Muscle RING-Finger Protein MuRF1 as a Connector of Muscle
Energy Metabolism and Protein Synthesis

Suguru Koyama1,2,#, Shoji Hata1,#, Christian Witt3,#, Yasuko Ono1, Stefanie
Lerche3, Koichi Ojima1,4, Tomoki Chiba5, Naoko Doi1,4, Fujiko Kitamura1, Keiji
Tanaka5, Keiko Abe2, Stephanie Witt3, Vladimir Rybin6, Alexander Gasch3,
Thomas Franz6, Siegfried Labeit3, and Hiroyuki Sorimachi1,4,*

1Department of Enzymatic Regulation for Cell Functions (Calpain Project), and 5Department of

Molecular Oncology, Tokyo Metropolitan Institute of Medical Science (Rinshoken), Tokyo 113-8613,
2

Graduate school of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, 3Institut fürAnästhesiologie und Operative Intensivmedizin, Medical Faculty Mannheim, Mannheim 68167,
Germany, 4CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan, 6EMBL,
Proteomic Core Facility, Meyerhofstr. 1, Heidelberg 69012, Germany
Running title: MuRF1 and Muscle Protein Metabolism#These authors contributed equally to this study.
*Corresponding author: Hiroyuki Sorimachi, Ph.D., Department of Enzymatic Regulation for Cell
Functions (Calpain PT), Tokyo Metropolitan Institute of Medical Science (Rinshoken), Tokyo 113-8613,
Tel. +81-3-3823-2181; FAX. +81-3-3823-2359; E-mail: sorimach@rinshoken.or.jp
Abbreviations used: A340, absorbance at 340 nm; BCAA, branched-chain amino acid; CC, coiled
coil domain; CK, creatine kinase; CSA, cross section area; D5-F, D-5-phenylalanine; GMEB1,
glucocorticoid modulatory element binding protein-1; GST, Glutathione-S-transferase; HIBADH,
3-hydroxyisobutyrate dehydrogenase; KO, knock out; MARP1, muscle ankyrin repeat protein 1; MFC,
MuRF family highly conserved domain; μCL, μ-calpain large subunit; PCA, perchloric acid; PMSF,
phenylmethylsulfonyl fluoride; SUMO, small ubiquitin related modifier; TA, tibialis anterior; ttk,
connectin/titin kinase region; WT, wild type.

1

Summary
During pathophysiological muscle-wasting, a family of ubiquitin ligases, including MuRF1(Muscle RING-Finger protein-1), has been proposed to trigger muscle protein degradation via
ubiquitination. Here, we characterized skeletal muscles from wild-type (WT) and MuRF1-KO mice
under amino acid (AA) deprivation as a model for physiological protein degradation, where skeletal
muscles altruistically waste themselves to provide AA to other organs. When WT and MuRF1-KO mice
were fed a diet lacking AA, MuRF1-KO mice were less susceptible to muscle wasting, both formyocardium and skeletal muscles. Under AA depletion, WT mice had reduced muscle protein synthesis,
while MuRF1-KO mice maintained non-physiologically elevated levels of skeletal muscle protein denovo synthesis. Consistent with a role of MuRF1 for muscle protein turnover during starvation, theconcentrations of essential AA, especially branched-chain AA, in the blood plasma significantly
decreased in MuRF1-KO mice under AA deprivation.
To clarify the molecular roles of MuRF1 for muscle metabolism during wasting, we searched for
MuRF1-associated proteins using pull-downs and mass-spectrometry. Muscle-type creatine kinase(M-CK), an essential enzyme for energy metabolism, was identified among the interacting proteins.
Co-expression studies revealed that M-CK interacts with the central regions of MuRF1 including its
B-box domain, and that MuRF1 ubiquitinates M-CK, which triggers the degradation of M-CK via
proteasomes. Consistent with a role of MuRF1 for adjusting CK activities in skeletal muscles byregulating its turnover in vivo, we found that CK levels were significantly higher in the MuRF1-KO
mice

than

in

WT

mice.

Glucocorticoid-modulatory-element-binding

protein-1

and

3-hydroxyisobutyrate-dehydrogenase, previously identified as potential MuRF1 interacting proteins,were also ubiquitinated MuRF1-dependently.
Taken together, these data suggest that MuRF1 multifacetedly participates in the regulation of AA
metabolism, including the control of free AA and their supply to other organs under catabolic conditions,and in the regulation of ATP synthesis under metabolic stress conditions where MuRF1 expression is
induced.

2

Keywords: MuRF1; RING-finger protein; titin/connectin; creatine kinase; muscle atrophy; E3ubiquitin ligases; Glucocorticoid modulatory element binding protein-1; 3-hydroxyisobutyrate
dehydrogenase3

