Actin dynamics are implicated in various cellular processes, not only through the regulation of cytoskeletal organization, but also via the control of gene expression. In the present study we show that the Src family kinase substrate p130Cas (Cas is Crk-associated substrate) influences actin remodelling and concomitant muscle-specific gene expression, thereby regulating myogenic differentiation. In C2C12 myoblasts, silencing of p130Cas expression by RNA interference impaired F-actin (filamentous actin) formation and nuclear localization of the SRF (serum-response factor) co-activator MAL (megakaryocytic acute leukaemia) following the induction of myogenic differentiation. Consequently, formation of multinucleated myotubes was abolished. Re-introduction of wild-type p130Cas, but not its phosphorylation-defective mutant, into p130Cas-knockdown myoblasts restored F-actin assembly, MAL nuclear localization and myotube formation. Depletion of the adhesion molecule integrin β3, a key regulator of myogenic differentiation as well as actin cytoskeletal organization, attenuated p130Cas phosphorylation and MAL nuclear localization during C2C12 differentiation. Moreover, knockdown of p130Cas led to the activation of the F-actin-severing protein cofilin. The introduction of a dominant-negative mutant of cofilin into p130Cas-knockdown myoblasts restored muscle-specific gene expression and myotube formation. The results of the present study suggest that p130Cas phosphorylation, mediated by integrin β3, facilitates cofilin inactivation and promotes myogenic differentiation through modulating actin cytoskeleton remodelling.

INTRODUCTION

The differentiation of muscle precursor cells or myoblasts into myotubes, which plays a central role in skeletal myogenesis, is a complex multi-step process involving cell-cycle withdrawal, muscle-specific gene expression, and cell alignment and fusion [1,2]. These steps are orchestrated by various signal transduction molecules, such as MAPKs (mitogen-activated protein kinases) and Akt, cell-cycle regulators and transcription factors [35]. The expression of muscle-specific genes, including α-actin, MyoD (myogenic differentiation factor D), MHC (myosin heavy chain) and myogenin, is dependent upon SRF (serum-response factor) binding to the CArG box motif of the SRE (serum-response element) [69]. Extracellular stimuli, such as serum and growth factors, induce SRF transcriptional activation via two distinct signalling cascades [8,10]. One pathway is through complex formation of SRF with the TCF (ternary complex factor) family of SRF cofactors activated by ERK (extracellular-signal-regulated kinase). The other involves modulation of actin cytoskeletal dynamics and concomitant nuclear translocation of another SRF cofactor MAL (megakaryocytic acute leukaemia), also known as MKL1 and MRTF-A. Monomeric actin [G-actin (globular actin)] directly binds to MAL and mitigates SRF transcriptional activity by sequestering MAL from the nucleus [11]. At the onset of myogenic differentiation, both ERK down-regulation and MAL/SRF activity are required for the expression of skeletal-muscle-specific genes [12,13].

The adaptor molecule p130Cas (Cas is Crk-associated substrate), which localizes to focal adhesions, is involved in various cellular processes, including migration, survival, transformation and invasion [14]. It is composed of multiple functional domains, including an N-terminal SH3 (Src homology 3) domain (termed CasSH3), a central substrate domain (termed CasSD) and a C-terminal Src-binding domain (termed CasSBD). CasSH3 interacts with various proteins, including FAK (focal adhesion kinase). CasSD comprises 15 YxxP motifs that are the major sites of tyrosine phosphorylation by Src family kinases. We previously reported that mechanical extension of CasSD potentiates its susceptibility to phosphorylation, which provides docking sites for Crk, Nck and Ship2, and facilitates downstream signalling [1517]. p130Cas−/− mice are embryonic lethal at 11.5–12.5 dpc (days post-coitum), exhibiting systemic congestion and growth retardation [18]. The hearts of p130Cas−/− mice exhibited disorganized myofibrils with disrupted Z discs, indicating significant roles for p130Cas in cardiac myocyte development. However, the mechanism of how p130Cas regulates skeletal muscle differentiation remains elusive.

The integrins, which form functional heterodimers of α and β subunits, constitute a family of transmembrane receptors for ECM (extracellular matrix) proteins. They play a major role in cell–matrix adhesion and adhesion-initiated signal transduction [19]. Integrin β3 has been reported to be involved in myogenic differentiation [20]. We find that phosphorylation of p130Cas, which is integrin-β3-dependent, plays an essential role in myotube formation via modulation of the actin cytoskeleton and muscle-specific gene expression. During the initial phase of myogenic differentiation, cells become elongated and aligned, involving a decrease in stress fibre formation [2]. On the other hand, integrin β3/p130Cas signalling, which positively regulates F-actin (filamentous actin) formation [18,19,21,22], is up-regulated in differentiating myoblasts and is required for muscle-specific gene expression. In the present study, we demonstrate that the integrin β3/p130Cas axis restricts the amount of G-actin available and concomitantly facilitates the nuclear localization of MAL through suppression of cofilin activity. These findings provide novel insights into the regulatory mechanisms behind myogenesis.

EXPERIMENTAL

Cell culture and retroviral infection

C2C12 myoblasts were cultured in GM [growth medium; DMEM (Dulbecco's modified Eagle's medium, Nissui Pharmaceutical) supplemented with 10% FBS (fetal bovine serum)]. To induce differentiation of C2C12 myoblasts, the culture medium was replaced with DM (differentiation medium; DMEM containing 2% horse serum). Retroviral infection was performed as described previously [23,24]. Depending on the retroviral vectors used, infected cell populations were selected in either hygromycin (300 μg/ml) or puromycin (4 μg/ml) for 3 days, or neomycin (G418; 400 μg/ml) for 14 days.

Antibodies

Anti-phospho-p130Cas (Tyr165) polyclonal (Cell Signaling Technology), anti-p130Cas polyclonal (αCas3) [17], anti-myogenin monoclonal (BD Biosciences), anti-α-actin monoclonal (5C5, Sigma), anti-integrin β3 polyclonal (Cell Signaling Technology), anti-phospho-cofilin (S3) polyclonal (Cell Signaling Technology), anti-cofilin polyclonal (Cell Signaling Technology), anti-MHC monoclonal (MY32, Sigma), anti-MAL polyclonal (H-140, Santa Cruz Biotechnology), anti-ILK (integrin-linked kinase) rabbit monoclonal (EP1593Y, Merck Millipore), anti-ILK mouse monoclonal (65.1.9, Upstate Biotechnology) and anti-phospho-paxillin (Tyr118) polyclonal (Cell Signaling Technology) antibodies were used for immunoblot, immunoprecipitation and immunofluorescence analyses.

