia Sarcopenia Muscle 2016;7:181–192. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Lehmann S, Bass JJ, Szewczyk NJ. Knockdown of the C. elegans kinome identifies kinases required for normal protein homeostasis, mitochondrial network structure, and sarcomere structure in muscle. Cell Commun Signal 2013;11:71, https://doi.org/10.1186/1478‐811X‐11‐71. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Plowman GD, Sudarsanam S, Bingham J, Whyte D, Hunter T. The protein kinases of Caenorhabditis elegans: a model for signal transduction in multicellular organisms. Proc Natl Acad Sci U S A 1999;96:13603–13610. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Stein L, Sternberg P, Durbin R, Thierry‐Mieg J, Spieth J. WormBase: network access to the genome and biology of Caenorhabditis elegans . Nucleic Acids Res 2001;29:82–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 2003;421:231–237. [DOI] [PubMed] [Google Scholar]
- 24. Rual JF, Ceron J, Koreth J, Hao T, Nicot AS, Hirozane‐Kishikawa T, et al. Toward improving Caenorhabditis elegans phenome mapping with an ORFeome‐based RNAi library. Genome Res 2004;14:2162–2168. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25. Lehmann S, Shephard F, Jacobson LA, Szewczyk NJ. Using multiple phenotype assays and epistasis testing to enhance the reliability of RNAi screening and identify regulators of muscle protein degradation. Genes (Basel) 2012;3:686–701. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Mostafavi S, Ray D, Warde‐Farley D, Grouios C, Morris Q. GeneMANIA: a real‐time multiple association network integration algorithm for predicting gene function. Genome Biol 2008;9: https://doi.org/10.1186/gb‐2008‐9‐s1‐s4.S4 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Yang CY, Chang CH, Yu YL, Lin TC, Lee SA, Yen CC, et al. PhosphoPOINT: a comprehensive human kinase interactome and phospho‐protein database. Bioinformatics 2008;24:i14–i20. [DOI] [PubMed] [Google Scholar]
- 28. Shaye DD, Greenwald I. OrthoList: a compendium of C. elegans genes with human orthologs. PLoS One 2011;6: https://doi.org/10.1371/journal.pone.0020085.e20085 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. Manning G. Genomic overview of protein kinases. WormBook 2005;1–19, https://doi.org/10.1895/wormbook.1.60.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30. Smoot ME, Ono K, Ruscheinski J, Wang PL, Ideker T. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics 2011;27:431–432. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31. Burdine RD, Chen EB, Kwok SF, Stern MJ. egl‐17 encodes an invertebrate fibroblast growth factor family member required specifically for sex myoblast migration in Caenorhabditis elegans . Proc Natl Acad Sci U S A 1997;94:2433–2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32. Wissmann A, Ingles J, Mains PE. The Caenorhabditis elegans mel‐11 myosin phosphatase regulatory subunit affects tissue contraction in the somatic gonad and the embryonic epidermis and genetically interacts with the Rac signaling pathway. Dev Biol 1999;209:111–127. [DOI] [PubMed] [Google Scholar]
- 33. Huang da W, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 2009;37:1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34. Drummond MJ, Fry CS, Glynn EL, Dreyer HC, Dhanani S, Timmerman KL, et al. Rapamycin administration in humans blocks the contraction‐induced increase in skeletal muscle protein synthesis. J Physiol 2009;587:1535–1546. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35. Lee I, Lehner B, Crombie C, Wong W, Fraser AG, Marcotte EM. A single gene network accurately predicts phenotypic effects of gene perturbation in Caenorhabditis elegans . Nat Genet 2008;40:181–188. [DOI] [PubMed] [Google Scholar]
- 36. Zhong W, Sternberg PW. Genome‐wide prediction of C. elegans genetic interactions. Science 2006;311:1481–1484. [DOI] [PubMed] [Google Scholar]
- 37. Warde‐Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P, et al. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res 2010;38:W214–W220. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38. Andrabi S, Gjoerup OV, Kean JA, Roberts TM, Schaffhausen B. Protein phosphatase 2A regulates life and death decisions via Akt in a context‐dependent manner. Proc Natl Acad Sci U S A 2007;104:19011–19016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39. Schiaffino S, Mammucari C. Regulation of skeletal muscle growth by the IGF1‐Akt/PKB pathway: insights from genetic models. Skelet Muscle 2011;1:4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40. Andres‐Mateos E, Brinkmeier H, Burks TN, Mejias R, Files DC, Steinberger M, et al. Activation of serum/glucocorticoid‐induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy. EMBO Mol Med 2013;5:80–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41. Anderson NG, Maller JL, Tonks NK, Sturgill TW. Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 1990;343:651–653. [DOI] [PubMed] [Google Scholar]