Introduction

Skeletal muscle, the largest organ in vertebrates, is important not only for motor function andsustaining the body shape, but also in metabolic homeostasis.1 For instance, under conditions ofmalnutrition, muscle proteins are rapidly degraded to amino acids, which are supplied through the bloodto other tissues to supplement nutritional sources.1 Therefore, muscle protein degradation, when underappropriate control, is beneficial and necessary for the whole organism to maintain metabolic
homeostasis. Muscle wasting is induced not only by malnutrition, particularly a lack of proteins, butalso by muscle disuse and various pathological conditions, such as diabetes, sepsis, cancer cachexia, andsarcopenia.2-5 In these cases, muscle atrophy and the underlying disease states can exacerbate each otherin vicious cycles. Therefore, the elucidation of the molecular mechanisms of muscle atrophy is also
very important for improving the treatment of certain diseases.
Muscle proteins, especially myofibril proteins, are thought to be degraded by the calpain systemand the ubiquitin(Ub)-proteasome system.6-10 Under conditions promoting muscle atrophy, such as
immobilization, denervation, and fasting, the expression levels of two muscle-specific Ub-ligases,
MuRF1 (muscle RING-finger protein 1) and atrogin-1/MAFbx (muscle atrophy F-box), areup-regulated. 11-14 Furthermore, mice lacking either of these genes show partial resistance to the muscle
atrophy caused by denervation, suggesting that both of these Ub-ligases participate in the induction ofatrophy.11; 15
At present it is unclear how the different E3 ubiquitin ligases that are co-expressed in striatedmuscle tissues (such as MuRF1 and two paralogues, MuRF2 and MuRF3, which form a distinct
conserved subgroup in mammals within the RBCC (RING-finger–B-box–Coiled-coil)/TRIM (Tripartite
motif) protein family16; 17) functionally cooperate, for example, if they target similar or distinct sets ofmuscle proteins, and which muscle proteins could represent rate limiting targets for muscle turnover.

4

Our previous studies on the interactomes recognized by MuRFs demonstrated that MuRF1 and MuRF2(but not MuRF3) interact with the giant muscle proteins, nebulin and connectin/titin.18; 19 Titin is a giant
filamentous protein that forms an intra-sarcomeric filament system by connecting the Z-band and the
M-line of the sarcomere.20; 21 It should be noted that MuRF1 interacts with the titin domain A169,22
which positions MuRF1 immediately N-terminal to the connectin/titin kinase domain (ttk) and close to
the binding site for skeletal-muscle-specific calpain, p94/calpain 3, of another neighboring
connectin/titin molecule that is headed in the other direction.23-25 This raises the possibility that MuRF1
is in the triad of ubiquitin-proteasome, calpain, and ttk signaling pathways at the M-line.18; 26
So far, most molecular insights into the signaling pathways regulated by MuRFs have beenobtained from studies on cultured myocytes.27-33 These studies have suggested that MuRF1 may
ubiquitinate troponin I,29 and may also recognize many other myofibrillar proteins and energy metabolic
enzymes.19

Furthermore, MuRF1 interacts with several proteins involved in SUMOylation andtranscriptional regulation.30; 34 Therefore, MuRF1 may have multiple functions in the regulation ofmuscle metabolism. However, so far, these models need to be tested in vivo. Here, we attempted todetermine MuRF1’s role for muscle metabolism by nutritionally depleting MuRF1 KO mice(constitutively lacking MuRF1 protein) and WT mice for amino acids. Results indicate that bothmuscle-type creatine kinase activities, identified as one of the substrates ubiquitinated by MuRF1, andmuscle protein synthesis are regulated by MuRF1, suggesting that MuRF1 connects the regulation ofenergy metabolism and protein synthesis in muscle.

Results

Identification of muscle-type creatine kinase as a MuRF1-interacting protein. We screened for5

MuRF1-interacting proteins by a GST-pull-down assay using N-terminally GST-tagged recombinant
MuRF1 (GST-MuRF1) and muscle lysates prepared from MuRF1 KO mice (i.e., lacking endogenous
MuRF1). Several proteins were specifically co-precipitated with GST-MuRF1 (Figure 1, lane 4).
Among these proteins, a 40-kDa species (Figure 1, arrowhead) was identified by mass-spectrometry
analysis as M-CK (creatine kinase, muscle type), a muscle enzyme that is critical for energy
metabolism.35; 36 An interaction between MuRF1 and M-CK was suggested previously by yeast
two-hybrid analysis,19 raising the possibility that these two proteins form a complex within thesarcomeric M-line region, the main site of their in vivo localization in myocytes.37; 38
To confirm the interaction between MuRF1 and M-CK in living cells, an immunoprecipitation
analysis was performed. When Flag-M-CK was co-expressed with myc-MuRF1 in COS7 cells, they
were co-precipitated (Figure 2B, lane 7). To examine whether the MuRF1 paralogues, MuRF2 and
MuRF3, interact with M-CK, they were also co-expressed with Flag-M-CK.

The myc-MuRF3

interacted with Flag-M-CK, although this interaction was weaker than that with myc-MuRF1 (Figure
2B, lane 9). No interaction between myc-MuRF2 and Flag-M-CK was detectable under the conditions
used (Figure 2B, lane 8).
MuRF1 ubiquitinates M-CK, leading to M-CK degradation via proteasomes. To determine thesite of MuRF1 that interacts with M-CK, Flag-M-CK and various deletion constructs of myc-MuRF1
were co-expressed in COS7 cells. As shown in Figure 2C, all the constructs containing B-box domain
were co-precipitated with Flag-M-CK (lanes 13-15, 20), while MuRF1 B-box deletion mutant was not(lane 16), indicating that the B-box of MuRF1 is important for contributing to or mediating thisinteraction (Figure 2A). The co-precipitates contained ubiquitinated proteins probably corresponding toubiquitinated M-CK and/or self-ubiquitinated MuRF1 (data not shown).
Since it was not possible to distinguish between self-ubiquitination of MuRF1 andMCK-ubiquitination in the above experiments, we examined whether M-CK was ubiquitinated in a

6

MuRF1-dependent fashion: N-terminally HA-tagged ubiquitin (HA-Ub) was co-expressed with
Flag-M-CK and myc-MuRF1 in the presence of MG132, a proteasome inhibitor. Flag-M-CK was then
immunoprecipitated with anti-Flag agarose after denaturation. Both mono- and oligo-ubiquitinatedFlag-M-CKs were detected with anti-Flag and anti-HA antibodies when myc-MuRF1 was co-expressed(Figures 3A and 3B, lane 9). When the procedures above were performed in the absence of MG132,
ubiquitinated Flag-M-CK was almost undetectable (Figures 3A and B, lane 3).
Deletion of RING domain of MuRF1 abolished ubiquitination of Flag-M-CK (Figures 3A and 3B,lanes 4 and 10), even though the expression levels of the proteins were in similar levels (Figure 3C, lanes
3, 4, 9, and 10).

