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. 2011 Sep 21;92(3):409–419. doi: 10.1093/cvr/cvr247

Serine-910 phosphorylation of focal adhesion kinase is critical for sarcomere reorganization in cardiomyocyte hypertrophy

Miensheng Chu 1, Rekha Iyengar 2, Yevgeniya E Koshman 2, Taehoon Kim 2, Brenda Russell 3, Jody L Martin 1, Alain L Heroux 2, Seth L Robia 1, Allen M Samarel 1,2,*
PMCID: PMC3246880  PMID: 21937583

Abstract

Aims

Tyrosine-phosphorylated focal adhesion kinase (FAK) is required for the hypertrophic response of cardiomyocytes to growth factors and mechanical load, but the role of FAK serine phosphorylation in this process is unknown. The aims of the present study were to characterize FAK serine phosphorylation in cultured neonatal rat ventricular myocytes (NRVM), analyse its functional significance during hypertrophic signalling, and examine its potential role in the pathogenesis of human dilated cardiomyopathy (DCM).

Methods and results

Endothelin-1 (ET-1) and other hypertrophic factors induced a time- and dose-dependent increase in FAK-S910 phosphorylation. ET-1-induced FAK-S910 phosphorylation required ETAR-dependent activation of PKCδ and Src via parallel Raf-1 → MEK1/2 → ERK1/2 and MEK5 → ERK5 signalling pathways. Replication-deficient adenoviruses expressing wild-type (WT) FAK and a non-phosphorylatable, S910A-FAK mutant were then used to examine the functional significance of FAK-S910 phosphorylation. Unlike WT-FAK, S910A-FAK increased the half-life of GFP-tagged paxillin within costameres (as determined by total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching) and increased the steady-state FAK–paxillin interaction (as determined by co-immunoprecipitation and western blotting). These alterations resulted in reduced NRVM sarcomere reorganization and cell spreading. Finally, we found that FAK was serine-phosphorylated at multiple sites in non-failing, human left ventricular tissue. FAK-S910 phosphorylation and ERK5 expression were dramatically reduced in patients undergoing heart transplantation for end-stage DCM.

Conclusion

FAK undergoes S910 phosphorylation via PKCδ and Src-dependent pathways that are important for cell spreading and sarcomere reorganization. Reduced FAK-S910 phosphorylation may contribute to sarcomere disorganization in DCM.

Keywords: Signal transduction, Src, Protein kinase C, Costamere, Dilated cardiomyopathy

1. Introduction

Cardiomyocytes adapt to increased haemodynamic load and growth factor stimulation by the addition and reorganization of their sarcomeres. An important site for new sarcomere formation is the costamere, which provides a physical coupling of the cardiomyocyte to its extracellular matrix, thereby linking the sarcolemmal membrane to peripheral Z-discs.1 In cultured cardiomyocytes, costameres reorganize to form adhesive structures that are similar to typical focal adhesions found in non-muscle cells.2 Because of the similarities between costamere formation in cardiomyocytes and focal adhesion formation in non-muscle cells, we and others have proposed that the non-receptor protein tyrosine kinase focal adhesion kinase (FAK) plays an important role in both processes.3 FAK localizes to costameres in the intact heart in vivo,4 and to focal adhesion sites and costameres in cultured cardiomyocytes.5 Cardiomyocytes isolated from mice with tissue-specific deletion of FAK show increased length but not width and display disorganized myofibrils with increased non-myofibrillar space filled with swollen mitochondria.6 FAK deletion also causes dilated cardiomyopathy (DCM) with ageing and produces eccentric rather than concentric left ventricular (LV) hypertrophy with angiotensin II (AngII) infusion or transverse aortic coarctation, suggesting an intrinsic abnormality in sarcomere assembly.6–8 Overexpression of FAK-related non-kinase (FRNK) or ‘knocking-down’ FAK by siRNA prevents normal costamerogenesis and myofibrillogenesis during skeletal muscle differentiation.9 Furthermore, overexpression of FRNK in cardiomyocytes also prevents the endothelin-1 (ET-1)-induced increase in total protein/DNA, and the assembly of newly synthesized myofibrillar proteins into sarcomeres.10 These findings suggest that FAK is required for the normal assembly of sarcomeres in response to both biomechanical and neurohormonal stimuli that induce cardiomyocyte hypertrophy.

FAK serves both catalytic and scaffolding functions during biomechanical and neurohormonal signalling. FAK contains a central catalytic domain flanked by large N- and C-terminal non-catalytic domains. The C-terminal region encodes its focal adhesion targeting (FAT) domain, as well as proline-rich regions involved in protein–protein interactions. The C-terminal domain also contains several serine and tyrosine phosphorylation sites, whose phosphorylations differ depending on cell type. Tyrosine phosphorylations at Y861 and Y925 are rapidly induced in cultured rat cardiomyocytes upon stimulation with hypertrophic agonists, leading to subsequent phosphotyrosine-dependent interactions with other signalling proteins such as Src, p130Cas, and Grb2. FAK also contains four serine phosphorylation sites within its C-terminal domain (S722, S843, S846, and S910).11 These serine residues are in close proximity to protein–protein interaction sites, such as the binding site for p130Cas.12 The FAT domain interacts with paxillin and talin, which is responsible for targeting FAK to newly formed and existing focal adhesion complexes and for promoting downstream signalling.13 Very little is known about the role of FAK serine phosphorylation in cardiomyocytes, but one report indicates that FAK serine phosphorylation increases dramatically in hypertensive rats, with different sites of phosphorylation appearing to regulate FAK subcellular localization.14

The objectives of the present study were to (i) characterize FAK serine phosphorylation in cultured NRVM, in non-failing human LV myocardium, and in myocardium obtained from patients with end-stage DCM; (ii) identify the responsible signalling pathways; and (3) determine its functional significance during hypertrophic signalling. Data are presented to indicate that FAK-S910 phosphorylation appears to be an important event in cell spreading and sarcomere reorganization. Furthermore, FAK is serine-phosphorylated in non-failing, human LV tissue, but its phosphorylation is dramatically reduced in end-stage DCM, perhaps contributing to the sarcomere disorganization observed in these failing hearts.

2. Methods

2.1. Reagents

A detailed description of the reagents used in this study is provided in the Supplementary material online.