- 42. Alessi DR, Gomez N, Moorhead G, Lewis T, Keyse SM, Cohen P. Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines. Curr Biol 1995;5:283–295. [DOI] [PubMed] [Google Scholar]
- 43. Sawyer TK, Shakespeare WC, Wang Y, Sundaramoorthi R, Huang WS, Metcalf CA 3rd, et al. Protein phosphorylation and signal transduction modulation: chemistry perspectives for small‐molecule drug discovery. Med Chem 2005;1:293–319. [DOI] [PubMed] [Google Scholar]
- 44. Wang YC, Peterson SE, Loring JF. Protein post‐translational modifications and regulation of pluripotency in human stem cells. Cell Res 2014;24:143–160. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45. Rapundalo ST. Cardiac protein phosphorylation: functional and pathophysiological correlates. Cardiovasc Res 1998;38:559–588. [DOI] [PubMed] [Google Scholar]
- 46. Fabian MA, Biggs WH 3rd, Treiber DK, Atteridge CE, Azimioara MD, Benedetti MG, et al. A small molecule‐kinase interaction map for clinical kinase inhibitors. Nat Biotechnol 2005;23:329–336. [DOI] [PubMed] [Google Scholar]
- 47. Davis MI, Hunt JP, Herrgard S, Ciceri P, Wodicka LM, Pallares G, et al. Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol 2011;29:1046–1051. [DOI] [PubMed] [Google Scholar]
- 48. Fingar DC, Blenis J. Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 2004;23:3151–3171. [DOI] [PubMed] [Google Scholar]
- 49. Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 2011;12:21–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50. Lokireddy S, Kukushkin NV, Goldberg AL. cAMP‐induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins. Proc Natl Acad Sci U S A 2015;112:E7176–E7185. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51. Gross S, Rahal R, Stransky N, Lengauer C, Hoeflich KP. Targeting cancer with kinase inhibitors. J Clin Invest 2015;125:1780–1789. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52. He RJ, Yu ZH, Zhang RY, Zhang ZY. Protein tyrosine phosphatases as potential therapeutic targets. Acta Pharmacol Sin 2014;35:1227–1246. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53. Zhang SH, Liu J, Kobayashi R, Tonks NK. Identification of the cell cycle regulator VCP (p97/CDC48) as a substrate of the band 4.1‐related protein‐tyrosine phosphatase PTPH1. J Biol Chem 1999;274:17806–17812. [DOI] [PubMed] [Google Scholar]
- 54. Piccirillo R, Goldberg AL. The p97/VCP ATPase is critical in muscle atrophy and the accelerated degradation of muscle proteins. EMBO J 2012;31:3334–3350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55. Zhao Y, Adjei AA. The clinical development of MEK inhibitors. Nat Rev Clin Oncol 2014;11:385–400. [DOI] [PubMed] [Google Scholar]
- 56. Laporte J, Kress W, Mandel JL. Diagnosis of X‐linked myotubular myopathy by detection of myotubularin. Ann Neurol 2001;50:42–46. [DOI] [PubMed] [Google Scholar]
- 57. Tosch V, Rohde HM, Tronchere H, Zanoteli E, Monroy N, Kretz C, et al. A novel PtdIns3P and PtdIns(3,5)P2 phosphatase with an inactivating variant in centronuclear myopathy. Hum Mol Genet 2006;15:3098–3106. [DOI] [PubMed] [Google Scholar]
- 58. Romero‐Suarez S, Shen J, Brotto L, Hall T, Mo C, Valdivia HH, et al. Muscle‐specific inositide phosphatase (MIP/MTMR14) is reduced with age and its loss accelerates skeletal muscle aging process by altering calcium homeostasis. Aging (Albany NY) 2010;2:504–513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59. Phillips BE, Williams JP, Gustafsson T, Bouchard C, Rankinen T, Knudsen S, et al. Molecular networks of human muscle adaptation to exercise and age. PLoS Genet 2013;9: https://doi.org/10.1371/journal.pgen.1003389.e1003389 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 60. Narayan KM, Ali MK, Koplan JP. Global noncommunicable diseases—where worlds meet. N Engl J Med 2010;363:1196–1198. [DOI] [PubMed] [Google Scholar]
- 61. von Haehling S, Morley JE, Coats AJS, Anker SD. Ethical guidelines for publishing in the Journal of Cachexia, Sarcopenia and Muscle: update 2015. J Cachexia Sarcopenia Muscle 2015;6:315–316. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.