Skeletal muscle atrophy remains a complication occurring both as a natural response to muscle disuse and as a pathophysiological response to illness such as diabetes mellitus and nerve injury, such as traumatic muscle denervation. The ubiquitin–proteasome system (UPS) is the predominant proteolytic machinery responsible for atrophy of skeletal muscle, and Nedd4-1 (neural precursor cell-expressed developmentally down-regulated 4-1) is one of a series of E3 ubiquitin ligases identified to mediate inactivity-induced muscle wasting. Targets of Nedd4-1 mediated ubiquitination in skeletal muscle remain poorly understood. In the present study, we identified PDLIM7 (PDZ and LIM domain 7, Enigma), a member of the PDZ–LIM family of proteins, as a novel target of Nedd4-1 in skeletal muscle. The PDZ–LIM family of proteins is known to regulate muscle development and function. We show that Nedd4-1 expression in muscle atrophied by denervation is co-incident with a decrease in PDLIM7 and that PDLIM7 protein levels are stabilized in denervated muscle of Nedd4-1 skeletal muscle-specific knockout mice (SMS-KO). Exogenous PDLIM7 and Nedd4-1 transfected into human embryonic kidney (HEK)293 cells co-immunoprecipitate through binding between the PY motif of PDLIM7 and the second and third WW domains of Nedd4-1 and endogenous PDLIM7 and Nedd4-1 interact in the cytoplasm of differentiated C2C12 myotubes, leading to PDLIM7 ubiquitination. These results identify PDLIM7 as a bona fide skeletal muscle substrate of Nedd4-1 and suggest that this interaction may underlie the progression of skeletal muscle atrophy. This offers a novel therapeutic target that could be potentially used to attenuate muscle atrophy.

INTRODUCTION

Cellular ubiquitination is a proteolytic pathway, whereby ubiquitin moieties are covalently attached to specific proteins, often directing them to degradation by the 26S proteasome [1,2]. Specific targets of ubiquitin-mediated proteolysis are largely determined by the E3 ligase involved in the cascade, which selectively binds to substrate proteins via interaction domains and facilitates the attachment of ubiquitin. Four families of E3 ligases exist: HECT (homologous with E6-AP C-terminus) domain, U-box, PHD (plant homoeodomain) finger and RING (really interesting new gene) finger ubiquitin ligases [3,4]. The ubiquitin-mediated proteolytic pathway plays a predominant role in the process of skeletal muscle atrophy resulting from illness or muscle disuse [510] and the E3 ubiquitin ligase Nedd4-1 (neural precursor cell-expressed developmentally down-regulated 4-1) has been shown to increase in models of skeletal muscle atrophy [11,12]. Nedd4-1 is a ubiquitously expressed HECT domain E3 ubiquitin ligase that regulates stability and organization of a variety of proteins through interactions between its WW domain(s) and PY motifs of its substrates [1316].

Nedd4-1 has been previously shown to mediate skeletal muscle atrophy; Nedd4-1 increases in expression in muscle atrophied by inactivity (i.e. denervation, unloading) and Nedd4-1 skeletal muscle-specific knockout (SMS-KO) mice exhibit partial protection against muscle wasting [12,17]. The mechanisms by which Nedd4-1 mediates this process remain unknown; as such, we sought to identify novel protein substrates of Nedd4-1 in muscle. Using a mass spectrophotometry (MS) approach, we subjected Nedd4-1 SMS-KO and wild-type (WT) mice to the tibial nerve transection model of denervation-induced muscle atrophy and pursued proteins that were differentially expressed in the gastrocnemius muscle of the knockout compared with control WT mice. We identified a PY-motif-containing protein, PDLIM7 (PDZ and LIM domain 7, Enigma), that was significantly increased in the Nedd4-1-knockout muscle, suggesting that the absence of Nedd4-1 was affecting (stabilizing) its expression.

PDLIM7 contains an N-terminal PDZ domain, three C-terminal LIM domains and a canonical PY (PPXY) motif [18] and belongs to the PDZ–LIM family of proteins which act as scaffolds, binding actin-associated proteins including tropomyosin and a range of signalling proteins [19]. PDLIM7 associates with, and localizes to, actin filaments in fibroblasts via its PDZ domain [19] and is known to regulate skeletal muscle development in zebrafish; PDLIM7 deficiency induces disorganized tail muscle fibres and muscle contractile dysfunction [20,21].

We found that PDLIM7 and Nedd4-1 co-immunoprecipitate, interact via a WW domain–PY motif interaction and co-localize in differentiated myotubes. We show that increased Nedd4-1 expression in atrophying muscle is coincident with a decrease in PDLIM7, that Nedd4-1-ubiquitinates PDLIM7 and mutation of its PY motif inhibits Nedd4-1-mediated ubiquitination, suggesting that PDLIM7 is a true Nedd4-1 muscle substrate. We propose that PDLIM7 may play a role in the development of Nedd4-1-mediated skeletal muscle atrophy.

EXPERIMENTAL

Muscle atrophy model

Nedd4-1 SMS-KO (myoCre; Nedd4-1flox/flox) and sibling WT control mice (myoCre;Nedd4-1+/+), generated in our laboratory [12], were subjected to the tibial nerve transection model of denervation-induced skeletal muscle atrophy, as previously described [22]. All procedures involving animals were carried out in accordance with the Canadian Council on Animal Care guidelines and were approved by the Animal Research Ethics Board of McMaster University. Briefly, the right tibial nerve was transected in Nedd4-1 SMS-KO and control mice pairs under inhalational halothane anaesthesia, resulting in complete denervation of the gastrocnemius muscle. The contralateral leg served as an internal control in each animal. Mice were maintained under conditions of routine care for 7 days, after which they were killed and the gastrocnemius muscles were harvested from the operated experimental limb and non-operated contralateral control limb. After rapid, atraumatic dissection, the muscle was snap-frozen in liquid nitrogen and stored at–80°C for subsequent protein extraction.