2.2. Neonatal rat ventricular myocyte isolation and culture

Animals used in these experiments were handled in accordance with the Guiding Principles in the Care and Use of Laboratory Animals, published by the US National Institutes of Health and approved by the American Physiological Society. Two-day old Sprague–Dawley rats were subjected to hypothermic cardiac arrest, followed by decapitation into liquid N2. Following removal of the heart, neonatal rat ventricular myocytes (NRVM) were isolated by collagenase digestion as previously described,10 with additional details provided in the Supplementary material online.

2.3. Site-directed mutagenesis

3xFLAG-tagged wild-type (WT) FAK cDNA was kindly provided by Dr Joseph C. Loftus, Mayo Clinic Arizona. A serine-910 to alanine (S910A) mutation was created with the USB Change-ITTM Multiple Mutation Site-Directed Mutagenesis Kit (Santa Clara, CA, USA). The primer used for mutagenesis was: 5′-/phos/CTT-CAG-CCC-CAG-GAA-ATC-GCC-CCC-CCT-CCC-ACT-GCC-AAC-3′ for S910A, where the underline represents the mutated bases. The mutation within FAK cDNA was confirmed by DNA sequencing.

2.4. Adenoviral constructs

A detailed description of the adenoviruses used in these experiments is provided in the Supplementary material online.

2.5. Co-immunoprecipitation, SDS–PAGE, and western blotting

For co-immunoprecipitation, cells were lysed with non-denaturing lysis buffer and used to co-immunoprecipitate FLAG-tagged FAK and paxillin, as described in detail in the Supplementary material online. For SDS–PAGE and western blotting, NRVM and human LV tissue samples (∼100 mg wet weight) were sonicated in denaturing lysis buffer. Equal amounts of extracted cellular proteins (50–75 µg) were separated by SDS–PAGE and western blotting, as described in detail in the Supplementary material online.

2.6. Total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching

NRVM grown on four-well, borosilicate glass-bottomed chamber slides coated with fibronectin were infected (10 MOI, 24 h) with Adv-WT-FAK or Adv-S910A-FAK, and then infected with Adv-GFP-paxillin (10 MOI) for an additional 24 h. GFP-paxillin was imaged within costameres located at the cell membrane–substratum interface using an inverted total internal reflection fluorescence (TIRF) microscope and subjected to fluorescence recovery after photobleaching (FRAP). Methodological details are provided in the Supplementary material online. Data were fit to the following single-exponential function:

2.6.

where F/F0 was the normalized fluorescence intensity, Y0 the best-fitting value for F/F0 at t = 0 (i.e. immediately after the flash), and kFRAP was the first-order rate constant (s−1) describing the rate of rise in F/F0. Half-life (t1/2, s) was derived from kFRAP as:

2.6.

2.7. Immunofluorescent microscopy

NRVM were infected (10 MOI, 24 h) with Adv-WT-FAK or Adv-S910A-FAK and then stimulated with ET-1 (100 nM) for 48 h. Cells were fixed, permeabilized, and stained for FLAG and rhodamine phalloidin as described in the Supplementary material online.

2.8. Cell surface area measurements

NRVM were infected (10 MOI, 24 h) with Adv-WT-FAK or Adv-S910A-FAK and then treated with ET-1 100 nM for 48 h. Live cells were then dye-loaded with BCECF-AM, with details provided in the Supplementary material online.

2.9. Left ventricular tissue from non-failing and failing human hearts

Samples of LV tissue were obtained from Loyola University Medical Center's (LUMC's) Cardiovascular Institute Tissue Repository and from the Gift of Hope Organ and Tissue Donor Network. The investigation conformed to the principles outlined in the Declaration of Helsinki. A detailed protocol and informed consent document were reviewed by LUMC's Institutional Review Board prior to tissue procurement. Following informed consent, explanted human LV tissue was obtained from patients undergoing heart transplantation for non-ischaemic, DCM. Tissue samples were quick-frozen in liquid N2 in the operating room and stored at −80°C. Following informed consent from organ donor family members, donor hearts judged unsuitable for cardiac transplantation were stored in cardioplegic solution on ice and were delivered within 4 h of cardiac extirpation by the Gift of Hope Organ and Tissue Donor Network. Tissue samples were then quickly frozen in liquid N2 and stored at −80°C.

2.10. Data analysis

Results were expressed as means ± SEM. Normality was assessed using the Kolmogorov–Smirnov test, and homogeneity of variance was assessed using Levene's test. Data were compared by one-way ANOVA or one-way ANOVA on Ranks, followed by the Dunnett' test or Student–Newman–Keuls test (multiple groups); or unpaired t-test or Wilcoxin's rank-sum test (two groups), where appropriate. Differences among means were considered significant at P< 0.05. Data were analysed using the SigmaStat Statistical Software Package, Ver. 3.11 (Jandel Scientific, San Rafael, CA, USA).

3. Results

3.1. Hypertrophic agonists induce FAK serine phosphorylation in NRVM

FAK serine phosphorylation was detectable under basal conditions at all four serine residues examined. Stimulation of cells with AngII, insulin-like growth factor-1 (IGF-1), ET-1, and phenylephrine (PE) (all at concentrations known to induce NRVM hypertrophy10,15–17) all increased FAK-S910 phosphorylation over time (5–30 min), with ET-1 being the most potent (Figure 1A and B). In contrast, S722 was constitutively phosphorylated, and S732 and S843 phosphorylation were only modestly increased in response to ET-1 (Figure 1) and other agonists (data not shown). The time-course, dose–response, and ETAR dependency of ET-1-induced FAK-S910 phosphorylation were similar to those of FAK-Y925 phosphorylation, but occurred later than FAK-Y397 autophosphorylation (see Supplementary material online, Figures S1–S3 for further details). Therefore, we focused on the signalling pathways and functional significance of FAK-S910 phosphorylation in subsequent experiments.

Figure 1.