Plasmids

To generate retroviral vectors pSuper-p130Cas and pSuper-integrin β3, mouse p130Cas target sequence (5′-GCATGACATCTACCAAGTT-3′), mouse integrin β3 target sequence (5′-CAGCTCATTGTTGATGCTT-3′) [25], mouse TESK (testicular protein kinase) 1 target sequence (5′-GTGCCTGCTTTCCGAACTTTG3′) or mouse TESK2 target sequence (5′-CCTGAGTTCTTGCATCA-3′) were cloned into pSuper-puro vector (Oligoengine) respectively. The wild-type or phosphorylation-defective mutant 15YF [17] of mouse p130Cas was subcloned into pBabe-hygro vector. To protect from shRNA (short hairpin RNA)-mediated knockdown, these pBabe-p130Cas-hygro constructs contained several nucleotide mismatch mutations (5′-GCATGATATATATCAAGTT-3′) compared with the p130Cas shRNA sequence without amino acid substitution. A full-length mouse Cfl (cofilin) cDNA was obtained from NIH 3T3 cDNA pools and cloned into a pBabe-HA vector. pBabe-HA-cofilin-S3E encoded a mutant cofilin in which the serine residue at amino acid position 3 was replaced with glutamic acid. The bacterial expression vector for GST (glutathione transferase)–cofilin was constructed by subcloning wild-type cofilin into pGEX-4T-1. pMX-IlkS343D-neo was generated as described previously [24].

Immunoblot analysis

C2C12 cells were solubilized with an ice-cold cell lysis buffer [50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% SDS, 10 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, protease inhibitor cocktail (Roche) and 1 mM DTT (dithiothreitol)], sonicated and centrifuged at 20000 g for 20 min. The supernatants were then subjected to SDS/PAGE.

Luciferase assay

pSRE–Luc (Luc is luciferase) was obtained from Clontech Laboratories. The control plasmid pRL (Renilla luciferase)-TK (thymidine kinase) was obtained from Toyobo. The reporter construct α-actin–Luc was generated by subcloning the 1.2 kbp fragment encompassing the promoter region (−1200 to +10) of the mouse skeletal muscle Acta (α-actin) gene into the pGL3-basic vector. The luciferase activity was scaled to the average value of the cells cultured in GM set at 1. Data represent the means±S.D. for three independent assays. A P value of <0.02 was considered statistically significant and is indicated by an asterisk on Figures.

Immunofluorescence

Immunofluorescence staining was performed as described previously [26]. Cells were fixed with 4% PFA (paraformaldehyde), permabilized with 0.1% Triton X-100, and then blocked with 2% BSA in cytoskeletal stabilizing buffer [137 mM NaCl, 5 mM KCl, 1.1 mM sodium phosphate (dibasic), 0.4 mM monopotassium phosphate, 4 mM sodium bicarbonate, 2 mM MgCl2, 5.5 mM glucose, 2 mM EGTA and 5 mM Pipes (pH 6.1)]. Alexa Fluor® 488- or 546-conjugated goat anti-mouse and anti-rabbit IgG (Molecular Probes) were used as secondary antibodies. DAPI (4′,6-diamidino-2-phenylindole, Vector Laboratories), Alexa Fluor® 488–phalloidin (Molecular Probes) and Alexa Fluor® 594–DNase I (Molecular Probes) were used to stain the nucleus, F-actin and G-actin respectively. Cells were observed with an inverted microscope (Nikon Eclipse TE2000-S) or a confocal microscope comprising an inverted microscope (IX81, Olympus) equipped with a spinning disc confocal unit (PerkinElmer Life Sciences), a water-immersion objective [NA (numerical aperture) 1.20, 60×; PLFL Olympus] and an EMCCD (electron multiplying charge-coupled device) camera (C9100-50, Hamamatsu Photonics) or a confocal microscope (A1Rsi, Nikon) equipped with oil-immersion (NA 1.40, 100×; Plan Apochromat VC, Nikon) and air (NA 0.95, 40×; Plan Apochromat, Nikon) objectives. Acquired images were analysed off-line with the public domain ImageJ program (version 1.45f; NIH).

Ratio of F-actin to G-actin

F-actin and G-actin were visualized with Alexa Fluor® 488–phalloidin and Alexa Fluor® 594–DNase I respectively. The intensity ratio of F-actin to G-actin was calculated for each pixel and the calculated values were averaged within individual cell regions. Data represent the means±S.D. for more than ten independent cells.

In-gel kinase assay

An in-gel kinase assay of ILK was performed as described previously with slight modifications [27]. Briefly, C2C12 cells were solubilized with an ice-cold cell lysis buffer [50 mM Tris/HCl (pH 7.4), 1% Nonidet P40, 0.5% SDS, 50 mM NaCl, 5 mM EDTA, 0.1 mM sodium orthovanadate, 5 mM 2-glycerophosphate, 2.5 mM sodium fluoride and a protease inhibitor cocktail], and immunoprecipitated with an anti-ILK antibody (Upstate Biotechnology). The precipitated complexes were subjected to SDS/PAGE using a gel containing 0.25 mg/ml GST–cofilin. After electrophoresis, the gel was incubated in a kinase assay buffer [20 mM Hepes (pH 7.4), 10 mM MgCl2, 50 mM NaCl, 100 mM sodium orthovanadate, 20 mM 2-glycerophosphate, 1 mM DTT and 100 μM ATP] at 30°C for 30 min after denaturation and renaturation, and then transferred on to a PVDF membrane. The level of GST–cofilin phosphorylation was assessed by Western blotting using an anti-phospho-cofilin (S3) antibody.

RT (reverse transcription)–PCR

Total RNA extraction and cDNA preparation were performed as described previously [23]. RT–PCR analysis of hILK-S343D (the S343D mutant of human ILK) and ACTB (β-actin) was carried out using the following primer pairs: hILK (762 bp), 5′-GCAGTCGCCGTTCGCCT-3′ (forward) and 5′-AGTAGGATGAGGAGCAGGT-3′ (reverse), ACTB (mouse β-actin) (313 bp), 5′-ATGGATGACGATATCGCTGCGC-3′ (forward) and 5′-GCAGCACAGGGTGCTCCTCA-3′ (reverse).