Mass spectrometry

Gastrocnemius muscle (10 mg) was minced and solubilized in 0.5–1.0 ml of lysis buffer (20 mM HEPES, pH 8.0, and 8 M urea). The solution was sonicated and centrifuged at 20000 g for 15 min. The supernatant was collected and reduced with 4.1 DTT mM at 60°C for 20 min and cooled to room temperature. The solution was alkylated by addition of 8.3 mM iodoacetamide to the supernatant at room temperature in the dark for 15 min, diluted with 20 mM HEPES to a concentration of 2 mM urea and subsequently digested with bovine trypsin treated with tosylphenylalanylchloromethane (TPCK)/trypsin overnight at room temperature. Trifluoroacetic acid (TFA) was added to a final concentration of 1% and the solution was left at room temperature for 10 min. The digested peptides were collected in the supernatant after centrifugation at 2000 g for 5 min at room temperature.

The peptides were loaded on a 7-cm pre-column (150 μm i.d.) containing a Kasil frit packed with 3.5 cm, 5 μm Magic C18 100 Å (1 Å=0.1 nm) reversed-phase material (Michrom Bioresources) followed by 3.5 cm Luna 5 μm SCX 100 Å strong cation exchange resin (Phenomenex). The samples were automatically loaded from a 96-well microplate autosampler using the EASY-nLC system (Thermo Fisher Scientific) at 3 μl/min. The pre-column was connected to an 8 cm fused silica analytical column (75 μm i.d.) via a microsplitter tee to which a distal 2.3 kV spray voltage was applied. The analytical column was pulled to a fine electrospray emitter using a laser puller. For the peptide separation on the analytical column, a water/acetonitrile gradient was applied at an effective flow rate of 400 nl/min, controlled by the EASY-nLC. Ammonium acetate salt bumps (8 μl) were applied at ratios of 0%, 10%, 20%, 30%, 40% and 100% of 500 mM ammonium acetate, using the 96-well microplate autosampler at a flow rate of 3 ml/min in a vented-column setup. The eluted peptides were electrosprayed directly into the MS. The MS operated in a cycle of one full-scan mass spectrum (400–1800 m/z), followed by six data-dependent MS/MS spectra at 35% normalized collision energy, which was continuously repeated throughout the entire MudPIT separation. The MS functions and the HPLC solvent gradients were controlled by the Xcalibur data system (Thermo Fisher Scientific). The raw data files were searched using Sequest (Thermo Fisher Scientific) using a parent ion accuracy of 5 p.p.m and a fragment accuracy of 0.5 Da. A fixed modification of carbamidomethyl cysteine and variable modification of oxidized methionine were included in the search. Exclusive unique peptide sequences from hits in the knockout samples and not the WT samples were then entered into the NCBI BLAST algorithm to identify proteins.

Cell culture

All cells were cultured at 37°C in 5% CO2.

For biochemical characterization of the Nedd4-1–PDLIM7 interaction, plasmids expressing WT (human) hNedd4-1 (accession number NM006154) and haemagglutinin (HA)-tagged hPDLIM7 (accession number AF265209 from the SIDNET MGC collection), WT or PY motif hPDLIM7 mutant (whereby the second invariant proline of the PPXY motif was mutated to alanine using a QuikChange Mutagenesis Kit; Stratagene), were transfected into human embryonic kidney (HEK)293 cells grown in Dulbecco's modified Eagle's medium (DMEM), 10% FBS, penicillin/streptomycin (Pen–Strep) using standard CaCl2 transfection. At 48 h post-transfection, cells were harvested and ruptured in lysis buffer (150 mM NaCl, 50 mM HEPES, 1% Triton X-100, 10% glycerol, 1 mM MgCl2 and 1 mM EGTA) with a protease inhibitor cocktail, vortex-mixed for 10 s and incubated on ice for 10 min. Lysate was then cleared by centrifugation at 12000 g for 10 min and the protein concentrations in the supernatant were quantified using a Pierce BCA protein assay.

Protein subcellular localization and characterization of the Nedd4-1–PDLIM7 interaction was determined in the mouse C2C12 skeletal muscle cell line. Differentiation of myoblasts into myotubes was achieved by growing C2C12 cells in DMEM, 10% FBS and Pen–Strep until confluency (48 h), followed by treatment with differentiation medium (2% horse serum and 1% Pen–Strep in DMEM) for 2–7 days. Cells were either immunostained or whole-cell protein lysates were harvested. At serial time points after plating myoblasts and differentiating into myotubes, cells were ruptured in lysis buffer (150 mM NaCl, 50 mM HEPES, 1% Triton X-100, 10% glycerol, 1 mM MgCl2 and 1 mM EGTA) with a protease inhibitor cocktail, vortex-mixed for 10 s and incubated on ice for 10 min. Lysate was then cleared by centrifugation at 12000 g for 10 min and the protein concentrations in the supernatant were quantified using a Pierce BCA protein assay. This produced the soluble fraction of whole-cell lysates containing predominantly cytosolic proteins. Pellets were solubilized in 150 μl of a 1% solution of Triton X-100, sonicated and quantified using a Pierce BCA protein assay to provide the insoluble fraction of whole-cell lysates, containing mostly cytoskeletal proteins, including actin.

To assess the ubiquitination of transfected or endogenous PDLIM7 protein in HEK293 and C2C12 cells respectively, 25 μM 26S proteasome inhibitor MG132 (Sigma–Aldrich) was added to transfected HEK293 cells or C2C12 myoblasts and myotubes, 6 h prior to harvesting lysates.