Figure 1

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PKC-dependent FAK serine phosphorylation in NRVM. (A) FAK-S910 phosphorylation was assessed in NRVM maintained in control medium (0 min), or stimulated with AngII (30 μM), IGF-1 (50 ng/mL), ET-1 (100 nM), and PE (10 μM) for varying time periods (5–30 min). (B) NRVM were stimulated with 100 nM ET-1 for varying time periods (0–180 min). Quantitative analysis was performed by scanning densitometry, and FAK-S910 phosphorylation intensity was normalized to total FAK intensity at each time point. Data are means ± SEM of at least four independent experiments and were plotted relative to 0 min (i.e. without ET-1 stimulation). *P< 0.05 vs. ET-1 stimulated, control cells. (C) NRVM were pre-treated (1 h) with the non-selective PKC inhibitor GF102903X (GFX; 10 µM), the classical PKC inhibitor Gö6983 (Gö; 10 µM), or the PKCδ-selective inhibitor rottlerin (Rot; 10–20 µM), and then stimulated with ET-1 (100 nM; 30 min). (D) NRVM were infected with Adv-β-gal, Adv-dnPKCα, Adv-dnPKCɛ, and Adv-dnPKCδ (100 MOI, 48 h) and then stimulated with ET-1 (100 nM; 30 min). Cell extracts (50 µg total protein) were separated by SDS–PAGE followed by western blotting with FAK-S910 and total anti-FAK antibodies, and PKCα-, PKCδ-, and PKCɛ-specific antibodies, as indicated. Data are means ± SEM at least four independent experiments and are expressed as the percentage of ET-1 stimulation in the absence of antagonists. *P< 0.05 vs. ET-1-stimulated control cells.

3.2. PKC activation is necessary to induce FAK-S910 phosphorylation

NRVM express both classical and novel PKC isoenzymes, including PKCα, PKCδ, and PKCɛ, and PKC activation is an important downstream effector of ETAR stimulation.18 Therefore, PKC inhibitors were used to determine whether ET-1-induced PKC activation was necessary for FAK-S910 phosphorylation. As seen in Figure 1C, the non-selective PKC inhibitor GF102903X (GFX; 10 µM; 1 h pre-treatment) significantly reduced but did not eliminate FAK-S910 phosphorylation. In contrast, the classical PKC inhibitor Gö6983 (Gö; 10 µM, 1 h pre-treatment) was ineffective, suggesting that a Ca2+-independent, rather than a Ca2+-dependent PKC was required. In support of this observation, the PKCδ-selective inhibitor rottlerin (Rot; 10–20 µM, 1 h pre-treatment) also reduced FAK-S910 phosphorylation to approximately the same extent as GFX. None of the small-molecule inhibitors alone increased FAK-S910 phosphorylation.

3.3. PKCδ is the PKC isoenzyme involved in FAK-S910 phosphorylation

To further support the PKC inhibitor studies, adenoviral-mediated overexpression of dominant-negative (dn) PKCα, PKCɛ, and PKCδ were used to block ET-1-induced FAK-S910 phosphorylation. As seen in Figure 1D, both dnPKCα and dnPKCδ, but not dnPKCɛ, demonstrated inhibitory activity. The dnPKCα data were somewhat surprising, as the classical PKC inhibitor Gö was ineffective (Figure 1C). However, dnPKCα may not be entirely specific for PKCα inhibition,19,20 perhaps by interfering with the activation of other PKC isoenzymes. Additional evidence demonstrating that PKCδ is both necessary and sufficient to induce FAK-S910 phosphorylation is provided in the Supplementary material online, Figures S4 and S5.

3.4. ET-1 activates the ERK cascade and phosphorylates FAK-S910 via Raf-1

Hunger-Glaser et al.21,22 demonstrated that stimulation of Swiss-3T3 cells with bombesin, lysophosphatidic acid, and epidermal growth factor promoted a striking increase in FAK-S910 phosphorylation in a PKC- and ERK1/2-dependent manner. Therefore, we next examined the time course of ERK1/2 activation and FAK-S910 phosphorylation in response to ET-1 (100 nM; 0–60 min). As seen in Figure 2A, ERK1/2 phosphorylation increased approximately six-fold within 5 min of ET-1 stimulation. ERK1/2 activation preceded the peak of FAK-S910 phosphorylation, which occurred at 30–90 min. ET-1-induced ERK1/2 activation was partially dependent on PKCδ, as dnPKCδ overexpression blocked ERK1/2 phosphorylation over the same time period (Figure 2B). ET-1-induced ERK1/2 and FAK-S910 phosphorylation were also blocked by two different inhibitors of MEK1/2 (i.e. PD98059 and U0126), the upstream regulators of ERK1/2 (Figure 2C). In contrast, SB203580, a highly specific p38MAPK inhibitor, was without effect. The involvement of Raf-1, an upstream regulator of the ERK cascade in cardiomyocytes, was examined by adenoviral overexpression of dnRaf-1;23 and by pharmacological inhibition with sorafenib (1–10 µM; 1 h pre-treatment). As seen in Figure 2D and Supplementary material online, Figure S6, both approaches reduced ERK1/2 and FAK-S910 phosphorylation in response to ET-1 stimulation. Of note, ET-1-induced ERK1/2 activation was not dependent on FAK kinase activity (see Supplementary material online, Figure S7 for further details).

Figure 2.

Figure 2

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ET-1-induced FAK-S910 phosphorylation is through a Raf1–MEK1/2–ERK1/2 pathway. (A) NRVM were stimulated with 100 nM ET-1 for varying time periods (0–60 min). Western blots were probed with FAK-S910, pERK1/2, and anti-FAK and anti-ERK2 antibodies. Quantitative analysis was performed as in Figure 1. (B) NRVM were infected with empty control Adv, or Adv-dnPKCδ (100 MOI, 48 h) and then stimulated with ET-1 (100 nM; 10 min). Data are expressed as the percentage of ET-1 stimulation for the control virus. (C) NRVM were pre-treated (1 h) with SB203580 (SB; 10 μM), PD98059 (PD; 30 μM), or U0126 (1–10 μM) and then stimulated with ET-1 (100 nM; 30 min). Data are expressed as the percentage of ET-1 stimulation in the absence of inhibitors. *P< 0.05 vs. unstimulated cells. (D) NRVM were infected with dnRaf-1 (50 MOI, 48 h) and then stimulated with ET-1 (100 nM; 10 or 30 min). Cell lysates were separated by SDS–PAGE, followed by western blotting with various antibodies as indicated. Data are means ± SEM of at least four independent experiments. *P< 0.05 vs. Adv-β-gal-infected control cells.