Under the same conditions, myc-MuRF2 and myc-MuRF3 also ubiquitinatedFlag-M-CK (Figures 3A and 3B, lanes 11 and 12), although the levels of these expressed proteins andubiquitination varied.
Taken together, these data from COS7 cells indicate that MuRF1’s B-box is essential forinteraction between M-CK and MuRF1. Furthermore, this interaction is coupled to the ubiquitination of
M-CK. Finally, MG132-sensitivity indicates its role for proteasome-dependent degradation. Further
studies are needed to clarify whether MuRF2 and MuRF3 can also function as Ub-ligases for M-CK invivo (the analysis of MuRF2 has been hampered by the existence of at least two isoforms, see ref 30).
MuRF1 also ubiquitinates GMEB1 and HIBADH. Previously, we have identified glucocorticoid
modulatory element binding protein-1 (GMEB1) and 3-hydroxyisobutyrate dehydrogenase (HIBADH)
as interacting molecules of MuRF1.19; 30 To investigate whether GMEB1 and HIBADH are alsoubiquitinated by MuRF1, they were co-expressed with MuRF1 in COS7 cells. As shown in Figure 3D,
co-expression of GMEB1 or HIBADH and MuRF1, but not MuRF1ΔRING, showed ubiquitionation
signals significantly in the presence of MG132 (lanes 5 and 11). These results indicate that GMEB1 andHIBADH are also targets of MuRF1-dependent proteasome-mediated degradation.
MuRF1 is up-regulated under amino acid deprivation. To investigate the physiological relevance7

of M-CK’s ubiquitination by MuRF1, we compared WT and MuRF1 KO mice under conditions
provoking muscle atrophy. MuRF1 is reported to be up-regulated under all muscle atrophy-inducing
conditions examined so far.11-14 In this study, mice were fed only 10% glucose solution and waterinstead of normal food for one week, to induce muscle atrophy by stimulating protein turnover. Since
this condition provides mice with carbohydrates freely, it is much milder for mice than starvation,
providing more focus on muscle protein turnover with less affection of total body energy homeostasis.
Consistently, body weights of mice under this condition were about 90 % of the initial state on day 7(Figure 4A), whereas they decreased to almost 80% in 3 days under starvation conditions (data notshown). We call this feeding condition the “amino acid deprivation (–AA) condition”. Under the –AA
condition, the weight and cross-sectional area (CSA) of the TA fibers of WT mice became significantly
smaller (p < 0.05) than those of control WT mice fed normal food (Figures 4B and 4C), confirming thewasting of muscle under the –AA condition. Also, under this condition, the mRNA levels of MuRF1
and atrogin-1/MAFbx were elevated (Figure 4D).
When MuRF1 KO mice were fed under the same –AA condition, the weight and CSA of their TA
fibers were significantly larger (p < 0.05) than those of the –AA WT mice (Figures 4B and 4C). Thus,
MuRF1 plays a critical role in the muscle atrophy induced by the –AA condition, as it does in othermuscle-wasting conditions.11-14 Atrogin-1/MAFbx was upregulated similarly in WT and MuRF1 KO
mice, suggesting that MuRF1 and MAFbx are regulated independently, and, furthermore, that elevatedatrogin-1 expression were unable to induce atrophy in the MuRF1 KO skeletal muscles (Figure 4D).
Similar as in skeletal muscle, MuRF1 deficient myocardium tended to be also protected from wastingunder the –AA feeding protocol (Figures 4E and 6B).
MuRF1 KO alleviates decrease of creatine kinase levels under the –AA condition. Next, thephysiological significance of the induction of MuRF1 under AA starvation as well as MuRF1-M-CK
interaction was investigated by comparing the expression of M-CK in the MuRF1 KO and WT mice. To

8quantify CK, the CK activity in the presence of excess phosphocreatine was measured. In skeletal
muscle, there are two isoforms of CK, muscle-type (M-CK) and mitochondria-type (Mi-CK). Although
this assay cannot distinguish the activities of these two isoforms, M-CK predominates in skeletal
muscle.39 Therefore, the measured activity in skeletal muscle is considered to reflect the quantity of
M-CK. Under normal feeding conditions, the level of CK in WT and MuRF1 KO mice did not differ
significantly. In contrast, under the –AA condition, in which MuRF1 was induced in WT, the CK levelin the MuRF1 KO mice was significantly higher (p < 0.05) than in WT mice (161% of the level in –AA
WT) (Figure 5). These results demonstrate that M-CK activity is down-regulated by MuRF1 in vivo
under muscle-wasting conditions such as AA starvation, in which MuRF1 is up-regulated.
Concentrations of amino acids in the blood plasma are significantly decreased in –AA MuRF1 KO
mice. When insufficient amounts of amino acids are supplied in the diet, the degradation of muscle
proteins can alternatively provide amino acids to other organs via the bloodstream.1 Therefore, weinvestigated whether the disruption of MuRF1 affects the concentrations of amino acids in the bloodplasma. Under normal feeding conditions, no difference in any of the plasma amino acid concentrations
was detected between WT and MuRF1 KO mice (Table 1). Under the –AA feeding protocol, the WT
mice showed a significant increase in the total and non-essential amino acid concentrations (Table 1, p <0.05), but not in the levels of circulating essential amino acids. In contrast, in the –AA MuRF1 KO mice,the total amino acid serum concentrations did not change significantly, but the level of essential aminoacids decreased (in comparison with both WT and MuRF1 KO mice; see Table 1, p < 0.05). In particular,
the concentrations of branched-chain amino acids (BCAA), which are very important for protein
turnover and the energy homeostasis of muscle,1; 40 were lower in the –AA MuRF1 KO mice than in the–AA WT mice (Table 1, p < 0.05). Therefore, under the –AA condition, the bloodstream is depleted ofessential amino acids, especially BCAA, in MuRF1 KO mice, probably due at least in part to impaired
MuRF1-mediated protein turnover.