Western blotting and immunoprecipitation

Gastrocnemius total protein was extracted by homogenizing (Polytron PT 1200E, Kinematica) the muscle in muscle lysis buffer [5 mM Tris/HCl, pH 8.0, 1 mM EDTA, 1 mM EGTA, 1 mM 2-mercaptoethanol, 1% glycerol, PMSF (1 mM) and leupeptin and aprotinin (10 μg/ml each)] three times for 30 s and homogenates were centrifuged at 1600 g for 10 min at 4°C. The supernatant was cleared by centrifuging further for 10 min at 4°C at 10000 g. Protein lysates were quantified using a Pierce (Rockford) BCA Protein Assay Kit and normalized for equal loading. Then 25 μg of the muscle lysate, 60 μg of HEK293 cells transfected with various constructs or 30 μg of C2C12 lysate was separated by SDS/PAGE (8% gel). Western blotting was performed using the following primary antibodies: monoclonal anti-Nedd4-1 antibodies (BD Biosciences) at 1:500 dilution; polyclonal anti-PDLIM7 antibodies (Proteintech), 1:1000 dilution; monoclonal anti-HA antibodies (Covance), 1:1000 dilution; monoclonal anti-ubiquitin antibodies (Covance), 1:1000 dilution; and polyclonal anti-HPRT (hypoxanthine-guanine phosphoribosyltransferase) antibodies (Abcam), 1:1000 dilution. Protein bands were detected with horseradish peroxidase-linked goat anti-rabbit or anti-mouse secondary antibodies (Cell Signaling Technology) used at 1:10 000 dilution. The chemiluminescent signal was acquired using a charge-coupled device (CCD) camera (Bio-Rad Laboratories VersaDoc) and the total signal was quantified using Image Lab software (Bio-Rad Laboratories) with volume analysis.

For co-immunoprecipitation of PDLIM7 and Nedd4-1, equal amounts of lysate from hNedd4-1 or HA–hPDLIM7 WT or PY mutant transfected or untransfected HEK 293 cells were incubated with 25 μl of anti-HA–agarose beads (Abcam) for 6 h at 4°C, or equal amounts of C2C12 myoblast and myotube lysates were incubated with 5 μl of anti-PDLIM7 antibodies or anti-Nedd4-1 antibodies overnight at 4°C. Then 25 μl of Protein G–agarose beads (50% slurry) was then added to the C2C12 lysates and incubated for 1 h at 4°C while spinning. Anti-HA–agarose beads or Protein G–agarose beads were then collected by centrifugation (10000 g, 1 min, 4°C) and washed three times in high-salt HNTG (500 mM NaCl, 20 mM HEPES, pH 7.5, 10% glycerol and 0.1% Triton X-100) and three times in low-salt HNTG (the same, but with 150 mM NaCl) to remove non-specifically bound proteins. The beads were mixed with 35 μl of 1× sample buffer and boiled at 95°C for 5 min to elute proteins. Protein eluate was resolved by SDS/PAGE (8% gel) and transferred on to Protran 0.2 μm nitrocellulose (PerkinElmer) for Western blot analysis.

In vitro binding assays

Human Nedd4-1 WW domains in pQE-30 (Qiagen) were a gift from Daniela Rotin (Hospital for Sick Children, Toronto, Canada). The WW domains were cloned with the following boundaries: WW I 638–760 bp; WW II 1112–1231 bp; WW III 1318–1425 bp; WW IV 1487–1606 bp. His6-tagged proteins were produced in M15 (pREP4) bacteria and purified following the manufacturer's instructions (Qiagen). GST-fusion proteins of hPDLIM7 were produced by PCR of the PY motif-containing region (286–407 bp) and a proline-rich region (415–615 bp; as a negative control), TA cloning into pCR2.1-TOPO (Invitrogen), subsequent cloning into pGEX-5X1 vector and expression in BL21(DE3) pLys S Escherichia coli bacteria according to the manufacturer's protocol (Promega). GST- fusion proteins were bound on glutathione–agarose beads (50% slurry) by incubating for 1 h at 4°C. Proteins were eluted with 30 mM reduced glutathione by incubating for 1 h at 4°C. Eluted GST proteins and bound His6 proteins were quantified and equal amounts of GST–PY (PY motif) and GST–Pro (proline-rich region) hPDLIM7 fusion proteins (300 μM) were incubated with equal amounts of His6-bound WW proteins (500 μM) for 2 h at 4°C. The beads and bound proteins were washed three times with high-salt HNTG and twice with low-salt HNTG. Beads were then mixed with 20 μl of 1× sample buffer and boiled at 95°C for 5 min to elute proteins. Protein eluate was resolved by SDS/PAGE (15% gel) and transferred on to Protran 0.2 μm nitrocellulose and subjected to Western blotting with anti-His5 (Qiagen) and anti-GST antibodies (Sigma).

Immunostaining

C2C12 cells were plated on glass coverslips and immunostained as myoblasts or (differentiated) myotubes. Coverslips were fixed in 3.7% paraformaldehyde, permeabilized in 0.2% Triton X-100 and blocked in 3% BSA in PBS. Coverslips were incubated with primary antibodies for 1 h at room temperature and then were washed in PBS. Secondary antibodies conjugated to fluorophores were added to the coverslips for 45 min at room temperature, followed by Hoechst at a dilution of 1:12000 for 2 min at room temperature. Coverslips were washed in PBS and mounted on slides in mounting medium (Dako). Images were acquired using a point-scanning confocal microscope (LSM 700; Zeiss Canada) and analysed using ZEN software. Primary antibodies used were monoclonal anti-Nedd4-1 at 1:50 dilution [23] and polyclonal anti-PDLIM7 at 1:50 dilution [24]. Secondary antibodies used were Alexa Fluor 568-conjugated goat anti-mouse at 1:1000 dilution and Alexa Fluor 488-conjugated goat anti-rabbit at 1:1000 dilution (Molecular Probes). To detect the actin cytoskeleton, cells were immunostained with rhodamine phalloidin at 1:1000 dilution (Cytoskeleton). For quantification of co-localization, images were analysed with the FIJI coloc2 to determine Mander's correlation coefficients.