RESULTS

p130Cas phosphorylation is required for C2C12 myotube formation

To examine whether p130Cas was involved in myogenic differentiation, we silenced p130Cas expression in C2C12 myoblasts using a retroviral system. Expression of p130Cas was efficiently attenuated by shRNA-mediated gene knockdown at least up to day 4 after replacement of GM with DM (Figure 1A, top panel). Control-shRNA-expressing cells became elongated and aligned by day 2, and formation of multinucleated myotubes was observed on day 4 after medium replacement with DM (Figure 1B, top panels). In contrast, p130Cas-knockdown myoblasts (CasKD cells) did not form MHC-positive multinucleated myotubes by day 4, although cell elongation and alignment was observed by day 2 (Figure 1B, bottom panels). Expression of skeletal-muscle-specific proteins, including skeletal muscle α-actin, MHC and myogenin, increased with the period of time in culture with DM, but was hardly induced in CasKD cells (Figures 1A and 1B). These results indicate the pivotal role for p130Cas in myogenic differentiation.

p130Cas is required for myotube formation of C2C12 cells

Figure 1
p130Cas is required for myotube formation of C2C12 cells

(A and B) C2C12 myoblasts were infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses. (A) Cells were cultured in GM and then switched to DM. Cells were then solubilized on the indicated days and the expression levels of p130Cas and skeletal-muscle-specific proteins were evaluated by immunoblot analysis. An anti-tubulin blot provided a loading control. (B) Cells cultured in DM were fixed with 4% PFA on the indicated days and subjected to anti-MHC immunostaining (red) and DAPI staining (blue). Images were obtained with an inverted microscope (Nikon Eclipse TE2000-S). Scale bar=50 μm. (C) C2C12 myoblasts cultured in GM were solubilized and phosphorylation levels of p130Cas (p-p130Cas) were evaluated by immunoblot analysis. (DF) C2C12 myoblasts were co-infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses, together with the shRNA-resistant form of wild-type p130Cas- or mutant p130Cas15YF-expressing retroviruses. (D) Cells cultured in GM were solubilized and the expression and phosphorylation levels of p130Cas were evaluated by immunoblot analysis. (E) Cells were cultured in GM and then switched to DM. Cells were then solubilized on the indicated days and the expression of skeletal-muscle-specific proteins was evaluated by immunoblot analysis. (F) Cells were fixed with 4% PFA 4 days after medium replacement with DM and subjected to anti-MHC immunostaining (red) and DAPI staining (blue). Scale bar=50 μm. Images were obtained with a confocal microscope (A1R) equipped with an objective (40×). vec, vector; WT, wild-type.

Figure 1
p130Cas is required for myotube formation of C2C12 cells

(A and B) C2C12 myoblasts were infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses. (A) Cells were cultured in GM and then switched to DM. Cells were then solubilized on the indicated days and the expression levels of p130Cas and skeletal-muscle-specific proteins were evaluated by immunoblot analysis. An anti-tubulin blot provided a loading control. (B) Cells cultured in DM were fixed with 4% PFA on the indicated days and subjected to anti-MHC immunostaining (red) and DAPI staining (blue). Images were obtained with an inverted microscope (Nikon Eclipse TE2000-S). Scale bar=50 μm. (C) C2C12 myoblasts cultured in GM were solubilized and phosphorylation levels of p130Cas (p-p130Cas) were evaluated by immunoblot analysis. (DF) C2C12 myoblasts were co-infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses, together with the shRNA-resistant form of wild-type p130Cas- or mutant p130Cas15YF-expressing retroviruses. (D) Cells cultured in GM were solubilized and the expression and phosphorylation levels of p130Cas were evaluated by immunoblot analysis. (E) Cells were cultured in GM and then switched to DM. Cells were then solubilized on the indicated days and the expression of skeletal-muscle-specific proteins was evaluated by immunoblot analysis. (F) Cells were fixed with 4% PFA 4 days after medium replacement with DM and subjected to anti-MHC immunostaining (red) and DAPI staining (blue). Scale bar=50 μm. Images were obtained with a confocal microscope (A1R) equipped with an objective (40×). vec, vector; WT, wild-type.

Phosphorylation of p130Cas was increased during differentiation of C2C12 myoblasts (Figure 1C), as reported previously [28]. To address whether p130Cas phosphorylation was required for myogenic differentiation, the shRNA-resistant form of p130Cas, either wild-type (CasWT) or the phosphorylation-defective mutant (Cas15YF) [17], was introduced into CasKD cells. We confirmed that the expression level of exogenous p130Cas (CasWT or Cas15YF) was comparable with that of endogenous p130Cas (Figure 1D). As shown in Figures 1(E) and 1(F), myotube formation and the expression of muscle-specific proteins were restored in wild-type p130Cas-expressing cells (CasWT cells), but not in p130Cas15YF-expressing cells (Cas15YF cells). These results indicate that p130Cas phosphorylation is required for the differentiation of C2C12 myoblasts.

p130Cas is involved in the regulation of MAL localization

Since SRF regulates the expression of skeletal-muscle-specific genes [69], we tested whether p130Cas modulated SRF transcriptional activity through the use of a luciferase assay. We found that the decrease in SRF activity after the replacement of GM with DM was significantly greater in CasKD cells when compared with control cells (Figure 2A, left-hand panel). The activity of the skeletal muscle ACTA promoter, which contains SRE(s), was remarkably increased after medium replacement with DM (Figure 2A, right-hand panel). However, activation of the skeletal muscle ACTA promoter by medium replacement in CasKD cells was insignificant (Figure 2A, right-hand panel).

p130Cas regulates nuclear localization of MAL during myogenic differentiation

Figure 2
p130Cas regulates nuclear localization of MAL during myogenic differentiation

(AC) C2C12 myoblasts were infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses. (D) C2C12 myoblasts were co-infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses, together with the shRNA-resistant form of wild-type p130Cas- or mutant p130Cas15YF-expressing retroviruses. (A) Cells were transfected with SRE–Luc (left-hand panel) or ACTA promoter–Luc (right) reporter plasmid, together with pRL-TK (internal control). At 1 day after transfection, cells were kept in GM or switched to DM 1 day before analysis, then lysed and subjected to a luciferase assay. Values were normalized with the mean values of the cells cultured in GM set at 1. Data represent the means±S.D. for three independent assays. *P<0.02 (unpaired Student's t test.). (BD) Cells were cultured in GM or switched to DM 1 day before analysis and then subjected to anti-MAL immunostaining (green, B and D; red, C) and DAPI staining (blue). (B and D) Images were obtained with a confocal microscope (A1R) equipped with an oil-immersion objective (100×). Scale bar=20 μm. (C) The Z-stack images with 0.2 μm-intervals were obtained using a confocal microscope (Spinning disc) equipped with a water-immersion objective (60×). Projected images along the z-axis and XZ-section images along the yellow lines are shown. Plots of MAL (red lines) and DAPI (blue lines) intensities along dotted lines in the XZ-section images are also shown. Intensities were normalized by the maximum values in the individual plots.