9Comparison of protein synthesis in starved WT and MuRF1 KO mice implicates MuRF1 as an
inhibitor of de novo muscle protein synthesis. Depletion of amino acids from the blood stream might becaused by impaired protein degradation. Alternatively, altered muscle protein synthesis might also
affect serum amino acid levels. Therefore, we compared de novo skeletal muscle protein synthesis in
WT and MuRF1 KO mice by the flooding bolus injection method, using deuterium-labeled
D-5-phenylalanine (D5-F) as a tracer. Significant incorporation of D5-F was not observed in 7-day –AA
mice (data not shown), consistent with general deprivation of muscle protein synthesis after lack ofessential amino acids. Depriving mice only for two to four days for amino acids resulted in more
moderate weight losses (Figures 6A and 6B). Therefore, we injected D5-F after 48 hours amino-acid
deprivation, and sacrificed the mice after 96 hours to determine fractional muscle protein synthesis rates.
Under these conditions, about two-fold higher incorporation of D5-F into total TCA-insoluble
quadriceps skeletal muscle proteins was detected in MuRF1 KO mice when compared to WT quadriceps(Figure 6C). These data implicate MuRF1 as a negative regulator of muscle protein synthesis under
metabolic stress situations so that consumption of BCAA amino acids reservoir is confined.

Discussion

In this study, we identified M-CK as a target of MuRF1, i.e., M-CK interacts with and is
ubiquitinated by MuRF1. Mice lacking MuRF1 showed resistance to the down-regulation of M-CK
when fed an aproteinogenic diet, but the concentrations of essential amino acids in their bloodstream
decreased. These results strongly suggest that MuRF1 is involved in the homeostatic regulation ofenergy and amino acid metabolism, possibly through the degradation of M-CK, under muscle
atrophy-inducing conditions, such as malnutrition, as shown here (Figure 7). This is the first report that10

identifies a bona fide in vivo target of MuRF1, M-CK.
M-CK is a critical enzyme for energy metabolism that reversibly produces phosphocreatine andATP.35; 36 Therefore, the down-regulation of M-CK by protein degradation is predicted to suppress
energy consumption by muscle.

Under malnutrition conditions, skeletal muscle, as the largest

ATP-consuming organ, must down-regulate its energy consumption. Mechanistically, this could beachieved by the degradation of M-CK following its ubiquitination by MuRF1. Some other metabolic
enzymes, such as adenylate kinase and aldolase A, are also potential substrates for MuRF1, because they
have been found to interact with MuRF1 baits in the yeast two-hybrid system.19 Together, these data andours suggest the novel hypothesis that MuRF1 is an energy homeostasis regulator for muscle, althoughfurther studies are required to determine the precise roles of MuRF1 in the regulation of energy
homeostasis.
Skeletal muscle provides the largest protein reservoir in vertebrates, and under malnutrition
conditions the degradation of skeletal muscle proteins provides amino acids for other tissues, via the
bloodstream. In the present study, MuRF1 was shown to be important for maintaining physiologicallevels of essential amino acids, especially BCAA, in the serum when dietary protein was lacking.
Consistent with this, HIBADH, one of key enzymes involved in the Val catabolic pathway,41; 42 is
ubiquitinated by MuRF1. This most likely suppresses BCAA consumption as a source for energy
catabolism, and thus can explain the elevated free BCAA levels. These findings suggest in turn that
MuRF1 is involved in the regulation of amino acid homeostasis. One of the mechanisms could be that
MuRF1 marks muscle proteins for degradation, including M-CK, troponin I,29 and other myofibrillar
proteins, like telethonin/T-cap, myotilin, and connectin/titin.19 While previous studies have focused on
the roles of MuRF1 and atrogin for muscle protein degradation, here we also tested if MuRF1
participates in the control of protein synthesis. Indeed, about two-fold elevated protein synthesis foundin –AA MuRF1 KO mice demonstrated that MuRF1 is required to safeguard BCAA amino acid serum11levels also by inhibiting muscle protein synthesis. These findings implicate the coupling of synergistic
mechanisms, protein degradation and synthesis, by MuRF1. Moreover, degradation of GMEB1, atranscriptional regulator in response to changes in cellular glucocorticoid levels, is regulated by MuRF1,
suggesting that protein synthesis is also modulated by MuRF1 at the level of transcription as suggested
previously.30 The relative contributions of the stimulation of muscle protein degradation and theinhibition/regulation of protein synthesis by MuRF1 represent one of the important issues to beaddressed by future studies.
Our study showed some functional redundancy of MuRF paralogues in vitro. This is the firstevidence that MuRF2 and 3 also have ubiquitin ligase activity. This finding is consistent withobservations that some myofibrillar proteins are still ubiquitinated in MuRF1 KO mice19 and that
MuRF1 KO mice have a subtle phenotype under normal conditions.11; 19 However, the ubiquitin ligase
activity of MuRF2 and 3 did not completely compensate for the functions of MuRF1 undermuscle-wasting conditions in this study. This may be attributable to differences in the tissue-specificity
and subcellular localizations of the MuRFs.18; 28; 31-33 In adult skeletal muscle, transcripts of MuRF1 are
more abundant than those of MuRF2 and 3,18 suggesting MuRF1 plays the central roles over otherparalogues in skeletal muscle.
The B-box region of MuRF1 was identified as a binding site for M-CK (see Figure 2). The
importance of the B-box domain in other RBCC/TRIM proteins has been highlighted in previous
reports.43-45 Consistent with this, the B-box regions are highly conserved among MuRFs, with about
80% identity (Figure 2A). MuRF3 is, however, more similar to MuRF1 in this region than is MuRF2,
which may explain the distinct binding affinities of MuRFs for M-CK.
During the preparation of this manuscript, Zhao et al. reported the ubiquitination of M-CK by
MuRF1 in vitro.46 Interestingly, they showed that only the oxidized form, one of two forms of M-CK, issusceptible to ubiquitination by MuRF1. In our results, relatively small amounts of M-CK were12

