FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes

Article

FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes Graphical Abstract

Authors Alisson Campos Cardoso, Ana Helena Macedo Pereira, Andre Luis Berteli Ambrosio, ..., Marcio Chain Bajgelman, Sandra Martha Gomes Dias, Kleber Gomes Franchini

Correspondence [email protected]

In Brief Cardoso et al. report the crystal structure of the FAK FAT domain in complex with transcription factor MEF2, and present biochemical and cell-based data that FAK upregulates MEF2 transcriptional activity in cardiomyocytes to regulate the expression of the stress responsive gene Jun in response to biomechanical stimulation.

Highlights d

FAK accumulates into the nucleus of cardiomyocytes in response to mechanical stress

d

FAK physically interacts with the MEF2 transcription factor through its FAT domain

d

We report the crystal structures of the FAK FAT domain with the MEF2 dimer

d

FAK cooperates with MEF2 to control load-induced expression of Jun in cardiomyocytes

Cardoso et al., 2016, Structure 24, 1–10 August 2, 2016 ª 2016 Elsevier Ltd. http://dx.doi.org/10.1016/j.str.2016.06.003

Accession Numbers 5F28

Please cite this article in press as: Cardoso et al., FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.06.003

Structure

Article FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes Alisson Campos Cardoso,1 Ana Helena Macedo Pereira,1 Andre Luis Berteli Ambrosio,1 Silvio Roberto Consonni,1 Renata Rocha de Oliveira,1 Marcio Chain Bajgelman,1 Sandra Martha Gomes Dias,1 and Kleber Gomes Franchini1,2,* 1Brazilian

National Laboratory for Biosciences, Center for Research in Energy and Materials, Campinas, Sa˜o Paulo 13084-971, Brazil of Internal Medicine, School of Medicine, University of Campinas, Campinas, Sa˜o Paulo 13081-970, Brazil *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2016.06.003

2Department

SUMMARY

Focal adhesion kinase (FAK) has emerged as a mediator of mechanotransduction in cardiomyocytes, regulating gene expression during hypertrophic remodeling. However, how FAK signaling is relayed onward to the nucleus is unclear. Here, we show that FAK interacts with and regulates myocyte enhancer factor 2 (MEF2), a master cardiac transcriptional regulator. In cardiomyocytes exposed to biomechanical stimulation, FAK accumulates in the nucleus, binds to and upregulates the transcriptional activity of MEF2 through an interaction with the FAK focal adhesion targeting (FAT) domain. In the crystal structure (2.9 A˚ resolution), FAT binds to a stably folded groove in the MEF2 dimer, known to interact with regulatory cofactors. FAK cooperates with MEF2 to enhance the expression of Jun in cardiomyocytes, an important component of hypertrophic response to mechanical stress. These findings underscore a connection between the mechanotransduction involving FAK and transcriptional regulation by MEF2, with potential relevance to the pathogenesis of cardiac disease.

INTRODUCTION Focal adhesion kinase (FAK), an essential non-receptor tyrosine kinase, mediates signaling initiated by integrins and other cell surface receptors, including G protein-coupled receptors, cytokine receptors, and growth-factor receptors (Hall et al., 2011). FAK serves dual roles as both a kinase and a scaffold that function in cellular processes such as migration, proliferation, and survival, as well as biological properties associated with tissue homeostasis and disease (Schaller, 2010). Equally important, FAK has emerged as a key molecule transducing the effects of mechanical forces on intracellular signals, which is critical for such processes as tissue morphogenesis, integrity, and remodeling of many organs, including heart, bone, and lung (Hytonen and Wehrle-Haller, 2015; Tomakidi et al., 2014). In the case of the heart, FAK has been shown to be central for the development