Figure 2
p130Cas regulates nuclear localization of MAL during myogenic differentiation

(AC) C2C12 myoblasts were infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses. (D) C2C12 myoblasts were co-infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses, together with the shRNA-resistant form of wild-type p130Cas- or mutant p130Cas15YF-expressing retroviruses. (A) Cells were transfected with SRE–Luc (left-hand panel) or ACTA promoter–Luc (right) reporter plasmid, together with pRL-TK (internal control). At 1 day after transfection, cells were kept in GM or switched to DM 1 day before analysis, then lysed and subjected to a luciferase assay. Values were normalized with the mean values of the cells cultured in GM set at 1. Data represent the means±S.D. for three independent assays. *P<0.02 (unpaired Student's t test.). (BD) Cells were cultured in GM or switched to DM 1 day before analysis and then subjected to anti-MAL immunostaining (green, B and D; red, C) and DAPI staining (blue). (B and D) Images were obtained with a confocal microscope (A1R) equipped with an oil-immersion objective (100×). Scale bar=20 μm. (C) The Z-stack images with 0.2 μm-intervals were obtained using a confocal microscope (Spinning disc) equipped with a water-immersion objective (60×). Projected images along the z-axis and XZ-section images along the yellow lines are shown. Plots of MAL (red lines) and DAPI (blue lines) intensities along dotted lines in the XZ-section images are also shown. Intensities were normalized by the maximum values in the individual plots.

At the onset of differentiation, MAL/SRF activity is required for expression of skeletal-muscle-specific genes [13]. We therefore examined whether knockdown of p130Cas altered the subcellular localization of MAL in C2C12 myoblasts. When cells were cultured in GM, the distribution of MAL was diffuse in the cytoplasm and nuclei of both control and CasKD cells (Figure 2B, top panels). MAL was similarly distributed in the cytoplasm and nuclei after medium replacement with DM in control cells; however, it was eliminated from nuclei after medium change in CasKD cells (Figure 2B, bottom panels, and Figure 2C). We next tested whether p130Cas phosphorylation modulated the localization of MAL. The nuclear localization of MAL, which was diminished in CasKD cells, was restored by expression of CasWT, but not Cas15YF (Figure 2D), indicating that p130Cas phosphorylation is involved in the regulation of MAL localization.

p130Cas participates in the regulation of actin remodelling in myoblasts

It has been reported that MAL translocation is regulated by G-actin [11,29]. Since the actin cytoskeleton is disorganized in p130Cas-deficient MEFs (mouse embryonic fibroblasts) [18,21], we examined whether p130Cas regulated MAL localization in differentiating C2C12 myoblasts through its action on the actin cytoskeleton. Stress fibres appeared to be less developed in CasKD cells compared with control cells when cultured in GM (Figure 3A, top panels); however, the ratio of F- to G-actin was comparable between these cells (Figure 3B, top panels, and Figure 3C). After medium replacement with DM, stress fibre formation and the F-actin/G-actin ratio were decreased in both control and CasKD cells when compared with those cultured in GM (Figures 3A–3C). Notably, both stress fibre formation and the F-actin/G-actin ratio were significantly decreased in CasKD cells compared with control cells (Figures 3A and 3B, bottom panels, Figure 3C, and Supplementary Figures S1A–S1C at http://www.BiochemJ.org/bj/445/bj4450323add.htm). Re-introduction of CasWT, but not Cas15YF, restored actin stress fibre formation in CasKD cells under both culture conditions, i.e. DM and GM (Figure 3D and Supplementary Figure S1D). The decrease in the ratio of F-actin to G-actin after medium replacement with DM was moderated by the expression of CasWT, but not Cas15YF (Figures 3E and 3F). These results suggest that p130Cas phosphorylation plays a role in the regulation of actin remodelling and thereby regulates MAL nuclear localization in differentiating C2C12 myoblasts.

p130Cas regulates actin remodelling in myoblasts

Figure 3
p130Cas regulates actin remodelling in myoblasts

C2C12 myoblasts were cultured in GM or switched to DM 1 day before analysis. (AC) Cells were infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses. (DF) Cells were co-infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses, together with either vector- or shRNA-resistant form of wild-type p130Cas- or mutant p130Cas15YF-expressing retroviruses. (A and D) Cells stained for F-actin. Scale bar=20 μm. (B and E) Ratio images of F-actin to G-actin. Cells were stained for F-actin and G-actin with Alexa Fluor® 488–phalloidin and Alexa Fluor® 594–DNase I respectively, and their intensity ratio was calculated. Scale bar=20 μm. (C and F) F-actin/G-actin ratio. Values were normalized with the mean value of the control cells cultured in GM. Data represent the means±S.D. for more than ten independent cells. *P<0.01 (unpaired Student's t test.). WT, wild-type.

Figure 3
p130Cas regulates actin remodelling in myoblasts

C2C12 myoblasts were cultured in GM or switched to DM 1 day before analysis. (AC) Cells were infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses. (DF) Cells were co-infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses, together with either vector- or shRNA-resistant form of wild-type p130Cas- or mutant p130Cas15YF-expressing retroviruses. (A and D) Cells stained for F-actin. Scale bar=20 μm. (B and E) Ratio images of F-actin to G-actin. Cells were stained for F-actin and G-actin with Alexa Fluor® 488–phalloidin and Alexa Fluor® 594–DNase I respectively, and their intensity ratio was calculated. Scale bar=20 μm. (C and F) F-actin/G-actin ratio. Values were normalized with the mean value of the control cells cultured in GM. Data represent the means±S.D. for more than ten independent cells. *P<0.01 (unpaired Student's t test.). WT, wild-type.