ubiquitinated by MuRF1 in COS7 cells (see Figure 3A), and it is possible that the reduced form of
M-CK predominated in COS7 cells, with only a small proportion in the oxidized form. Since theoxidized form of M-CK is less active than the reduced form,46 it is reasonable to selectively degrade theoxidized form as a source of amino acids under malnutrition conditions.
In conclusion, our investigation in vivo using MuRF1 KO mice demonstrated two distinct andimportant functions of MuRF1 in skeletal muscle: as a modulator for energy homeostasis by regulating
M-CK via ubiquitination, and as a supplier of BCAA amino acids to other tissues by regulating muscle
protein turnover. Intriguingly, in addition to regulation of protein degradation, we also found evidence
that MuRF1 negatively regulates protein synthesis. Thus, MuRF1 may function as a potent regulator of
muscle turnover by controlling both degradation and synthesis (Figure 7). The regulation of MuRF1
activity in a clinical setting, for example, by using some as-yet-unknown inhibitor, might contribute toimproved treatments for pathological muscle wasting.

Materials and Methods
Expression Plasmids
Human MuRF1, MuRF2, M-CK, and mouse MuRF3 were amplified by PCR using Pfu DNA
polymerase (Stratagene) and a skeletal muscle cDNA library (Clontech), and the sequences wereverified by DNA sequencing. The cDNA fragments encoding MuRF1-RING and MuRF1-MFC (aminoacids 66-340, 101-340, respectively, see Figure 2) were also generated by using PCR amplification.
These cDNAs were inserted into the pGEX-6X vector containing an N-terminal GST-tag (GEHealthcare) or pcDNA3.1 containing an N-terminal Flag- or myc-tag47 (generous gifts from Dr. Tatsuya
Maeda). MuRF1-C.C2 and MuRF1-C.C1 (amino acids 1-137, 1-225, respectively, see Figure 2) wereconstructed by inserting BamHI and HindIII fragments of pcDNA3.1-N-myc MuRF1 into the pSRD
vector,48 respectively.
GST Pull-down experiment
GST-MuRF1 or GST was expressed in Escherichia coli BL21(DE3) and purified withglutathione-Sepharose 4B (GE Healthcare), according to the manufacturer’s instructions. Frozen

13sections from tibialis anterior (TA) muscles of MuRF1 KO mice (see “Mouse experiments” below) were
homogenized in 10 volumes (w/v) of lysis buffer (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA,
1 mM PMSF, 10 μg/ml aprotinin, 1% Nonidet P-40) for 1 hr. After ultra-centrifugation (20,000 x g for
20 min), the supernatant (1 mg of total protein amounts) was incubated for 3 hr with 3 μg of GST-tagged
MuRF1 immobilized on glutathione-Sepharose 4B. After three washes with lysis buffer, the precipitates
were boiled with SDS-PAGE sample buffer, and the solubilized proteins were electrophoresed andsilver-stained without adding glutaraldehyde (Silver Stain Kit, Protein, GE Healthcare).
Identification of MuRF1-Interacting ProteinsProtein bands above obtained were excised and subjected to in-gel digestion followed by tandem
mass-spectrometry analysis, as described previously.49-51 Trypsin digestion was performed overnight at
37oC. The peptides were extracted from the gel using 5% trifluoroacetic acid and 50% acetonitrile. The
eluate was deionized using a ZipTip (Millipore), recovered in matrix solution (2.5 mg/ml
α-cyano-4-hydroxycinnamic acid, 50% acetonitrile, and 0.1% trifluoroacetic acid), and spotted onto amatrix-assisted laser desorption ionization (MALDI) target plate. After the spots were air-dried,
MALDI-time-of-flight (TOF) mass-spectrometry was performed using an ABI 4800 analyzer. The
protein was identified using the Mascot program (Matrix Science).
Immunoprecipitation and detection of ubiquitination
COS7 cells were transiently transfected with expression vector(s) by electroporation, as previously
described.48 Forty-eight hours after transfection, the cells were treated with 25 μM MG132 added inculture medium for 2.5 hr. The cells were then harvested and lysed by sonication in TNE buffer (10 mM
Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM EDTA) supplemented with 1% Nonidet P-40 andinhibitors (10 μg/ml Aprotinin, 0.1 mM PMSF, and 25 μM MG132). The lysates were subjected toultracentrifugation at 20,000 x g for 30 min. The protein concentrations of the supernatants were
determined by the RC-DC protein assay (BioRad), and equal amounts of protein were used for
immunoprecipitation with an anti-myc antibody or anti-Flag (M2)-agarose (Sigma), as previously
described 47. For detection of ubiquitination status, the same procedures were performed with TNE
buffer supplemented with 0.1% SDS.
CK activity Assay
The amount of creatine kinase was represented by its activity measured, as previously described.52
Briefly, TA muscles (ca. 1 mg) were homogenized in 1,000 volume (v/w) of 26 mM Tris/Cl (pH 8.0), 0.3
M sucrose, 1% NP-40, and 20 mM 2-mercaptoethanol, aliquots of 50 μl were incubated with 1 ml of 10
mM Tris/Cl (pH 7.4), 130 mM KCl, 1 mM MgCl2, 2 mM AMP, 50 μM diadenosine pentaphosphate, 5mM glucose, 0.7 mM NADP, 1.5 mM ADP, 9 mM phosphocreatine, and 1.3 and 0.5 units of hexokinase14