Statistical analyses

Continuous data are reported as a means±S.D. and were compared using Student's t test or ANOVA followed by Tukey's post-test analysis, to compare multiple means when appropriate. Statistical significance was assumed if P<0.05.

RESULTS

PDLIM7 skeletal muscle expression is affected by Nedd4-1 in a murine model of denervation-induced muscle atrophy

As increased Nedd4-1 expression is correlated with and mediates the progression of atrophic processes in muscle cells, we sought to identify potential substrates that may be targeted by Nedd4-1 in muscle to elicit its effects. We hypothesized that any potential Nedd4-1 substrates would be protected from ubiquitin-mediated degradation in Nedd4-1-knockout muscle and thus be present in higher levels than in WT muscle. Since Nedd4-1 muscle expression increases upon denervation, we subjected Nedd4-1 SMS-KO (n=2) and control mice (WT) mice (n=2) to the tibial nerve transection model of muscle atrophy and at 1 week post-tibial nerve transection used MS to identify proteins that were differentially expressed in the experimental samples. The predominant peptides identified in both Nedd4-1 SMS-KO and WT samples were from cytoskeleton-associated proteins (such as actin, myosin and tropomyosin) and some were found to be differentially expressed (increased) in the knockout samples relative to WT. We chose to focus on peptides that were not associated with the cytoskeleton and were ‘lower-abundance’ peptides. Dozens of peptides fulfilling these criteria were enriched in the knockout samples compared with the WT and several of these enriched peptides corresponded to proteins that contained PY motifs. In particular, one set of spectra had 10-fold the number of peptides (14% sequence coverage) in the Nedd4-1 SMS-KO muscle compared with the WT samples. These enriched unique peptide sequences were used in a BLAST search and identified the PDZ domain-LIM domain- and PY motif-containing protein PDLIM7 (Figure 1A) which was significantly higher (95% peptide threshold) in the Nedd4-1 SMS-KO denervated muscle compared with the control, suggesting that its expression was influenced by Nedd4-1. mPDLIM7 is a family of proteins with several isoforms. The peptides identified in the MS and used in our subsequent BLAST search correspond to the murine PDLIM7 ‘a’ isoform, [which has the highest degree (94%) of identity to the human PDLIM7 isoform 1, used in our biochemical experiments below] and which contains a conserved PY motif in the linker region between the PDZ and LIM domains (Figure 1A).

PDLIM7 is a PY motif-containing protein whose expression is decreased in denervated atrophic muscle and retained in the absence of Nedd4-1

Figure 1
PDLIM7 is a PY motif-containing protein whose expression is decreased in denervated atrophic muscle and retained in the absence of Nedd4-1

(A) Schematic representation of the PDZ- and LIM-domain-containing protein PDLIM7 shows an N-terminal PDZ domain, three terminal LIM domains in addition to a conserved PY motif, PPXY, the canonical binding site for the Nedd4-1 WW domains. (B) Western blots of Nedd4-1, PDLIM7 and HPRT (loading control) in protein lysates from denervated gastrocnemius muscle (Den) and the contralateral control gastrocnemius (Con) muscle of myoCre;Nedd4-1flox/flox (Nedd4-1 SMS-KO, n=7) and myoCre;Nedd4-1+/+ (littermate control, n=7) mice at 1 week post-tibial nerve transection. Representative blots from four mice (two Nedd4-1 SMS-KO and littermate control pairs) are shown. Lysate from HEK293 cells transfected with PDLIM7 (293 Tx PDLIM7) and fully differentiated C2C12 myotubes (MT) serve as positive controls for PDLIM7 immunoblotting. Nedd4-1 is absent from the mature muscle of Nedd4-1 SMS-KO mice, but trace expression is evident upon denervation due to retained satellite cell Nedd4-1 expression [12]. (C) PDLIM7 protein levels were normalized to the corresponding HPRT levels (lane matched) and PDLIM7 expression is reported as the ratio of protein in denervated (Den) gastrocnemius to contralateral control (Con) muscle. PDLIM7 levels were significantly decreased in denervated gastrocnemius muscle of littermate control mice, but not in Nedd4-1 SMS-KO mice. Data are means±S.E.M.; *P<0.05. Abbreviation: 293 Untx, HEK293 cells untransfected.

Figure 1
PDLIM7 is a PY motif-containing protein whose expression is decreased in denervated atrophic muscle and retained in the absence of Nedd4-1

(A) Schematic representation of the PDZ- and LIM-domain-containing protein PDLIM7 shows an N-terminal PDZ domain, three terminal LIM domains in addition to a conserved PY motif, PPXY, the canonical binding site for the Nedd4-1 WW domains. (B) Western blots of Nedd4-1, PDLIM7 and HPRT (loading control) in protein lysates from denervated gastrocnemius muscle (Den) and the contralateral control gastrocnemius (Con) muscle of myoCre;Nedd4-1flox/flox (Nedd4-1 SMS-KO, n=7) and myoCre;Nedd4-1+/+ (littermate control, n=7) mice at 1 week post-tibial nerve transection. Representative blots from four mice (two Nedd4-1 SMS-KO and littermate control pairs) are shown. Lysate from HEK293 cells transfected with PDLIM7 (293 Tx PDLIM7) and fully differentiated C2C12 myotubes (MT) serve as positive controls for PDLIM7 immunoblotting. Nedd4-1 is absent from the mature muscle of Nedd4-1 SMS-KO mice, but trace expression is evident upon denervation due to retained satellite cell Nedd4-1 expression [12]. (C) PDLIM7 protein levels were normalized to the corresponding HPRT levels (lane matched) and PDLIM7 expression is reported as the ratio of protein in denervated (Den) gastrocnemius to contralateral control (Con) muscle. PDLIM7 levels were significantly decreased in denervated gastrocnemius muscle of littermate control mice, but not in Nedd4-1 SMS-KO mice. Data are means±S.E.M.; *P<0.05. Abbreviation: 293 Untx, HEK293 cells untransfected.