3.5. Src is also involved in ET-1-induced FAK-S910 phosphorylation

When NRVM were treated with GFX (Figure 1C), Rot (Figure 1C), or dnPKCδ (Figure 1D), ET-1-induced FAK-S910 phosphorylation was only reduced by ∼50%, suggesting that other signalling pathways might also be involved. ET-1 also stimulates Src family kinases (SFKs) and is important for ET-1-induced cardiomyocyte hypertrophy.24 Therefore, to investigate whether SFKs were also involved, the SFK-specific inhibitor PP2, along with its inactive analogue PP3, was used. NRVM were pre-treated (1 h) with PP2 (5–25 µM) or PP3 (25 μM) and then stimulated with ET-1 (100 nM; 30 min). ET-1-induced FAK-S910 phosphorylation was dose-dependently reduced by PP2, but not PP3 (Figure 3A). To confirm these inhibitor studies, we used adenoviral-mediated overexpression of dnSrc (10–50 MOI; Figure 3B), which also significantly reduced ET-1-induced FAK-S910 phosphorylation, suggesting that Src was the specific SFK.

Figure 3.

Figure 3

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ET-1-induced FAK-S910 phosphorylation is also through Src. (A) NRVM were pre-treated (1 h) with PP2 (5–25 μM) or PP3 (25 μM) and then stimulated with ET-1 (100 nM; 30 min). (B) NRVM were infected with Adv-dnSrc (10–50 MOI, 48 h) or empty Adv (50 MOI; 48 h) and then stimulated with ET-1 (100 nM; 10 or 30 min). (C) NRVM were pre-treated (1 h) with PP2 (25 μM), PP3 (25 μM), rottlerin (Rot; 20 μM), Rot + PP2, or Rot + PP3 and then stimulated with ET-1 (100 nM; 30 min). (D) NRVM were infected with empty Adv or Adv-dnSrc (50 MOI, 48 h) and then stimulated with ET-1 (100 nM; 10 min). Cell lysates were separated by SDS–PAGE, followed by western blotting with various antibodies as indicated. Data are means ± SEM from at least four independent experiments and are expressed as the percentage of ET-1 stimulation in the absence of inhibitors or dnSrc. *P< 0.05 vs. ET-1-stimulated cells; &num;P< 0.05 vs. ET-1-stimulated cells treated with Rot + PP2.

As further evidence for the involvement of two parallel signalling pathways, we noted that neither PP2 nor Rot alone was sufficient to eliminate FAK-S910 phosphorylation. However, when NRVM were pre-treated with both Rot and PP2, the inhibitor combination completely blocked ET-1-induced FAK-S910 phosphorylation (Figure 3C).

3.6. Src regulates FAK-S910 phosphorylation through MEK5/ERK5

ET-1-induced ERK1/2 phosphorylation was only partially inhibited by overexpression of dnSrc (Figure 3D) and was not significantly affected by PP2 (see Supplementary material online, Figure S8), suggesting that Src mediates downstream FAK-S910 phosphorylation by activation of serine protein kinase(s) other than ERK1/2. Villa-Moruzzi25 demonstrated that stimulation of Swiss-3T3 cells with PMA induced FAK-S910 phosphorylation via activation of ERK5. Furthermore, Src was involved in ERK5 activation in other cell types.26 Therefore, we examined the time course of ERK5 activation in response to ET-1 (100 nM; 0–60 min), and compared it with the time course of FAK-S910 phosphorylation. As seen in Figure 4A, ERK5 phosphorylation increased significantly within 5 min of ET-1 stimulation and preceded the peak of FAK-S910 phosphorylation, which occurred at 30–90 min. Next, we used adenoviral-mediated overexpression of dnERK5 (10–50 MOI), which dose-dependently reduced FAK-S910 phosphorylation (Figure 4B). Conversely, adenoviral-mediated overexpression of caMEK5 (the upstream activator of ERK5) was sufficient to induce FAK-S910 phosphorylation (see Supplementary material online, Figure S9A). Of note, ET-1-induced ERK5 activity was also partially dependent on Src, as overexpression of dnSrc (Figure 4C) or pre-treatment with PP2 (see Supplementary material online, Figure S9B) both blocked ET-1-induced ERK5 phosphorylation. Overexpression of dnPKCδ also reduced ET-1-induced ERK5 activation (Figure 4D). Overall, these results indicated that ET-1-induced FAK-S910 phosphorylation involved both Src → MEK5 → ERK5 and PKCδ → MEK5 → ERK5 signalling pathways.

Figure 4.

Figure 4

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MEK5/ERK5 is involved in ET-1-induced FAK-S910 phosphorylation. (A) NRVM were stimulated with 100 nM ET-1 for varying time periods (0–60 min). Quantitative analysis of ERK5 and FAK-S910 phosphorylation was performed as in Figure 1. (B) NRVM were infected with empty Adv or Adv-dnERK5 (10–50 MOI, 48 h) and then stimulated with ET-1 (100 nM; 30 min). (C) NRVM were infected (48 h) with dnSrc (50 MOI) or empty Adv (50 MOI) and then stimulated with ET-1 (100 nM; 10 min). (D) NRVM were infected (48 h) with dnPKCδ (50 MOI) or empty Adv (50 MOI) and then stimulated with ET-1 (100 nM; 10 min). Lysates were separated by SDS–PAGE, followed by western blotting with various antibodies as indicated. Data are means ± SEM of at least four independent experiments and are expressed as the percentage of ET-1 stimulation in the control cells. *P< 0.05 vs. ET-1-stimulated cells.

3.7. FAK-S910 phosphorylation regulates the interaction of FAK with paxillin

To determine whether FAK-S910 phosphorylation affects the FAK–paxillin interaction, we used TIRF microscopy and FRAP analysis to examine paxillin-binding kinetics at costameres located at the cell membrane–substratum interface. Adenoviral overexpression of an FLAG-tagged, non-phosphorylatable FAK mutant (S910A-FAK) significantly reduced the kFRAP of GFP-paxillin, when compared with NRVM expressing FLAG-tagged WT-FAK (Figure 5A). Thus, rendering FAK non-phosphorylatable at S910 increased the half-life of GFP-paxillin within costameres (5.4 ± 0.0.3 vs. 6.6 ± 0.3 s for WT-FAK- vs. S910A-FAK-expressing cells; P= 0.006). Thus, overexpression of S910A-FAK reduced the dynamic exchange of GFP-paxillin and increased its interaction with other focal adhesion proteins within cardiomyocyte costameres. These altered paxillin-binding kinetics resulted in increased steady-state interaction of endogenous paxillin with S910A-FAK, as determined by co-immunoprecipitation and western blotting with anti-FLAG or anti-paxillin antibodies (Figure 5B).