and glucose-6-phosphate dehydrogenase, respectively, at 25oC for 20 min, and A340 was measured. All
the reagents were purchased from Sigma. The values were normalized to the amount of protein, which
was determined by the RC-DC protein assay (BioRad).
Mouse experiments
All procedures using experimental animals were approved by the Experimental Animal Care and Use
Committee of the Tokyo Metropolitan Institute of Medical Science. For inactivation of MuRF1, thegene was targeted by homologous recombination (described more in detail in ref. 53). WT and MuRF1
KO mice (7-weeks old) were divided into two groups: mice in the control group were fed normal foodad libitum, and mice in the experimental group were fed only 10% glucose solution and water, for one
week. Whole blood collected from the heart with heparin was spun (10,000 x g, 5 min), and thesupernatant was used for the measurement of amino acid concentrations in the blood plasma (measured
by SRL Inc., Japan). The cross-sectional area of the TA muscle fibers was determined as the field area
divided by the number of myofibers in Hematoxylin-Eosin-stained transverse sections.
For the determination of D5-F incorporation, mice were fed with 10% glucose and water for 4 days.
Forty-eight hr after the start of starvation, 50 µmol/100g D5-F were injected intraperitoneally. After
another 48 hr, mice were sacrificed for the following analysis. The bound amino acids were basicallydetermined as described before.54; 55 The quadriceps was powdered with a mortar and homogenized in 8
volumes of 90 mM perchloric acid (PCA). Homogenates were centrifuged and the insoluble materials
were washed in 4 volumes 0.2 M PCA. The pellets were resuspended in 0.3 M NaOH and lysed for 2h at
37°C. Proteins were precipitated with 2 M PCA overnight at 4°C. The protein pellet was hydrolyzed in
6M HCl at 110°C for 24h. HCl was removed by evaporation. The pellet was resuspended in H2O andpurified with a cation-exchange column (AG50 W-X8, BioRad). The purified amino acids were eluted
from the column by 4 M NH4OH, dried, and resuspended in methanol. Relative content of D5-F to F
was determined by mass-spectrometry by comparing ion counts at m/Z of 171.24 (D5-F) and 166.24 (F)using D5-F and F as standards (Sigma).
RT-PCR
Total RNA was extracted from frozen muscles with TRIzol reagent (Invitrogen). First-strand cDNA wassynthesized with a First-strand cDNA synthesis kit (GE Healthcare), and used as a template for RT-PCR.
RT-PCR was performed using ExTaq DNA polymerase (TaKaRa) and the following primers:
5’-gactcctgcagagtgaccaag-3’ and 5’-cttctacaatgctcttgatgagc-3’ for MuRF1; 5’-gaatagcatccagatcagcag-3’
and5’-gagaatgtggcagtgtttgca-3’

foratrogin-1/MAFbx;

5’-tcaaaggtcacaacaccatccagg-3’ for μCL.

155’-gaattggaataccacattttacgagg-3’

andAcknowledgements
We would like to thank Dr Kenji Takehana (Ajinomoto Co., Inc.), Dr Ichiro Matsumoto and Dr Takumi
Misaka (The University of Tokyo), Dr Tatsuya Maeda (The University of Tokyo), Dr Shigeo Murata(Rinshoken), Dr Choji Taya (Rinshoken), and Dr Hiromichi Yonekawa (Rinshoken), and all of ourlaboratory members, for experimental support and valuable discussions. This work was supported inpart by JSPS Research Fellowships for Young Scientists 1811508 (to S.K.), by MEXT.KAKENHI
18076007 (to H.S.), by JSPS.KAKENHI 18770124 (to Y.O.), 18700392 (to K.O.), 17780115 (to S.H.),
18380085 and 19658057 (to H.S.), by the Sasagawa Scientific Research Grant from The Japan Science
Society (to S.H. and K.O.), by a Research Grant (17A-10) for Nervous and Mental Disorders from the
Ministry of Health, Labor and Welfare, by a Takeda Science Foundation research grant (to H.S.), by the
DFG (La668/10-1 and 11-1 to S.L.; Wi3278/2-1 to C.W.), and by the NAR-initiative of theLandesstiftung Baden-Württemberg.