To confirm the MS results, we performed Western blot analysis of protein lysates from denervated and contralateral control gastrocnemius muscle of Nedd4-1 SMS-KO and littermate (WT) control mice (Figure 1B). PDLIM7 protein levels in denervated gastrocnemius muscle of control mice were significantly decreased compared with Nedd4-1 SMS-KO mice (0.51±0.08 compared with 0.98±0.16 respectively; Figure 1C). The decreased levels of PDLIM7 occurred concomitant to increased levels of Nedd4-1 protein (Figure 1B), supporting the notion of PDLIM7 being a novel target of Nedd4-1-mediated degradation.

Nedd4-1 and PDLIM7 interact via a WW domain–PY motif interaction

PDLIM7 contains a PY-motif (PPRY) in its linker region (between the PDZ and LIM domains) that is conserved across mammalian species and adheres to the canonical WW domain-binding motif sequence for Nedd4-1, suggesting that the interaction between the two proteins may occur in a similar manner. To verify that the PDLIM7/Nedd4-1 interaction occurs by WW domain–PY motif binding, we expressed His6-tagged versions of the four WW domains of human Nedd4-1 and incubated equal amounts of the fusion proteins (bound to Ni2+ beads) with soluble purified GST-fusion proteins of regions of hPDLIM7 encompassing the PY motif (GST–PY) or a proline-rich region C-terminal to the PY motif (GST–Pro) as a negative control (Figure 2). We found that the GST–PY consistently bound to WW II and WW III of hNedd4-1, albeit the intensity of binding to WW III appeared much greater. GST–Pro did not bind to any of the His6–WW fusion proteins. These data suggest that the interaction between the two proteins in vivo is mediated in canonical fashion through a PY–WW domain interaction.

hPDLIM7 and hNedd4-1 binding is mediated through a PY motif–WW domain interaction

Figure 2
hPDLIM7 and hNedd4-1 binding is mediated through a PY motif–WW domain interaction

(A) GST-fusion proteins of the area encompassing the PY motif of hPDLIM7 (GST–hPDLIM7 PY) and of a non-PY-containing proline-rich region of hPDLIM7 (GST–hPDLIM7 PRO) as control were generated. Samples of 0.1 μg of soluble GST–PY or GST–Pro were incubated with 0.3 μg of His6-tagged hNedd4—WW I,–WW II,–WW III or–WW IV immobilized on Ni2+nitrilotriacetate beads (His–hNedd4 WW domain I–IV). Following thorough washes of the beads, bound proteins were eluted with sample buffer and separated by SDS/PAGE, and Western blotting was performed with anti-GST and anti-His antibodies, as indicated. Molecular masses are indicated in kDa. Binding of the PDLIM7 PY motif is evident with WW II and WW III domains of Nedd4-1. (B) Sequence alignment of the four WW domains of hNedd4-1 and percentage sequence identities derived using NCBI Protein BLAST. A colon represents an identical match with WW domain III. The XP groove of WW III is in bold.

Figure 2
hPDLIM7 and hNedd4-1 binding is mediated through a PY motif–WW domain interaction

(A) GST-fusion proteins of the area encompassing the PY motif of hPDLIM7 (GST–hPDLIM7 PY) and of a non-PY-containing proline-rich region of hPDLIM7 (GST–hPDLIM7 PRO) as control were generated. Samples of 0.1 μg of soluble GST–PY or GST–Pro were incubated with 0.3 μg of His6-tagged hNedd4—WW I,–WW II,–WW III or–WW IV immobilized on Ni2+nitrilotriacetate beads (His–hNedd4 WW domain I–IV). Following thorough washes of the beads, bound proteins were eluted with sample buffer and separated by SDS/PAGE, and Western blotting was performed with anti-GST and anti-His antibodies, as indicated. Molecular masses are indicated in kDa. Binding of the PDLIM7 PY motif is evident with WW II and WW III domains of Nedd4-1. (B) Sequence alignment of the four WW domains of hNedd4-1 and percentage sequence identities derived using NCBI Protein BLAST. A colon represents an identical match with WW domain III. The XP groove of WW III is in bold.

Nedd4-1-mediated PDLIM7 ubiquitination is dependent on an intact PY motif

To verify that hNedd4-1 and hPDLIM7 binding is mediated by a PY–WW domain interaction in living cells and to determine whether this interaction results in ubiquitination of PDLIM7, cDNA for HA-tagged hPDLIM7 or HA-tagged hPDLIM7 PY motif mutant was co-transfected into HEK293 cells along with hNedd4-1 cDNA. Lysates from these cells were subjected to immunoprecipitation with HA–agarose beads. Immunoprecipitates and cell lysates were separated by SDS/PAGE and probed with anti-PDLIM7, anti-ubiquitin, anti-Nedd4-1 and anti-HPRT (as a loading control) to detect expression of the respective proteins. Transfected WT hPDLIM7 was able to co-immunoprecipitate hNedd4-1, but transfected hPDLIM7 PY mutant was unable to do so, reinforcing the notion that interaction between the two proteins is probably mediated by the WW domain–PY motif binding (Figures 3A and 3B). Co-transfection with HA–hPDLIM7 and hNedd4-1 displayed a notable increase in ubiquitinated PDLIM7 compared with co-transfection with HA–hPDLIM7 PY mutant and hNedd4-1, as was evident by the high-molecular-mass smear present in the former and absent from the latter (Figures 3A and 3C). Furthermore, the addition of the 26S proteasome inhibitor MG132 results in a high-molecular-mass smear of the PDLIM7 band, in keeping with the accumulation of ubiquitinated forms of PDLIM7, when the WT protein is co-expressed with Nedd4-1 and, to a lesser extent, when expressed on its own (Figure 3D). This patterning is not evident with the PY mutant PDLIM7. These data together suggest that the interaction between Nedd4-1 and PDLIM7 results in ubiquitination of PDLIM7 and that PDLIM7 is a bona fide Nedd4-1 substrate. The smaller extent of apparent ubiquitinated PDLIM7 accumulation in the absence of transfected Nedd4-1, is probably due to the presence of other endogenous ubiquitin ligases or possibly low levels of endogenous Nedd4-1.