Figure 5.

Figure 5

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FAK-S910 phosphorylation regulates the stability of paxillin in costameres, ET-1-induced sarcomere reorganization, and cell spreading. (A) NRVM were infected with Adv-WT-FAK or Adv-S910A-FAK (10 MOI, 24 h) and then infected with Adv-GFP-paxillin (10 MOI) for an additional 24 h. GFP-paxillin localized to the cell membrane by TIRF microscopy was subjected to FRAP analysis. Data are means ± SEM for curve fitting analysis of WT-FAK or S910A-FAK-infected cells. Average kFRAP values for three independent experiments of 15 individual cells (i.e. 45 individual TIRF–FRAP recordings) were compared. *P< 0.05 vs. WT-FAK-infected cells. (B) NRVM were infected with Adv-WT-FAK or Adv-S910A-FAK (10 MOI, 48 h). Cell extracts (500 µg total protein) were co-immunoprecipitated with anti-FLAG or anti-paxillin antibodies, and immunoblots were probed with anti-FAK-S910, anti-paxillin, or anti-FLAG antibodies. (C) NRVM were infected with Adv-WT-FAK or Adv-S910A-FAK (10 MOI, 24 h) and then stimulated with ET-1 (100 nM, 48 h). Cells were fixed, permeabilized, and stained for F-actin (with rhodamine phalloidin, red) and for FLAG (green). Magnified images of boxed areas around bundles of sarcomeres are shown in the insets. Bar = 10 µm. (D) NRVM were infected with Adv-WT-FAK or Adv-S910A FAK (10 MOI, 24 h) and then stimulated with ET-1 (100 nM, 48 h). Cell surface area was measured by BCECF dye loading and image analysis. Data are means ± SEM of 250–400 cells from each treatment group, normalized to the surface area of untreated WT-FAK-expressing cells. *P< 0.05 vs. unstimulated, WT-FAK-infected cells; &num;P< 0.05 vs. ET-1-stimulated, WT-FAK-infected cells.

3.8. FAK-S910 phosphorylation is important for NRVM sarcomere assembly and spreading

Increased cell spreading, cytoskeletal reorganization, and sarcomere assembly are all characteristics of cardiomyocyte hypertrophy.27 Therefore, to determine the effect of FAK-S910 phosphorylation on these parameters, NRVM were infected with Adv-WT-FAK or Adv-S910A-FAK (10 MOI, 24 h) and then stimulated with ET-1 (100 nM; 48 h). ET-1-induced sarcomeric organization was first examined by double-label epifluorescent microscopy (Figure 5C). Although there was no apparent difference in sarcomeric organization in unstimulated cells, ET-1 stimulation induced an obvious increase in sarcomere assembly in cells expressing WT-FAK, but not in cardiomyocytes expressing S910A-FAK. These qualitative changes in sarcomeric organization were associated with a significant increase in the cell surface area of ET-1-treated WT-FAK-expressing cells, but there was no increase in the cell surface area of ET-1-stimulated cells expressing S910A-FAK (Figure 5D).

3.9. FAK is serine-phosphorylated in human hearts

Lastly, we examined the phosphorylation status of FAK serine residues in non-failing human LV tissue and in LV tissue from patients undergoing cardiac transplantation for end-stage DCM. As seen in Figure 6A, there was no significant difference in the amount of total FAK relative to total protein in tissue homogenates of both groups. FAK was serine-phosphorylated in non-failing LV at all four residues examined (S722, S732, S843, and S910). Interestingly, FAK-S843 phosphorylation was reduced by ∼63% (P= 0.175), and FAK-S910 phosphorylation was reduced by ∼91% (P< 0.05) in DCM when compared with non-failing, control hearts. These reductions in FAK serine phosphorylation were not associated with any significant change in ERK1/2 expression or phosphorylation (data not shown). In contrast, LV ERK5 expression was significantly reduced in patients with DCM undergoing heart transplantation, when compared with control, non-failing LV tissue samples (Figure 6B).

Figure 6.

Figure 6

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FAK serine phosphorylation in non-failing LV myocardium and in DCM. Human LV tissue extracts (75 µg total protein) from the non-failing (n= 11) and DCM (n= 6) LV myocardium were separated by SDS–PAGE and western blotting with phospho-specific and total anti-FAK antibodies, as indicated. (A) Representative western blots (left) and quantitative analysis of FAK serine phosphorylation at each site (right) are depicted. (B) Cell extracts from non-failing (n= 12) and DCM (n= 12) LV tissue samples were probed for total ERK5 and GAPDH. Upper panel depicts a representative western blot; lower panel depicts the quantitative analysis of relative ERK5 expression. (C) Schematic depiction of the dual signalling pathways responsible for ET-1-induced FAK-S910 phosphorylation.

4. Discussion

Previous studies addressing FAK's role in cardiomyocyte hypertrophy have focused on its tyrosine phosphorylation and kinase activity.10,17,28,29 However, it has been known for some time that FAK also undergoes serine phosphorylation at multiple residues within its C-terminus. For instance, Yi et al.30 showed that FAK-S722 and FAK-S910 phosphorylation were significantly increased in LV tissue and isolated cardiomyocytes of hypertensive heart failure rats. Despite this initial observation, however, no studies have addressed the responsible signalling pathways and functional significance of FAK serine phosphorylation in cardiomyocytes. In this report, we demonstrate that FAK is serine-phosphorylated in non-failing and failing human hearts. Furthermore, FAK serine phosphorylation is regulated by hypertrophic factors and plays a critical role in cell spreading and new sarcomere formation during cardiomyocyte hypertrophy.

We found that FAK-S910 phosphorylation was markedly reduced in DCM patients undergoing heart transplantation. These results suggested an important role for FAK serine phosphorylation in the pathological response of cardiomyocytes during end-stage heart failure. DCM is characterized by aberrant signal transduction, reduced contractile function, and cytoskeletal disarray,31 which was mimicked during hypertrophic stimulation of NRVM by overexpression of a non-phosphorylatable, S910A-FAK mutant. It is interesting to speculate that the failure to normally serine-phosphorylate FAK in DCM may have contributed to the cardiomyocyte structural abnormalities and reduced contractile function in these critically ill patients.