16

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21

FIGURE LEGENDS
Figure 1. Identification of MuRF1-interacting proteins. Recombinant GST-MuRF1 (lanes 4 and 5) or
GST (lanes 2 and 3) was incubated with (lanes 1, 2, and 4) or without (lanes 3 and 5) TA muscle lysate of
MuRF1 KO mice, and pulled down by glutathione Sepharose beads. Precipitates were subjected to
SDS-PAGE, and visualized with silver staining. Among the MuRF1-specific bands shown in lane 4, theband indicated by an open arrowhead was identified as creatine kinase muscle type (M-CK).
Figure 2. Interactions between MuRFs and M-CK. (A) Schematic structures of MuRFs.16; 18 The
regions of MuRF1 deletion mutants used in Figs 2C and 3 are represented by bars. (B) FLAG-tagged
M-CK and myc-tagged MuRF1, 2, or 3 (lanes 2 and 7, 3 and 8, or 4 and 9, respectively), or a mock
vector (lanes 1 and 6) or myc-tagged muscle ankyrin-repeat protein 1 (lanes 5 and 10) as negative
controls were co-expressed in COS7 cells. Cell lysates were immunoprecipitated (IP) with an anti-myc
antibody, and the precipitated proteins were subjected to western blot analysis using anti-FLAG or
anti-myc antibodies. (C) Myc-tagged MuRF1 deletion mutants and FLAG-tagged M-CK (lanes 5-8,
13-16, 18, and 20) or a mock vector (lanes 1-4, 9-12, 17, and 19) were co-expressed in COS7 cells.
Immunoprecipitation and western blots were performed as in B using the antibodies indicated. (B) and(C): Arrowheads and asterisks indicate M-CK and non-specific bands (immunoglobulin originatingfrom the antibody used for IP), respectively.
Figure 3. MuRF1 ubiquitinated M-CK, GMEB1, and HIBADH in COS7 cells. (A-C) HA-tagged
ubiquitin was co-expressed in COS7 cells with FLAG-tagged M-CK (lanes 2-6 and 8-12) andmyc-tagged MuRF1, MuRF1ΔRING, MuRF2, or MuRF3 (lanes 3 and 9, 4 and 10, 5 and 11, or 6 and 12,
respectively) or mock vector (lanes 2 and 8), or with mock vector alone (lanes 1 and 7). Cells wereseparated into two groups: after 48 hr of transfection, one group was treated with 25 mM MG132 for 2.5
hr (lanes 1-6), but the other was not (lanes 7-12). The cells were then harvested and lysed. Lysates wereanalyzed by western blots using an anti-myc antibody (C) or immunoprecipitated with an anti-FLAG
antibody under denaturing conditions (A, B). Precipitated proteins were subjected to western blot
analysis using anti-FLAG (A) or anti-HA antibodies (B). Open and closed arrowheads, open and closed
arrows indicate Ub-M-CK and M-CK, MuRF1-3 and MuRF1 ΔRING, respectively. (D) HA-tagged
ubiquitin, FLAG-tagged GMEB1 (lanes 1-6) or HIBADH (lanes 7-12), and myc-tagged MuRF1 (lanes 2,5, 8, 11) or MuRF1ΔRING (lanes 3, 6, 9, 12) or mock vector (lanes 1, 4, 7, 10) were co-expressed,
pulled-down, and tested for ubiqitination similarly as (A-C).
Figure 4. MuRF1 inactivation protects from muscle atrophy induced by –AA deprivation. (A) The bodyweight of each mouse was measured and represented as a percentage to the average body weight of the22

mice on day 0. Error bars indicate the average ± standard deviation (n = 3). (B) The weight of thetibialis anterior (TA) and gastrocnemius (GC) of WT or KO mouse under normal or –AA conditions.
Error bars indicate the average ± standard deviation (n = 3 x 2 (right and left legs)). (C) Cross-sectional
area of the TA muscle was measured and is shown as the average ± standard deviation (n = 2). (D)Amounts of mRNA for MuRF1, atrogin-1/MAFbx, and μCL as a standard were examined by RT-PCR.
Data are shown for two mice in each group. (E) The weight of the heart of each mouse was measured
and presented as ratios to the body weight on day 7. Error bars indicate the average ± standard deviation(n = 3). * indicates significant difference (p < 0.05) between WT –AA and KO –AA.
Figure 5. MuRF1 inactivation maintains creatine kinase activity during –AA deprivation. Significantly
higher creatine kinase level in MuRF1 KO than in WT mice under the –AA condition. The amount ofcreatine kinase (CK) in the TA muscles was measured as the activity in the presence of excess
phosphocreatine, and normalized to the amount of protein in the TA. Data are represented by arbitrary
units. *, p < 0.05.
Figure 6. Muscle protein synthesis in WT and MuRF1 KO skeletal muscles during amino aciddeprivation. Body (A) and muscle (B) weight loss during the 4-day amino acid deprivation. MuRF1
KO muscles are more resistant to the –AA condition. (C) After 4-day amino acid deprivation MuRF1
KO quadriceps muscles maintain two-fold higher protein synthesis rates when compared to WT mice.
Figure 7. Critical multiple roles of MuRF1 in muscle cells under malnutrition conditions. Signals formuscle atrophy such as malnutrition, immobilization, steroid administration, denervation, etc., lead tothe up-regulation of MuRF1, which ubiquitinates muscle proteins, including M-CK, GMEB1, HIBADH,
troponin I, and other myofibrillar proteins. At the same time, MuRF1 suppresses synthesis of muscle
proteins. The degradation of M-CK results in the suppression of energy consumption, while thedegradation of muscle proteins (including M-CK) generates free amino acids, which are transferred toother organs through the bloodstream. The degradation of GMEB1 and HIBADH alter expression ofgenes governed by glucocorticoid-responsive elements (GME) and Val catabolism, respectively. All
these functions are part of the emergency response of muscles to maintain homeostasis of the whole
body.