PDLIM7 ubiquitination is dependent on an intact PY motif

Figure 3
PDLIM7 ubiquitination is dependent on an intact PY motif

(A) HEK293 cell lysates transfected with HA-tagged hPDLIM7, hNedd4-1, HA-tagged hPDLIM7 PY motif mutant (HA–PDLIM7 mPY), HA-tagged hPDLIM7 with Nedd4-1, HA–PDLIM7 mPY with hNedd4-1 or untransfected lysate were subjected to immunoprecipitation with anti-HA-tagged agarose beads. Beads were washed and separated by SDS/PAGE, then subjected to Western blotting with anti-ubiquitin and anti-PDLIM7 antibodies and then stripped and re-probed with anti-Nedd4-1 antibodies. Ubiquitinated PDLIM7 is seen as a high-molecular-mass smear above 63 kDa in the HA-tagged hPDLIM7 and hNedd4-1 transfected lysate only. (B) Aliquots of whole-cell (WC) lysate were subjected to immunoblotting with anti-PDLIM7 and anti-Nedd4-1 antibodies and reprobed with anti-HPRT as a loading control. (C) The ubiquitination signal obtained via CCD camera acquisition in Figure 3A was quantified using ImageLab software (Bio-Rad Laboratories) and normalized to the immunoprecipitated PDLIM7 (lane matched). Co-transfection of hPDLIM7 and hNedd4-1 results in a significant increase in PDLIM7 ubiquitination. (D) Protein lysates of HEK293 transfected and untransfected cells treated with or without 25 μM MG132 for 6 h prior to harvesting, were subjected to immunoblotting with anti-PDLIM7 and anti-HPRT as a loading control. Molecular masses are indicated in kDa; *,& ,#P<0.05.

Figure 3
PDLIM7 ubiquitination is dependent on an intact PY motif

(A) HEK293 cell lysates transfected with HA-tagged hPDLIM7, hNedd4-1, HA-tagged hPDLIM7 PY motif mutant (HA–PDLIM7 mPY), HA-tagged hPDLIM7 with Nedd4-1, HA–PDLIM7 mPY with hNedd4-1 or untransfected lysate were subjected to immunoprecipitation with anti-HA-tagged agarose beads. Beads were washed and separated by SDS/PAGE, then subjected to Western blotting with anti-ubiquitin and anti-PDLIM7 antibodies and then stripped and re-probed with anti-Nedd4-1 antibodies. Ubiquitinated PDLIM7 is seen as a high-molecular-mass smear above 63 kDa in the HA-tagged hPDLIM7 and hNedd4-1 transfected lysate only. (B) Aliquots of whole-cell (WC) lysate were subjected to immunoblotting with anti-PDLIM7 and anti-Nedd4-1 antibodies and reprobed with anti-HPRT as a loading control. (C) The ubiquitination signal obtained via CCD camera acquisition in Figure 3A was quantified using ImageLab software (Bio-Rad Laboratories) and normalized to the immunoprecipitated PDLIM7 (lane matched). Co-transfection of hPDLIM7 and hNedd4-1 results in a significant increase in PDLIM7 ubiquitination. (D) Protein lysates of HEK293 transfected and untransfected cells treated with or without 25 μM MG132 for 6 h prior to harvesting, were subjected to immunoblotting with anti-PDLIM7 and anti-HPRT as a loading control. Molecular masses are indicated in kDa; *,& ,#P<0.05.

Nedd4-1 and PDLIM7 interaction and co-localization in C2C12 muscle cells occurs during myotube differentiation

To verify that PDLIM7 is a Nedd4-1 substrate in muscle, we assessed the subcellular co-localization of the endogenous proteins in C2C12 muscle cells, during differentiation from myoblasts to fully differentiated myotubes, with immunocytology and also evaluated protein binding with co-immunoprecipitation experiments. Using confocal microscopy, PDLIM7 was seen to localize both throughout the cytosol and (to a lesser degree) in association with the actin cytoskeleton in multi-nucleated, fully differentiated myotubes (Figure 4A). Co-localization of PDLIM7 with Nedd4-1 was evident in the myotube cytoplasm upon overlay (Figure 4B). In contrast, in undifferentiated myoblasts, PDLIM7 was predominantly associated with the actin cytoskeleton (Figure 4C), with minimal co-localization with Nedd4-1, whose expression was largely restricted to the cytoplasm (Figure 4D).

Nedd4-1 and PDLIM7 co-localize in muscle cells in vitro

Figure 4
Nedd4-1 and PDLIM7 co-localize in muscle cells in vitro

Immunofluorescence viewed on point-scanning confocal microscope of C2C12 differentiated multinucleated myotubes (A and B) or undifferentiated myoblasts (C and D), with DAPI (blue, to indicate nuclei), PDLIM7 (green) and Nedd4-1 or Rhodamine–Phalloidin (red). Overlay shows merging of the images and co-localization of proteins is reported using Mander's correlation coefficient generated using FIJI coloc2 software. Scale bar=20 μm.

Figure 4
Nedd4-1 and PDLIM7 co-localize in muscle cells in vitro

Immunofluorescence viewed on point-scanning confocal microscope of C2C12 differentiated multinucleated myotubes (A and B) or undifferentiated myoblasts (C and D), with DAPI (blue, to indicate nuclei), PDLIM7 (green) and Nedd4-1 or Rhodamine–Phalloidin (red). Overlay shows merging of the images and co-localization of proteins is reported using Mander's correlation coefficient generated using FIJI coloc2 software. Scale bar=20 μm.