Integrin β3 drives p130Cas phosphorylation in differentiating C2C12 myoblasts

It has been reported that integrin β3 is involved in myogenic differentiation [20], and that activation of integrin β3 signalling supports p130Cas phosphorylation [22]. We therefore tested whether integrin β3 was involved in the increase in p130Cas phosphorylation during myogenic differentiation. Consistent with the previous report [20], the expression of integrin β3 was increased during differentiation of C2C12 myoblasts (Figure 4A, top panel). The increase in p130Cas phosphorylation after medium replacement with DM was attenuated by silencing of integrin β3 (Figure 4A), suggesting that integrin β3 acts upstream of p130Cas in differentiating myoblasts. Furthermore, knock down of integrin β3 diminished C2C12 myotube formation (Figure 4B), as well as the expression of muscle-specific proteins (Figure 4C). Nuclear localization of MAL was also impaired by knock down of integrin β3 in C2C12 myoblasts cultured in DM (Figure 4D). These results suggest that integrin β3 plays a role in myogenic differentiation by facilitating p130Cas phosphorylation and subsequent MAL nuclear translocation and SRF activation.

Integrin β3 is involved in p130Cas phosphorylation during myogenic differentiation

Figure 4
Integrin β3 is involved in p130Cas phosphorylation during myogenic differentiation

C2C12 myoblasts were infected with shRNA vector- or integrin β3-shRNA-expressing retroviruses. (AC) Cells were cultured in GM and then switched to DM. (A) Cells were solubilized on the indicated day. The expression levels of integrin β3 (ITGβ3) and phosphorylated p130Cas (p-p130Cas) were evaluated by immunoblot analysis. (B) Cells were fixed with 4% PFA 4 days after medium replacement with DM, and subjected to anti-MHC immunostaining (red) and DAPI staining (blue). The images were obtained with a confocal microscope (A1R) equipped with an objective (40×). Scale bar=50 μm. (C) Cells were solubilized on the indicated days. The expression of skeletal-muscle-specific proteins was evaluated by immunoblot analysis. (D) Cells were cultured in GM or switched to DM 1 day before analysis and then subjected to anti-MAL immunostaining (green) and DAPI staining (blue). The images were obtained with a confocal microscope (A1R) equipped with an oil-immersion objective (100×). Scale bar=20 μm.

Figure 4
Integrin β3 is involved in p130Cas phosphorylation during myogenic differentiation

C2C12 myoblasts were infected with shRNA vector- or integrin β3-shRNA-expressing retroviruses. (AC) Cells were cultured in GM and then switched to DM. (A) Cells were solubilized on the indicated day. The expression levels of integrin β3 (ITGβ3) and phosphorylated p130Cas (p-p130Cas) were evaluated by immunoblot analysis. (B) Cells were fixed with 4% PFA 4 days after medium replacement with DM, and subjected to anti-MHC immunostaining (red) and DAPI staining (blue). The images were obtained with a confocal microscope (A1R) equipped with an objective (40×). Scale bar=50 μm. (C) Cells were solubilized on the indicated days. The expression of skeletal-muscle-specific proteins was evaluated by immunoblot analysis. (D) Cells were cultured in GM or switched to DM 1 day before analysis and then subjected to anti-MAL immunostaining (green) and DAPI staining (blue). The images were obtained with a confocal microscope (A1R) equipped with an oil-immersion objective (100×). Scale bar=20 μm.

Cofilin inactivation is important for myogenic differentiation via p130Cas

Active (unphosphorylated) cofilin stimulates actin depolymerization and increases the G-actin pool, thereby inhibiting MAL nuclear translocation and down-regulating SRF activity [29]. We found that phosphorylation of cofilin at Ser3, which leads to its inactivation, increased in control cells but not in CasKD cells when cultured in DM (Figure 5A). To examine the role of cofilin in p130Cas-mediated regulation of C2C12 differentiation, we expressed the phospho-mimetic mutant form of cofilin (cofilin-S3E) in CasKD cells (Figure 5B). The expression of cofilin-S3E restored stress fibre formation and MAL nuclear localization in CasKD cells (Figures 5C and 5D, and Supplementary Figure S1E). Accordingly, the expression of skeletal-muscle-specific proteins and myotube formation, which were attenuated by p130Cas gene silencing, were also restored upon cofilin-S3E expression (Figures 5E and 5F). These results suggest that cofilin is involved in p130Cas-mediated differentiation of C2C12 myoblasts.

Cofilin inactivation restores impaired myotube formation by knock down of p130Cas

Figure 5
Cofilin inactivation restores impaired myotube formation by knock down of p130Cas

(A) C2C12 myoblasts were infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses. The expression levels of cofilin and phosphorylation of cofilin (p-cofilin) were evaluated by immunoblot analysis. An anti-tubulin blot provided a loading control. (BF) C2C12 myoblasts were co-infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses, together with either vector- or HA-tagged mutant cofilin (cofilin-S3E)-expressing retroviruses. (B) The expression of HA-tagged cofilin-S3E was confirmed by immunoblot analysis. (C and D) Cells were cultured in GM or switched to DM 1 day before analysis. (C) Cells were stained for F-actin with phalloidin. (D) Anti-MAL immunostaining (green) and DAPI staining (blue). Images were obtained with a confocal microscope (A1R) equipped with an oil-immersion objective (100×). Scale bars=20 μm. (E and F) Cells were cultured in GM and then switched to DM. (E) Cells were solubilized on the indicated days and the expression levels of skeletal-muscle-specific proteins were evaluated by immunoblot analysis. (F) Cells were fixed with 4% PFA 4 days after medium replacement with DM and subjected to anti-MHC immunostaining (red) and DAPI staining (blue). Scale bar=50 μm. vec, vector.

Figure 5
Cofilin inactivation restores impaired myotube formation by knock down of p130Cas

(A) C2C12 myoblasts were infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses. The expression levels of cofilin and phosphorylation of cofilin (p-cofilin) were evaluated by immunoblot analysis. An anti-tubulin blot provided a loading control. (BF) C2C12 myoblasts were co-infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses, together with either vector- or HA-tagged mutant cofilin (cofilin-S3E)-expressing retroviruses. (B) The expression of HA-tagged cofilin-S3E was confirmed by immunoblot analysis. (C and D) Cells were cultured in GM or switched to DM 1 day before analysis. (C) Cells were stained for F-actin with phalloidin. (D) Anti-MAL immunostaining (green) and DAPI staining (blue). Images were obtained with a confocal microscope (A1R) equipped with an oil-immersion objective (100×). Scale bars=20 μm. (E and F) Cells were cultured in GM and then switched to DM. (E) Cells were solubilized on the indicated days and the expression levels of skeletal-muscle-specific proteins were evaluated by immunoblot analysis. (F) Cells were fixed with 4% PFA 4 days after medium replacement with DM and subjected to anti-MHC immunostaining (red) and DAPI staining (blue). Scale bar=50 μm. vec, vector.