We also demonstrated that neurohormonal agonists increase FAK-S910 phosphorylation, both through G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases. Our results are similar to studies conducted in Swiss-3T3 fibroblasts.21,22 In the case of ET-1, our data suggest that the ETAR is the responsible GPCR, which is consistent with studies, indicating that this receptor subclass is also responsible for other aspects of ET-1-induced NRVM hypertrophy.32 Of note, ET-1 stimulation caused the rapid, ETAR-mediated translocation of PKCδ and PKCɛ (but not PKCα), and subsequent stimulation of the Raf-1–MEK1/2–ERK1/2 cascade.16,33 However, ET-1-induced S910 phosphorylation was only reduced by ∼50% by PKCδ inhibition, suggesting that other signalling pathways activated by the ETAR are also operative. Our data indicating that PP2 and dnSrc also reduced, but did not eliminate FAK-S910 phosphorylation, suggested the existence of a parallel signalling pathway that involved SFKs. In this regard, several studies have provided evidence for GPCR-dependent activation of SFKs, and SFK activation is necessary for induction of atrial natriuretic factor transcription, a marker of NRVM hypertrophy. Indeed, the major SFK isoforms in NRVM are Src, Fyn, and Yes, and all three kinases are activated by ET-1.24

Our results indicate that SFK-dependent FAK-S910 phosphorylation is in part mediated by MEK5 and ERK5, and that PKCδ is also involved in this limb of the signalling pathways. Of note, Villa-Moruzzi25 demonstrated that ERK5 regulated FAK-S910 phosphorylation in Swiss-3T3 fibroblasts, and Src has been found to regulate ERK5 activation in other cell types.26 However, we now demonstrate that both SFKs and PKCδ are upstream regulators of the MEK5–ERK5 signalling cascade in NRVM. Despite these observations, the mechanisms responsible for Src activation by ET-1 are still not clear. Src can directly phosphorylate PKCδ and alter its substrate specificity in NRVM and other cell types.34–37 Conversely, PKCδ has been shown to regulate Src activity.38,39 Thus, the interaction between Src and PKCδ in regulating ET-1-induced FAK-S910 phosphorylation may be mediated by the substantial, bi-directional crosstalk between Src and PKCδ, and may also be responsible for the simultaneous activation of ERK1/2 and ERK5.

Our results indicate that both ERK1/2 and ERK5 can phosphorylate FAK at S910, and FAK-S910 phosphorylation appears necessary for new costamere formation during cardiomyocyte remodelling. A schematic depicting the dual signalling pathways is depicted in Figure 6C. Thus, we speculate that in addition to their roles in regulating gene transcription, ERK1/2 and ERK5 regulate costamere formation that is required for the addition of new sarcomeres, both in series and in parallel. Of note, these two distinct signalling pathways have also been implicated in regulating length vs. width remodelling of cardiomyocytes during ageing or stress stimulation.40,41 Although both kinase cascades converge on FAK, ERK1/2 and ERK5 may be capable of phosphorylating other focal adhesion protein substrates. It is conceivable that these other proteins are differentially phosphorylated by the two parallel pathways and are required to accomplish length vs. width remodelling by serving as modifiers of the assembly process.

FAK plays an important role in sarcomerogenesis, as previous studies have suggested that FAK is critical for costamerogenesis and sarcomeric reorganization in both cultured cardiomyocytes and skeletal muscle myotubes.9,10,42,43 Based on our results and those of other investigators, we propose that FAK-S910 phosphorylation is critical for regulating the conformation of the FAK FAT domain, and therefore its interaction with other focal adhesion proteins required for new sarcomere addition. Stabilizing the open conformation of the FAT domain may secondarily decrease FAK–paxillin interaction and thereby enhance the vinculin–paxillin interaction.22,44 These events could strengthen the costamere and promote sarcomere formation and reorganization.45,46 Overall, our findings provide a new mechanism to understand how FAK is involved in costamere assembly and sarcomere reorganization in response to neurohumoral factors that induce cardiomyocyte hypertrophy.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.

Funding

These studies were supported in part by NIH 2PO1 HL62426, NIH 1F32 HL096143, and a grant from the Ralph and Marian Falk Medical Research Trust. Y.E.K. was also an AHA Postdoctoral fellow during the time these studies were performed.

Supplementary Material

Supplementary Data
supp_92_3_409__index.html (2.2KB, html)