We subsequently performed immunoprecipitation experiments in C2C12 myoblasts differentiated into myotubes over a 7-day time course and similarly found that Nedd4-1 and PDLIM7 co-immunoprecipitated in fully differentiated myotubes, but not in undifferentiated myoblasts (Figure 5A). Furthermore, analysis revealed differential expression of PDLIM7 in the C2C12 muscle cell during the process of differentiation. Although PDLIM7 was highly expressed in the soluble cytosol-rich fraction of whole-cell lysates of myotubes, only trace expression was evident in the myoblasts. In contrast PDLIM7 was evident in the actin-rich insoluble fractions of the cellular lysate in undifferentiated myoblasts, but not in the myotubes (Figure 5B). Together, these results confirm binding between endogenous PDLIM7 and Nedd4-1 in C2C12 cells and suggest that this interaction may regulate the process of muscle differentiation as PDLIM7 translocates, in part, to the cytosol.

PDLIM7 ubiquitination occurs in muscle cells in vitro

Figure 5
PDLIM7 ubiquitination occurs in muscle cells in vitro

(A) C2C12 cells, subjected to a time course of differentiation, were lysed and the soluble portion of whole-cell (W.C.) lysates was subjected to immunoprecipitation with anti-Nedd4-1 antibodies and Protein G–agarose beads. Beads were washed, the immunoprecipitated proteins separated by SDS/PAGE and immunoblotted with anti-PDLIM7 and anti-Nedd4-1 antibodies. Aliquots of soluble lysate were subjected to immunoblotting with anti-PDLIM7, anti-Nedd4-1 and anti-HPRT as a loading control. (B) The insoluble fractions of the same lysates were solubilized in 1% Triton X- 100, sonicated and subjected to immunoblotting with anti-PDLIM7 and anti-actin as a loading control. (C) C2C12 cells were subjected to a time course of differentiation and were treated with or without 25 μM MG132 for 6 h prior to cell lysis. The soluble fraction collected from whole-cell lysate was subjected to immunoprecipitation with anti-PDLIM7 antibody and immunoblotted with anti-ubiquitin and anti-PDLIM7. Soluble lysates were subjected to immunoblotting with anti-PDLIM7 and anti-HPRT as a loading control. Molecular masses are indicated in kDa. Abbreviations: D, days; MB, myoblasts; MT, myotubes)

Figure 5
PDLIM7 ubiquitination occurs in muscle cells in vitro

(A) C2C12 cells, subjected to a time course of differentiation, were lysed and the soluble portion of whole-cell (W.C.) lysates was subjected to immunoprecipitation with anti-Nedd4-1 antibodies and Protein G–agarose beads. Beads were washed, the immunoprecipitated proteins separated by SDS/PAGE and immunoblotted with anti-PDLIM7 and anti-Nedd4-1 antibodies. Aliquots of soluble lysate were subjected to immunoblotting with anti-PDLIM7, anti-Nedd4-1 and anti-HPRT as a loading control. (B) The insoluble fractions of the same lysates were solubilized in 1% Triton X- 100, sonicated and subjected to immunoblotting with anti-PDLIM7 and anti-actin as a loading control. (C) C2C12 cells were subjected to a time course of differentiation and were treated with or without 25 μM MG132 for 6 h prior to cell lysis. The soluble fraction collected from whole-cell lysate was subjected to immunoprecipitation with anti-PDLIM7 antibody and immunoblotted with anti-ubiquitin and anti-PDLIM7. Soluble lysates were subjected to immunoblotting with anti-PDLIM7 and anti-HPRT as a loading control. Molecular masses are indicated in kDa. Abbreviations: D, days; MB, myoblasts; MT, myotubes)

To determine whether PDLIM7 ubiquitination occurs in C2C12 cells and is regulated during differentiation, we subjected C2C12 lysates, differentiated over a 7-day time course and treated with or without the proteasome inhibitor MG132, to immunoprecipitation with anti-PDLIM7 antibodies (Figure 5C). Substantially more PDLIM7 ubiquitination, as indicated by a high-molecular-mass smear on ubiquitin blotting, was evident in early differentiated myotubes (day 3 and day 5) treated with MG132 compared with levels in untreated myotubes. Most significantly, no ubiquitination was evident in undifferentiated myoblasts suggesting that the ubiquitination of PDLIM7 is associated with early stages of myotube development.

DISCUSSION

In the present study, we describe a novel interaction between the E3 ubiquitin ligase Nedd4-1 and the scaffolding protein PDLIM7. Although an interaction between PDLIM7 and Nedd4-1 has not yet been described, it is not entirely unexpected as both proteins are known to associate with and influence the actin cytoskeleton. Overexpression of human Nedd4-1 in yeast has previously been shown to negatively influence the dynamic organization of the actin cytoskeleton through inhibition of actin polymerization and has been suggested to target conserved actin-associated proteins [25]. PDLIM7 has also been previously identified to bind and localize to actin filaments in mammals via interaction with β-tropomyosin, suggesting a putative role in cytoskeleton organization [26].

Furthermore, we and others have previously shown a role for Nedd4-1 in skeletal muscle atrophy [12,17,27] and a role for PDZ–LIM proteins in skeletal muscle organization and maintenance has been well documented. Knockdown of Ldb3/Cypher, a member of the PDZ–LIM family in zebrafish, leads to abnormalities in somite compartmentalization and skeletal muscle organization [28]. In mice, Ldb3- null mutations are embryonic or perinatal lethal, with mice dying from a myopathy characterized by disorganized and fragmented Z lines in skeletal and cardiac muscle [29]. Upon knockdown of PDLIM7 in zebrafish by morpholino antisense oligonucleotides, Camarata et al. [30] show severe defects in growth and tail muscle development, a phenomenon that was suppressed by co-injection with Pdlim7 mRNA. In mice, global loss of PDLIM7 via a gene trap approach resulted in neonatal lethality in the majority of Pdlim7−/− mice, purported to result from unexpected alterations of haemostatic function [31]. Whereas the effect of PDLIM7 deficiency on skeletal muscle was not reported, surviving adult Pdlim7−/− mice exhibited a lower body weight compared with littermate controls despite similar birth weights [31], which may be contributed to by a reduced skeletal muscle mass.