ILK is involved in cofilin inactivation via p130Cas

It has been reported that cofilin is phosphorylated by several kinases, including LIMK (LIM domain kinase), TESK and ILK, whereas it is dephosphorylated by phosphatases such as Slingshot and Chronophin [30,31]. To address how p130Cas was linked to cofilin inactivation during C2C12 differentiation, we first tested whether LIMK activity was altered by silencing of p130Cas. Phosphorylation of LIMK decreased after medium replacement with DM, and no significant difference in LIMK phosphorylation was observed between control and CasKD cells (Supplementary Figure S2A at http://www.BiochemJ.org/bj/445/bj4450323add.htm). Phosphorylation of cofilin, but not of p130Cas, was decreased by treating the cells in GM with a LIMK inhibitor (Supplementary Figure S2B). However, LIMK inhibition affected neither p130Cas phosphorylation nor cofilin phosphorylation induced after medium replacement with DM (Supplementary Figure S2C). LIMK is therefore unlikely to be involved in p130Cas-mediated phosphorylation of cofilin in differentiating myoblasts.

TESK1 and TESK2 are expressed at high levels in testis and at low levels in other tissues [32,33], whereas their expression levels in skeletal muscle or myogenic cells remain undefined. The results of quantitative real-time PCR showed that both TESK1 and TESK2 were expressed in C2C12 myoblasts, although their expression levels were significantly lower compared with that in mouse testicular tissues (Supplementary Figure S3A at http://www.BiochemJ.org/bj/445/bj4450323add.htm). To examine the role of TESKs in the process of myogenic differentiation, we then silenced TESK1 and TESK2 expression in C2C12 myoblasts (Supplementary Figure S3B). Myotube formation, which was observed in the control cells on day 4, was impaired by knockdown of TESK2, but not of TESK1 (Supplementary Figures S3C and S3D). These results suggest that TESK1 is not involved in p130Cas-mediated myogenic differentiation. In the case of TESK2-knockdown myoblasts (TESK2KD cells), cell elongation/alignment on day 2, which was observed in both control and CasKD cells (Figure 1B, bottom panels), was prevented (Supplementary Figures S3C). Notably, unlike in CasKD cells, cell viability appeared remarkably decreased in TESK2KD cells when cultured in DM (Supplementary Figure S3E). Taken together, we suggest that silencing of TESK2 expression hampers myogenic differentiation by a mechanism unrelated to p130Cas-mediated signalling.

ILK has been reported to directly bind the integrin β subunit [34]. We analysed the distribution of ILK in C2C12 myoblasts by immunostaining. Although ILK moderately co-localized with phosphorylated paxillin, we were not able to clearly define ILK distribution to adhesions in control and CasKD cells cultured with GM (Figure 6A, top panels). However, there was a distinct difference in ILK distribution between control and CasKD cells after the induction of myogenic differentiation. ILK showed a more distinct accumulation and co-localization with phosphorylated paxillin in control cells (Figure 6A, bottom panels), but was diffusely distributed in the cytoplasm of CasKD cells. These results suggest that ILK plays a role in adhesion-associated p130Cas-mediated signalling events related to the induction of myogenic differentiation. To examine whether ILK was involved in p130Cas-dependent C2C12 differentiation, we expressed a human ILK mutant (hILK-S343D), which has been reported as constitutively active [34], in CasKD cells (Figure 6B). Similar to CasKD cells expressing the phospho-mimetic form of cofilin (cofilin-S3E) (Figures 5E and 5F), expression of hILK-S343D in CasKD cells restored myogenic differentiation (Figures 6C and 6D). These findings suggest that ILK is involved in the regulation of myogenic differentiation by p130Cas.

p130Cas regulates cofilin phosphorylation through ILK

Figure 6
p130Cas regulates cofilin phosphorylation through ILK

(A) C2C12 myoblasts were infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses. Cells were cultured in GM or switched to DM 1 day before analysis. Anti-ILK (red) and anti-phospho-paxillin (green) antibody immunostaining was performed. Images were obtained with a confocal microscope (A1R) equipped with an oil-immersion objective (100×). Scale bar=20 μm. (BD) C2C12 myoblasts were co-infected with p130Cas-shRNA-expressing retroviruses, with or without mutant ILK (ILK-S343D)-expressing retroviruses. (B) Cells cultured in GM were solubilized and the expression levels of p130Cas and mutant ILK were evaluated by immunoblot (I.B.) analysis and RT–PCR respectively. (C) Cells were cultured in GM and switched to DM. Cells were then solubilized on the indicated days and the expression levels of skeletal-muscle-specific proteins evaluated by immunoblot analysis. (D) Cells were fixed with 4% PFA 4 days after medium replacement with DM and subjected to anti-MHC immunostaining (red) and DAPI staining (blue). Scale bar=50 μm. (E) C2C12 myoblasts were infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses. Cells were solubilized on the indicated days and cell lysates were subjected to immunoprecipitation with an antibody against ILK. The activity of ILK-associated proteins in phosphorylating cofilin was estimated by an in-gel kinase (IGK) assay (top panel). Phosphorylated cofilin (p-cofilin) was evaluated by immunoblot analysis using an anti-phospho-cofilin antibody. Arrowheads indicate the position of ILK (~51 kDa). The molecular mass in kDa is given on the right-hand side. (F) A proposed model for p130Cas-mediated signalling that leads to skeletal-muscle-specific gene expression. IP, immunoprecipitation; vec, vector.

Figure 6
p130Cas regulates cofilin phosphorylation through ILK

(A) C2C12 myoblasts were infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses. Cells were cultured in GM or switched to DM 1 day before analysis. Anti-ILK (red) and anti-phospho-paxillin (green) antibody immunostaining was performed. Images were obtained with a confocal microscope (A1R) equipped with an oil-immersion objective (100×). Scale bar=20 μm. (BD) C2C12 myoblasts were co-infected with p130Cas-shRNA-expressing retroviruses, with or without mutant ILK (ILK-S343D)-expressing retroviruses. (B) Cells cultured in GM were solubilized and the expression levels of p130Cas and mutant ILK were evaluated by immunoblot (I.B.) analysis and RT–PCR respectively. (C) Cells were cultured in GM and switched to DM. Cells were then solubilized on the indicated days and the expression levels of skeletal-muscle-specific proteins evaluated by immunoblot analysis. (D) Cells were fixed with 4% PFA 4 days after medium replacement with DM and subjected to anti-MHC immunostaining (red) and DAPI staining (blue). Scale bar=50 μm. (E) C2C12 myoblasts were infected with shRNA vector- or p130Cas-shRNA-expressing retroviruses. Cells were solubilized on the indicated days and cell lysates were subjected to immunoprecipitation with an antibody against ILK. The activity of ILK-associated proteins in phosphorylating cofilin was estimated by an in-gel kinase (IGK) assay (top panel). Phosphorylated cofilin (p-cofilin) was evaluated by immunoblot analysis using an anti-phospho-cofilin antibody. Arrowheads indicate the position of ILK (~51 kDa). The molecular mass in kDa is given on the right-hand side. (F) A proposed model for p130Cas-mediated signalling that leads to skeletal-muscle-specific gene expression. IP, immunoprecipitation; vec, vector.