23

Figure 1
GST
musclelysate ＋
1

(kDa)
97
66
45
30
20

＋
2

－
3

GSTMuRF1＋
4－
5

Figure 2

A1

340 (aar)
1066

100 109

143

178

226

254

RING-Finger domain

310

MuRF family highly
conserved domain

MuRF1
80.7

88.2

78.8

61.1

41.4

50.0

55.0

B-box domain

MuRF2
82.5

91.2

81.8

66.7

51.7

42.9

MuRF3
MuRF1 ∆MFC

100

acidic region

137

MuRF1 B-box

102

142

MuRF1 ∆B-box

101

143

I.P: anti-myc(MuRFs)

MuRF1
MuRF2
MuRF3
cont

input

MuRF1
MuRF2
MuRF3
cont

B

coiled coil domain
C-terminus subgroup
One Signature box(COS domain)

MuRF1 ∆CC

myc-MuRFs

31.8 (% identity
to MuRF1)

1 2 3 4 5 6 7 8 9 10(kDa) 9766
45

I.B: anti-myc (MuRFs)*

45
I.B: anti-FLAG (M-CK)C

myc-MuRF1
FLAG-M-CK

I.P: anti-FLAG (M-CK)
I.P:
antiFLAG
input (M-CK)

WT
∆MFC
∆CC
∆B-boxWT∆MFC∆CC∆B-boxWT∆MFC∆CC∆B-boxWT∆MFC∆CC∆B-box

input

−

+

−

+

30

∆MFC

GFP-MuRF1 + + + +-B-boxFLAG-M-CK − + − +1718 19 20(kDa) 45

20

∆CC

30

(kDa)
45

1 2 3 4 5 6 7 8 9 10 1112 131415 16

I.B: anti-myc (MuRF1)

* WT

GFP
-B-boxI.B: anti-GFP (MuRF1)
45

45

I.B: anti-FLAG (M-CK)

I.B: anti-FLAG (M-CK)Figure 3
1
W
1 T
∆
2 RIN
W
3 T G
W
T

1
W
1 T
∆
2 RIN
W
3 T G
W
T

+MG132

myc-MuRF- - FLAG- M-CK - + + + + + - + + + + +1 2 3 4 5 6 7 8 9 10 11 12
(kDa)
A
97
(Ub)n-CK
66
Ub-M-CK
M-CK
43
I.B: anti-FLAG (M-CK)

I.P:
anti-FLAG(M-CK)

B(Ub)n-CK

97
66

Ub-M-CK
I.B: anti-HA (Ub)
97
66
43

C

MuRF2
MuRF3
MuRF1
MuRF1 ∆RING

30
I.B: anti-myc (MuRFs)

G
W
T
∆R
IN
G

W
T
∆R
IN

NG
W
T
∆R
I

W
T
∆R
I

NG

D
myc-MuRF1 +
+ +
MG132
7 8 9 10 11 12(kDa)

myc-MuRF1 MG132 - - - + + +1 2 3 4 5 6(Ub)n-GMEB1
(kDa)

97
66

(Ub)n-HIBADH

Ub-HIBADH
45
Ub-GMEB1
HIBADH
GMEB1
I.P:
I.P:
I.B: anti-FLAG (GMEB1)
I.B:
anti-FLAG(HIBADH)anti-FLAG
anti-FLAG(GMEB1)(HIBADH)97(Ub)n-GMEB1

9766(Ub)n-HIBADH

Ub-GMEB1

45

Ub-HIBADH

97
I.B: anti-HA (Ub)45

I.B: anti-HA (Ub)
MuRF1 WTMuRF1 ∆RING

30
I.B: anti-myc (MuRF1)

45

MuRF1 WTMuRF1 ∆RING

30
I.B: anti-myc (MuRF1)

Figure 4
A
relative body weight (% to day 0)

110

100

90
KO cont
WT cont
KO -AA
WT -AA

1

2

3

B

*
120
100

KO cont
WT cont
KO -AA
WT -AA

5

6

7

C
3

*

2.5
2

1.5

80

*

60

1

0.5
0

GC

E
6

5
0
nt

T

control -AA

co

µCL

control -AA

Watrogin1/
MAFbx

KO

KO

MuRF1

WT

heart/body weight (%)

D

W

TA

KO

0

T

co

nt

40

co
KO nt
-A
W .A
T
-A
.A

muscle weight (mg)

4
day

co
KO nt
-A
W .A
T
-A
.A

0

cross section area (x 103 µm2)

80

T

W

KO

T

W

KO

A

-A

A

-A

nt

co

nt

co

CK amount (arbitrary unit)

Figure 5

*

2

1.5

1

0.5

0

Figure 6A
relative body weight (% to day 0)

110

WT -AA
MuRF1 KO -AA

100

90

80
0

1

2
Day

B

3

C
4

140

MuRF1 KO
incorporation (%)

muscle weight (mg)

4

WT
120

100

3

2

1
80
0

0
Heart

Quad.

MuRF1
KO

WT

Figure 7

Signals for muscle atrophymuscleproteins

proteinsynthesis

ADP
M-CK
MuRF1

muscle Ubproteins

GMEB-1

HIBADHUb

HIBADHamino acidcatabolism

GMEB-1Ub

amino acids
Gene

GME

nucleusBlood stream

ATP
M-CK
Ub
energy consumption

transcriptional
regulation

Other organs

Musclecells