We have shown differential localization of PDLIM7 in developing myotubes ex vivo, as the protein translocates, in part, from the actin cytoskeleton in C2C12 myoblasts to a diffuse cytoplasmic staining in multi-nucleated myotubes. This was further confirmed by Western blot analysis, revealing an increase in cytoplasmic PDLIM7 in myotubes compared with myoblasts. Interestingly, Guy et al. [19] were unable to demonstrate differential protein expression of PDLIM7 (Enigma) in C2C12 myoblasts and myotubes differentiated for 5 days. The reason for this discrepancy is unclear, but may be due to the analysis of a combined soluble and insoluble fraction of cell lysates, which was not clearly reported. Our experiments indicate a definitive increase in cytoplasmic PDLIM7 with myotube formation, suggesting that the translocation of the protein may be associated with the process of myotube development. This finding has not been previously reported; however, Enigma homologue 1 (ENH1/PDLIM5) is an anchoring protein homologous with PDLIM7, which has been shown to promote myogenic genes and initiate C2C12 myotube formation [32].

Evidence of a dynamically localized PDLIM7 is well documented, as PDLIM7 has been shown to bind signalling molecules and direct their subcellular localization. PDLIM7 is known to bind to receptor tyrosine kinases such as the insulin and Ret (rearranged during transfection) receptor, through interaction with its LIM domains and transport them to the actin cytoskeleton providing a possible mechanism for this scaffolding protein to elicit its effects, although the significance of its involvement in these signalling cascades is not fully understood [29,33]. Previously, PDZ–LIM family members have also been recognized to bind key nuclear proteins, tethering them and re-localizing them to the actin cytoskeleton. PDLIM7, specifically, has been shown to bind the transcription factors Tbx4 (T box 4) and Tbx 5, preventing their activation of target genes in the nucleus [21,34]. It is possible then that the differential subcellular localization of PDLIM7 influences myoblast proliferation, differentiation and/or myotube formation through initiation of signalling cascade pathways or alternatively regulating transcriptional activity of myogenic factors and other proteins.

Our experiments suggest that PDLIM7 is a novel substrate to the E3 ubiquitin ligase Nedd4-1, interacting in mature myotubes and binding via its PY motif in canonical fashion to Nedd4-1 WW II and WW III domains, albeit the binding by WW III appears to be much stronger. This is in keeping with previous reports of Nedd4-1 WW III demonstrating the highest affinity binding to several other Nedd4-1 substrates including ENaC and Commissureless [15,35]. This enhanced affinity appears to be due to a broad XP groove that exists in WW III, facilitating contact between the substrate and the domain. The biological significance of this interaction in the context of muscle atrophy has not yet been identified, but can be speculated upon, as we have now shown that Nedd4-1 overexpression correlates with progression of atrophic processes in skeletal muscle, concomitant to decreases in PDLIM7 protein expression. Furthermore, the absence of Nedd4-1 in denervated muscle stabilizes PDLIM7 expression and partially inhibits muscle atrophy. Thus, PDLIM7 appears to be a likely target of Nedd4-1-mediated ubiquitination and subsequent degradation in atrophying muscle. This interaction may inhibit cellular processes initiated by PDLIM7 that are required for maintenance of muscle size, such as binding, translocation and possible recycling of growth factor receptors/signalling molecules to the actin cytoskeleton. Alternatively, PDLIM7 regulation of gene expression by binding and tethering transcription factors such as Tbx5/Tbx4 to the actin cytoskeleton, preventing their nuclear translocation and initiation of transcription, could provide an alternative mechanism for the regulation of muscle size [21]. Deletion of Tbx5 and Tbx4 in mice is known to lead to equivalent disruptions of normal skeletal muscle splitting patterns during development [36]. Further studies analysing myogenic transcription factor expression, for example, in the context of PDLIM7 localization and interaction with Nedd4-1, can shed light on to this possible mechanism.

In conclusion, we have identified a novel Nedd4-1 substrate in muscle, the scaffolding protein PDLIM7, which associates with the actin cytoskeleton and regulates muscle structure and function. The mechanism by which this interaction induces muscle loss remains unknown, yet the present work distinguishes PDLIM7 as a candidate for future studies examining the biological role of Nedd4-1 action in atrophying muscle and as a potential therapeutic target for muscle weakness.

AUTHOR CONTRIBUTION

Jane Batt and Pamela Plant conceived and designed the study. All authors completed experiments. James Bain performed all animal surgery. Robert D'Cruz, Pamel Plant and Jane Batt analysed the data and co-wrote the manuscript.

We gratefully acknowledge Caterina Di Ciano-Oliveira for her technical expertise and assistance, and Darquise Denis and Maribeth Mitri for their technical assistance.

FUNDING

This work was supported by the Canadian Institutes of Health Research [grant number JNM-90959] to J.B.

Abbreviations

     
  • CCD

    charge-coupled device

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • HA

    haemagglutinin

  •  
  • HECT

    homologous with E6-AP C-terminus

  •  
  • HEK

    human embryonic kidney

  •  
  • HPRT

    hypoxanthine-guanine phosphoribosyltransferase

  •  
  • i.d.

    internal diameter

  •  
  • MS

    mass spectrophotometry

  •  
  • Nedd4-1

    neural precursor cell-expressed developmentally down-regulated 4-1

  •  
  • Pen–Strep

    penicillin/streptomycin

  •  
  • SCX

    strong cation exchange

  •  
  • SMS-KO

    skeletal muscle-specific knockout

  •  
  • Tbx

    T box

  •  
  • WT

    wild-type

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Author notes

1

These authors contributed equally to this work.