References

  • 1.Terracio L, Simpson DG, Hilenski L, Carver W, Decker RS, Vinson N, et al. Distribution of vinculin in the Z-disk of striated muscle: analysis by laser scanning confocal microscopy. J Cell Physiol. 1990;145:78–87. doi: 10.1002/jcp.1041450112. [DOI] [PubMed] [Google Scholar]
  • 2.Decker ML, Simpson DG, Behnke M, Cook MG, Decker RS. Morphological analysis of contracting and quiescent adult rabbit cardiac myocytes in long-term culture. Anat Rec. 1990;227:285–299. doi: 10.1002/ar.1092270303. [DOI] [PubMed] [Google Scholar]
  • 3.Samarel AM. Costameres, focal adhesions, and cardiomyocyte mechanotransduction. Am J Physiol Heart Circ Physiol. 2005;289:H2291–H2301. doi: 10.1152/ajpheart.00749.2005. [DOI] [PubMed] [Google Scholar]
  • 4.Domingos PP, Fonseca PM, Nadruz W, Jr, Franchini KG. Load-induced focal adhesion kinase activation in the myocardium: role of stretch and contractile activity. Am J Physiol Heart Circ Physiol. 2002;282:H556–H564. doi: 10.1152/ajpheart.00534.2001. [DOI] [PubMed] [Google Scholar]
  • 5.Seko Y, Takahashi N, Sabe H, Tobe K, Kadowaki T, Nagai R. Hypoxia induces activation and subcellular translocation of focal adhesion kinase (p125FAK) in cultured rat cardiac myocytes. Biochem Biophys Res Commun. 1999;262:290–296. doi: 10.1006/bbrc.1999.1185. [DOI] [PubMed] [Google Scholar]
  • 6.Peng X, Kraus MS, Wei H, Shen TL, Pariaut R, Alcaraz A, et al. Inactivation of focal adhesion kinase in cardiomyocytes promotes eccentric cardiac hypertrophy and fibrosis in mice. J Clin Invest. 2006;116:217–227. doi: 10.1172/JCI24497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.DiMichele LA, Doherty JT, Rojas M, Beggs HE, Reichardt LF, Mack CP, et al. Myocyte-restricted focal adhesion kinase deletion attenuates pressure overload-induced hypertrophy. Circ Res. 2006;99:636–645. doi: 10.1161/01.RES.0000240498.44752.d6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Peng X, Wu X, Druso JE, Wei H, Park AY, Kraus MS, et al. Cardiac developmental defects and eccentric right ventricular hypertrophy in cardiomyocyte focal adhesion kinase (FAK) conditional knockout mice. Proc Natl Acad Sci USA. 2008;105:6638–6643. doi: 10.1073/pnas.0802319105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Quach NL, Rando TA. Focal adhesion kinase is essential for costamerogenesis in cultured skeletal muscle cells. Dev Biol. 2006;293:38–52. doi: 10.1016/j.ydbio.2005.12.040. [DOI] [PubMed] [Google Scholar]
  • 10.Eble DM, Strait JB, Govindarajan G, Lou J, Byron KL, Samarel AM. Endothelin-induced cardiac myocyte hypertrophy: role for focal adhesion kinase. Am J Physiol Heart Circ Physiol. 2000;278:H1695–H1707. doi: 10.1152/ajpheart.2000.278.5.H1695. [DOI] [PubMed] [Google Scholar]
  • 11.Grigera PR, Jeffery ED, Martin KH, Shabanowitz J, Hunt DF, Parsons JT. FAK phosphorylation sites mapped by mass spectrometry. J Cell Sci. 2005;118:4931–4935. doi: 10.1242/jcs.02696. [DOI] [PubMed] [Google Scholar]
  • 12.Ma A, Richardson A, Schaefer EM, Parsons JT. Serine phosphorylation of focal adhesion kinase in interphase and mitosis: a possible role in modulating binding to p130Cas. Mol Biol Cell. 2001;12:1–12. doi: 10.1091/mbc.12.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol. 2005;6:56–68. doi: 10.1038/nrm1549. [DOI] [PubMed] [Google Scholar]
  • 14.Yi XP, Zhou J, Huber L, Qu J, Wang X, Gerdes AM, et al. Nuclear compartmentalization of FAK and FRNK in cardiac myocytes. Am J Physiol Heart Circ Physiol. 2006;290:H2509–H2515. doi: 10.1152/ajpheart.00659.2005. [DOI] [PubMed] [Google Scholar]
  • 15.Sadoshima J, Izumo S. Molecular characterization of angiotensin II-induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. Critical role of the AT1 receptor subtype. Circ Res. 1993;73:413–423. doi: 10.1161/01.res.73.3.413. [DOI] [PubMed] [Google Scholar]
  • 16.Clerk A, Bogoyevitch MA, Anderson MB, Sugden PH. Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts. J Biol Chem. 1994;269:32848–32857. [PubMed] [Google Scholar]
  • 17.Taylor JM, Rovin JD, Parsons JT. A role for focal adhesion kinase in phenylephrine-induced hypertrophy of rat ventricular cardiomyocytes. J Biol Chem. 2000;275:19250–19257. doi: 10.1074/jbc.M909099199. [DOI] [PubMed] [Google Scholar]
  • 18.Sugden PH, Clerk A. Endothelin signalling in the cardiac myocyte and its pathophysiological relevance. Curr Vasc Pharmacol. 2005;3:343–351. doi: 10.2174/157016105774329390. [DOI] [PubMed] [Google Scholar]
  • 19.Garcia-Paramio P, Cabrerizo Y, Bornancin F, Parker PJ. The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation. Biochem J. 1998;333:631–636. doi: 10.1042/bj3330631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Whelan RD, Parker PJ. Loss of protein kinase C function induces an apoptotic response. Oncogene. 1998;16:1939–1944. doi: 10.1038/sj.onc.1201725. [DOI] [PubMed] [Google Scholar]
  • 21.Hunger-Glaser I, Fan RS, Perez-Salazar E, Rozengurt E. PDGF and FGF induce focal adhesion kinase (FAK) phosphorylation at Ser-910: dissociation from Tyr-397 phosphorylation and requirement for ERK activation. J Cell Physiol. 2004;200:213–222. doi: 10.1002/jcp.20018. [DOI] [PubMed] [Google Scholar]
  • 22.Hunger-Glaser I, Salazar EP, Sinnett-Smith J, Rozengurt E. Bombesin, lysophosphatidic acid, and epidermal growth factor rapidly stimulate focal adhesion kinase phosphorylation at Ser-910: requirement for ERK activation. J Biol Chem. 2003;278:22631–22643. doi: 10.1074/jbc.M210876200. [DOI] [PubMed] [Google Scholar]
  • 23.Iijima Y, Laser M, Shiraishi H, Willey CD, Sundaravadivel B, Xu L, et al. c-Raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells. J Biol Chem. 2002;277:23065–23075. doi: 10.1074/jbc.M200328200. [DOI] [PubMed] [Google Scholar]
  • 24.Kovacic B, Ilic D, Damsky CH, Gardner DG. c-Src activation plays a role in endothelin-dependent hypertrophy of the cardiac myocyte. J Biol Chem. 1998;273:35185–35193. doi: 10.1074/jbc.273.52.35185. [DOI] [PubMed] [Google Scholar]