To test whether ILK phosphorylated cofilin in C2C12 myoblasts, we conducted an in-gel kinase assay of anti-ILK immuno-precipitates using GST–cofilin as a substrate (Supplementary Figure S4 at http://www.BiochemJ.org/bj/445/bj4450323add.htm). Cofilin phosphorylation by protein(s) of 51 kDa, the molecular mass of ILK, was not apparent (Figure 6E, top panel, arrowhead). However, we found that cofilin was phosphorylated by ILK-associated proteins of several different molecular masses larger than 51 kDa, suggesting that ILK itself may not directly phosphorylate cofilin. The activity of cofilin phosphorylation was significantly increased in control cells after medium replacement with DM (Figure 6E, top panel, lanes 2 and 3) and was attenuated by silencing of p130Cas (Figure 6E, top panel, lanes 5 and 6). Although an increase in ILK expression was observed after the induction of differentiation (Figure 6E, bottom panel, lane 1 compared with lanes 2 and 3), it did not appear to fully account for the remarkable increase in cofilin phosphorylation (Figure 6E, top panel, lane 1 compared with lanes 2 and 3). Furthermore, ILK expression levels were comparable between control and CasKD cells (Figure 6B, bottom panel). It is therefore likely that p130Cas modulated the activity of ILK complexes, but not ILK expression. On the basis of these findings, we conclude that p130Cas regulates myogenic differentiation through cofilin inactivation by ILK complexes.

DISCUSSION

Myogenesis is involved not only in development, but also during maintenance and repair of adult skeletal muscle tissues. The actin cytoskeleton exhibits striking rearrangements during the differentiation of myoblasts to multinucleated muscle fibres. Remodelling of the actin cytoskeleton is required for elongation, alignment and fusion of myoblasts. p130Cas acts as a cytoskeletal mechanosensor through stretch-dependent tyrosine phosphorylation [17] and regulates actin dynamics [18]. In the present study we investigated the molecular function of p130Cas in myogenic differentiation, focusing on myotube formation and actin remodelling.

SRF activity is regulated by two distinct cofactors, namely TCF that is activated by the ERK pathway, and MAL that is regulated by the modulation of actin cytoskeletal dynamics [13]. MAL/SRF activity is required for the expression of skeletal-muscle-specific genes. As reported previously [12], ERK activity decreased in control cells after the induction of differentiation, i.e. medium replacement with DM, and was similarly decreased in CasKD cells (Supplementary Figure S5 at http://www.BiochemJ.org/bj/445/bj4450323add.htm). Concomitantly, F-actin formation also decreased (Figures 3A–3C). These results suggest that both TCF- and MAL-mediated SRF activities are mitigated at the onset of myogenic differentiation. The decrease in F-actin formation after medium replacement with DM was enhanced by silencing of p130Cas (Figures 3A–3C and Supplementary Figures S1A–S1C). Correspondingly, MAL was eliminated from the nuclei of CasKD cells cultured in DM (Figures 2B and 2C). This was consistent with the decrease in the SRE–Luc activity observed in CasKD cells under the same culture conditions (Figure 2A, left-hand panel). In contrast, the expression of skeletal-muscle-specific genes (Figure 1A) and the activity of the skeletal muscle ACTA promoter (Figure 2A, right-hand panel) were remarkably increased after medium replacement with DM. Previous studies have demonstrated that the myogenic transcriptional repressor YY1 (Yin and Yang 1) binds to the ACTA promoter and suppresses ACTA expression in proliferating C2C12 myoblasts [35,36]. It has been proposed that, at the onset of myogenic differentiation, YY1, which recruits the polycomb protein Ezh2 and HDAC1 (histone deacetylase 1), is replaced by SRF, MyoD and the histone acetyltransferases CBP [CREB (cAMP-response-element-binding protein)-binding protein] or PCAF (p300/CBP-associated factor) [35,36]. The ACTA promoter contains not only SREs, but also E-boxes, the consensus sequence for the binding of basic helix–loop–helix transcription factors including MyoD [37]. Taken together, the increase in ACTA promoter activity after medium replacement with DM (Figure 2A, right-hand panel) may be due to the replacement of the YY1–Ezh2–HDAC1 repressive complex with SRF, MyoD and histone acetyltransferase(s). We found that silencing of p130Cas abrogated the increase in ACTA promoter activity after medium replacement with DM (Figure 2A, right-hand panel). Since SRF activity is regulated by p130Cas at the onset of differentiation (Figure 2A, left-hand panel) and MyoD expression can be induced by SRF [7], the suppression of ACTA promoter activity by silencing of p130Cas may be a synergistic effect of inhibition of SRF and MyoD activities.

Previous reports demonstrate that integrin β3 is involved in myogenic differentiation through the activation of p38 MAPK, mediated by Rac1 [20,38]. Integrin β3 silencing attenuates the activity of p38-dependent myogenesis regulators such as p21CIP1 and myogenin [20], whereas knock down of p130Cas did not interfere with p38 MAPK activity (Supplementary Figure S2). In contrast, actin cytoskeleton reorganization during myogenic differentiation, which is reportedly mediated by integrin β3 [20], did involve p130Cas phosphorylation (Figures 3D–3F and Supplementary Figure S1D). Together with the decrease in p130Cas phosphorylation by silencing of integrin β3 (Figure 4A), we suggest that integrin β3 regulates myogenic differentiation by facilitating phosphorylation of p130Cas, which potentiates actin remodelling. In addition, MAL nuclear localization in differentiating myoblasts was attenuated by silencing of either p130Cas or integrin β3 (Figures 2B–2D and 4D). These results support the notion that the integrin β3/p130Cas pathway regulates MAL nuclear localization through the modulation of actin cytoskeletal dynamics.

Cofilin promotes actin filament disassembly and contributes towards supplying an abundant pool of cytoplasmic actin monomers, i.e. G-actin [39]. Subsequently, G-actin binds to MAL and inhibits its nuclear localization [29]. In the case of epidermal stem cells, cofilin inactivation and concomitant MAL/SRF activation are required for their differentiation into keratinocytes [40]. The results of the present study suggest that p130Cas is involved in cofilin inactivation during skeletal muscle differentiation. Cofilin inactivation lowers the concentration of G-actin and enhances MAL/SRF activity, which is critical for muscle-specific gene expression [10,41]. p130Cas-dependent inactivation of cofilin (Figure 5A) and restoration of myogenic differentiation of CasKD cells by the expression of a dominant-negative form of cofilin (cofilin-S3E) (Figures 5E and 5F) indicate that cofilin acts as a downstream mediator of p130Cas in the context of C2C12 differentiation. We suggest that p130Cas tempers the decrease in F-actin formation through inactivation of cofilin, and thereby contributes to the maintenance of SRF activity required for muscle-specific gene expression.

ILK has been reported to play a critical role in skeletal and cardiac muscle development and maintenance [34]. A previous study has shown that Kindlin-2, which is an integrin-associated adaptor protein and regulates the recruitment of ILK to focal adhesions [42], is required for C2C12 myotube formation [43]. However, detailed molecular mechanisms behind ILK-dependent regulation of myogenic differentiation remain unclear. The expression level of ILK appears important, as its overexpression impairs C2C12 myotube formation via a defect in ERK inactivation at the onset of differentiation [44]. Although the expression of mutant ILK (S343D) restored differentiation of CasKD cells (Figures 6C and 6D), cofilin phosphorylation by ILK itself was not apparent (Figure 6E). Taken together, ILK may be a crucial scaffold molecule that determines ERK-mediated proliferation and p130Cas-mediated differentiation of myoblasts. Moreover, it has been reported that ILK is required for skeletal muscle fibre stability [45] and we have found that p130Cas regulates sarcomeric organization in myotubes (K. Kawauchi, W. W. Tan, K. Araki, F. Abu Bakar, H. Fujita, H. Hirata and Y. Sawada unpublished work). From the mechanical implications of p130Cas and ILK functions [17,46,47], the interplay between p130Cas and ILK may be a key element for the maintenance of muscles.

Myoblasts elongate and align prior to their fusion, with a decrease in RhoA activity involved in these cell morphological changes [2]. Reduced F-actin formation after medium replacement with DM (Figures 3A–3C) may therefore result from the down-regulation of RhoA activity. The concomitant increase in G-actin was tempered by p130Cas-dependent inactivation of cofilin via ILK complexes (Figures 5A and 6E). Taken together, we therefore propose a novel model for the molecular mechanism underlying myogenic differentiation mediated by integrin β3/p130Cas signalling, in which skeletal-muscle-specific gene expression is induced by MAL/SRF through ILK-dependent cofilin inactivation (Figure 6F).

Differentiation is strictly programmed by integrin-mediated mechanosensing machinery [48]. p130Cas-dependent actin remodelling during myogenic differentiation may facilitate adhesion–cytoskeleton organization and serve as a mediator for the mechanical regulation of cell and tissue differentiation.

Abbreviations

     
  • Cas

    Crk-associated substrate

  •  
  • CasKD

    p130Cas-knockdown myoblast

  •  
  • CasSD

    p130Cas central substrate domain

  •  
  • CasSH3

    p130Cas N-terminal SH3 (Src homology 3) domain

  •  
  • CasWT

    wild-type shRNA-resistant form of p130Cas

  •  
  • Cas15YF

    mutant shRNA-resistant form of p130Cas

  •  
  • CBP

    CREB (cAMP-response-element-binding protein)-binding protein

  •  
  • DAPI

    4′,6-diamidino-2-phenylindole

  •  
  • DM

    differentiation medium

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • DTT

    dithiothreitol

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • F-actin

    filamentous actin

  •  
  • G-actin

    globular actin

  •  
  • GM

    growth medium

  •  
  • GST

    glutathione transferase

  •  
  • HDAC1

    histone deacetylase 1

  •  
  • ILK

    integrin-linked kinase

  •  
  • LIMK

    LIM domain kinase

  •  
  • MAL

    megakaryocytic acute leukaemia

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MHC

    myosin heavy chain

  •  
  • MyoD

    myogenic differentiation factor D

  •  
  • NA

    numerical aperture

  •  
  • PFA

    paraformaldehyde

  •  
  • RL

    Renilla luciferase

  •  
  • RT

    reverse transcription

  •  
  • SH3

    Src homology 3

  •  
  • shRNA

    short hairpin RNA

  •  
  • SRE

    serum-response element

  •  
  • SRF

    serum-response factor

  •  
  • TCF

    ternary complex factor

  •  
  • TESK

    testicular protein kinase

  •  
  • TESK2KD cell

    TESK2-knockdown myoblast

  •  
  • TK

    thymidine kinase

  •  
  • YY1

    Yin and Yang 1

AUTHOR CONTRIBUTION

Keiko Kawauchi and Yasuhiro Sawada designed the study. Wee Wee Tan and Keiko Kawauchi performed all the experiments with support from Hiroaki Hirata, Farhana Binte Abu Bakar, Keigo Araki, Hideaki Fujita and Minsoo Kim. Keiko Kawauchi, Wee Wee Tan, Keigo Araki, Minsoo Kim, Hiroaki Hirata and Yasuhiro Sawada wrote the paper.

We thank Dr Michael P. Sheetz, Dr Nobuyuki Tanaka, Dr G.V. Shivashankar, Dr Toshio Kitamura, Dr Kensaku Mizuno, Dr Kazumasa Ohashi, Dr Lisa Tucker-Kellogg, Dr Ichiro Harada, Dr Hiroaki Machiyama, Dr Sri Ram Krishna Vedula and Dr Qingsen Li for discussions. Dr Ryuichi Sakai provided an anti-p130Cas antibody (αCas3). Mr Lubin Chen assisted with data analysis.

FUNDING

This work was supported by the Ministry of Education [grant numbers R-154-000-406-112 and R-154-000-430-112]; the Biomedical Research Council [grant number R-154-000-423-305] and the Seed Fund of the Mechanobiology Institute, Singapore.

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Author notes

1

These authors contributed equally to this work.

Supplementary data