  • 25.Villa-Moruzzi E. Targeting of FAK Ser910 by ERK5 and PP1δ in non-stimulated and phorbol ester-stimulated cells. Biochem J. 2007;408:7–18. doi: 10.1042/BJ20070058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Sun W, Wei X, Kesavan K, Garrington TP, Fan R, Mei J, et al. MEK kinase 2 and the adaptor protein Lad regulate extracellular signal-regulated kinase 5 activation by epidermal growth factor via Src. Mol Cell Biol. 2003;23:2298–2308. doi: 10.1128/MCB.23.7.2298-2308.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Chien KR, Knowlton KU, Zhu H, Chien S. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J. 1991;5:3037–3046. doi: 10.1096/fasebj.5.15.1835945. [DOI] [PubMed] [Google Scholar]
  • 28.Pham CG, Harpf AE, Keller RS, Vu HT, Shai SY, Loftus JC, et al. Striated muscle-specific β1D-integrin and FAK are involved in cardiac myocyte hypertrophic response pathway. Am J Physiol Heart Circ Physiol. 2000;279:H2916–H2926. doi: 10.1152/ajpheart.2000.279.6.H2916. [DOI] [PubMed] [Google Scholar]
  • 29.Torsoni AS, Constancio SS, Nadruz W, Hanks SK, Franchini KG. Focal adhesion kinase is activated and mediates the early hypertrophic response to stretch in cardiac myocytes. Circ Res. 2003;93:140–147. doi: 10.1161/01.RES.0000081595.25297.1B. [DOI] [PubMed] [Google Scholar]
  • 30.Yi XP, Wang X, Gerdes AM, Li F. Subcellular redistribution of focal adhesion kinase and its related nonkinase in hypertrophic myocardium. Hypertension. 2003;41:1317–1323. doi: 10.1161/01.HYP.0000072772.74183.5F. [DOI] [PubMed] [Google Scholar]
  • 31.Hein S, Kostin S, Heling A, Maeno Y, Schaper J. The role of the cytoskeleton in heart failure. Cardiovasc Res. 2000;45:273–278. doi: 10.1016/s0008-6363(99)00268-0. [DOI] [PubMed] [Google Scholar]
  • 32.Ito H, Hiroe M, Hirata Y, Fujisaki H, Adachi S, Akimoto H, et al. Endothelin ETA receptor antagonist blocks cardiac hypertrophy provoked by hemodynamic overload. Circulation. 1994;89:2198–2203. doi: 10.1161/01.cir.89.5.2198. [DOI] [PubMed] [Google Scholar]
  • 33.Chiloeches A, Paterson HF, Marais R, Clerk A, Marshall CJ, Sugden PH. Regulation of Ras.GTP loading and Ras-Raf association in neonatal rat ventricular myocytes by G protein-coupled receptor agonists and phorbol ester. Activation of the extracellular signal-regulated kinase cascade by phorbol ester is mediated by Ras. J Biol Chem. 1999;274:19762–19770. doi: 10.1074/jbc.274.28.19762. [DOI] [PubMed] [Google Scholar]
  • 34.Hall KJ, Jones ML, Poole AW. Coincident regulation of PKCδ in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling. Biochem J. 2007;406:501–509. doi: 10.1042/BJ20070244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Rybin VO, Guo J, Gertsberg Z, Elouardighi H, Steinberg SF. Protein kinase Cɛ (PKCɛ) and Src control PKCδ activation loop phosphorylation in cardiomyocytes. J Biol Chem. 2007;282:23631–23638. doi: 10.1074/jbc.M701676200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Sumandea MP, Rybin VO, Hinken AC, Wang C, Kobayashi T, Harleton E, et al. Tyrosine phosphorylation modifies protein kinase Cδ-dependent phosphorylation of cardiac troponin I. J Biol Chem. 2008;283:22680–22689. doi: 10.1074/jbc.M802396200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Rybin VO, Guo J, Harleton E, Feinmark SJ, Steinberg SF. Regulatory autophosphorylation sites on protein kinase C-δ at threonine-141 and threonine-295. Biochemistry. 2009;48:4642–4651. doi: 10.1021/bi802171c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Brandt DT, Goerke A, Heuer M, Gimona M, Leitges M, Kremmer E, et al. Protein kinase Cδ induces Src kinase activity via activation of the protein tyrosine phosphatase PTPα. J Biol Chem. 2003;278:34073–34078. doi: 10.1074/jbc.M211650200. [DOI] [PubMed] [Google Scholar]
  • 39.Amos S, Martin PM, Polar GA, Parsons SJ, Hussaini IM. Phorbol 12-myristate 13-acetate induces epidermal growth factor receptor transactivation via protein kinase Cδ/c-Src pathways in glioblastoma cells. J Biol Chem. 2005;280:7729–7738. doi: 10.1074/jbc.M409056200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Nicol RL, Frey N, Pearson G, Cobb M, Richardson J, Olson EN. Activated MEK5 induces serial assembly of sarcomeres and eccentric cardiac hypertrophy. EMBO J. 2001;20:2757–2767. doi: 10.1093/emboj/20.11.2757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Kehat I, Davis J, Tiburcy M, Accornero F, Saba-El-Leil MK, Maillet M, et al. Extracellular signal-regulated kinases 1 and 2 regulate the balance between eccentric and concentric cardiac growth. Circ Res. 2011;108:176–183. doi: 10.1161/CIRCRESAHA.110.231514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Kovacic-Milivojevic B, Roediger F, Almeida EA, Damsky CH, Gardner DG, Ilic D. Focal adhesion kinase and p130Cas mediate both sarcomeric organization and activation of genes associated with cardiac myocyte hypertrophy. Mol Biol Cell. 2001;12:2290–2307. doi: 10.1091/mbc.12.8.2290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Mansour H, deTombe PP, Samarel AM, Russell B. Restoration of resting sarcomere length after uniaxial static strain is regulated by protein kinase Cɛ and focal adhesion kinase. Circ Res. 2004;94:642–649. doi: 10.1161/01.RES.0000121101.32286.C8. [DOI] [PubMed] [Google Scholar]
  • 44.Subauste MC, Pertz O, Adamson ED, Turner CE, Junger S, Hahn KM. Vinculin modulation of paxillin-FAK interactions regulates ERK to control survival and motility. J Cell Biol. 2004;165:371–381. doi: 10.1083/jcb.200308011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Conti FJ, Monkley SJ, Wood MR, Critchley DR, Muller U. Talin 1 and 2 are required for myoblast fusion, sarcomere assembly and the maintenance of myotendinous junctions. Development. 2009;136:3597–3606. doi: 10.1242/dev.035857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Zemljic-Harpf AE, Miller JC, Henderson SA, Wright AT, Manso AM, Elsherif L, et al. Cardiac-myocyte-specific excision of the vinculin gene disrupts cellular junctions, causing sudden death or dilated cardiomyopathy. Mol Cell Biol. 2007;27:7522–7537. doi: 10.1128/MCB.00728-07. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data
supp_92_3_409__index.html (2.2KB, html)
supp_cvr247_cvr247supp.doc (104.5KB, doc)
supp_cvr247_cvr247supp_fig1.tif (1.1MB, tif)
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supp_cvr247_cvr247supp_fig5.tif (2MB, tif)
supp_cvr247_cvr247supp_fig6.tif (736.3KB, tif)
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supp_cvr247_cvr247supp_fig8.tif (827.2KB, tif)
supp_cvr247_cvr247supp_fig9.tif (1MB, tif)

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