of hypertrophy in response to increased workload, a major pathophysiological process that underlies prevalent and deadly diseases like hypertension and heart failure. To date, cardiacspecific ablation of FAK function protects the heart from loadinduced hypertrophy, while cardiac-specific transgenic FAK expression is sufficient to promote spontaneous hypertrophy of the mouse heart (Clemente et al., 2012; DiMichele et al., 2006; Peng et al., 2006). Phosphatidylinositol 3-kinase/AKT/ mammalian target of rapamycin- and extracellular signal-regulated kinases 1/2-dependent signaling are potential downstream mediators of FAK-induced hypertrophy (Franchini, 2012). Typically regarded as functional in the cytoplasm, FAK can shuttle to the nucleus under appropriate conditions and in a number of different cell types, including cardiomyocytes (Fonseca et al., 2005; Lim et al., 2008, 2012; Luo et al., 2009; Serrels et al., 2015). Emerging evidence in cancer cells indicates that in the nucleus FAK is implicated in the control of gene expression and cell survival by interfering in the function of specific transcription factors, such as p53 and GATA4, and by controlling chromatin structure (Lim, 2013). In cardiomyocytes, we established that signaling mediated by FAK controls the activity of myocytes enhancer factor 2 (MEF2) transcription factors, a coupling that drives the MEF2-induced Jun promoter (Nadruz et al., 2005). Interestingly, downregulation of MEF2A has been attributed a critical role in the cardiac phenotype displayed by FAK knockout mice (Peng et al., 2008), further attesting to functionally relevant connections between FAK and MEF2 in cardiomyocytes. These data led us to consider a possible role for nuclear FAK in regulating MEF2 transcriptional activity in cardiomyocytes. In this paper, we report that mechanical stress triggers nuclear localization and a functionally important interaction of FAK with MEF2 transcription factor in cardiomyocytes. Through a combination of protein crystallography, small-angle X-ray scattering (SAXS) measurements, mutagenesis, reporter gene assays, chromatin immunoprecipitation, and imaging analysis, we show that when cardiomyocytes are exposed to biomechanical stimulation, FAK interacts directly with and upregulates MEF2 transcriptional activity leading to the regulation of the key stress responsive gene Jun. A special aspect of this work is the high-resolution crystal structure showing that the FAK C-terminal focal adhesion targeting (FAT) domain interacts with the MADS-box/MEF2 domain of MEF2. Our results also support that MEF2 serves as a FAK effector to the regulation of the primary stress response gene Jun in cardiomyocytes. Structure 24, 1–10, August 2, 2016 ª 2016 Elsevier Ltd. 1

Please cite this article in press as: Cardoso et al., FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.06.003

Figure 1. FAK Expression, Activity and Localization in Non-stretched and Stretched NRVMs (A) Representative pY397-FAK, FAK, MEF2, and GAPDH-specific immunoblots from extracts of non-stretched (NS) or 1-hr stretched (ST) NRVMs. (B) Representative FAK, MEF2, GAPDH, and Sm-D1 immunoblots from cytosolic (c) and nuclear (n) extracts of NS or ST NRVMs. (C) Confocal maximum-intensity z-projections of NS or ST NRVMs stained with anti-FAK (green) and DAPI (blue). Merged images are composed of FAK and DAPI. (D) Confocal maximum-intensity z-projections of ST NRVMs in which the primary antibody was omitted and used as a negative control. The merge image is composed by Alexa 488 and DAPI. The scale bar represents 20 mm. See also Figure S1. The densitometric readings of immunoblots are shown in Figure S2 and uncropped blots are shown in Figure S6A.

RESULTS FAK Translocates to the Nucleus and Interacts with MEF2 in Cardiomyocytes upon Mechanical Stress To determine the interaction between FAK and MEF2 we used neonatal cardiomyocytes derived from 2-day-old rats (neonatal rat ventricular myocytes [NRVMs]) subjected to cyclic stretch. As observed, controlled biaxial cyclic stretch (10% from the slack length; 60 Hz; 1 hr) causes a substantial increase in Y397 phosphorylation (Figure 1A), which is a hallmark of FAK activation (Schaller et al., 1994). No changes in the FAK, MEF2, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein expression levels were observed in the stretched NRVMs, compared with the non-stretched NRVMs (Figure 1A). A crucial step for FAK interaction with MEF2 would be its translocation to the cell nucleus. Thus, we started by studying the subcellular distribution of FAK by both differential ultracentrifugation and confocal imaging of NRVMs subjected to cyclic stretch. AntiFAK immunoblotting of the cytosolic and nuclear extracts of non-stretched cardiomyocytes indicated a cytosolic distribution of FAK, while in the stretched cells FAK was increased in the nuclear extracts (Figure 1B). Western blots of cardiomyocyte fractions show even loading of GAPDH in the cytosolic and MEF2 and the small nuclear ribonucleoprotein Sm D1 (Sm-D1) in the nuclear extracts. Changes in subcellular distribution of endogenous FAK in NRVMs were further evaluated by immunocytochemistry. Cells were also stained with DAPI (blue) to denote the nucleus. Cyclic stretch enhanced the FAK staining concentrated in the nucleus when compared with non-stretched cells (Figures 1C and 1D). Parallel studies examined changes in the distribution of FAK tagged with the mKO2 fluorophore following cyclic stretch of NRVM. As expected, mKO2-FAK displayed 2 Structure 24, 1–10, August 2, 2016

prominent nuclear localization in the stretched NRVM, in contrast to the non-stretched cells, which showed no significant accumulation of the fused protein in the nucleus (Figure S1). To explore whether nuclear FAK might associate with MEF2, cytosolic and nuclear extracts obtained from non-stretched or stretched NRVM were subjected to immunoprecipitation using anti-MEF2 antibody. As shown in Figure 2A, the anti-MEF2 antibody precipitated FAK from nuclear extracts, preferentially from those obtained from stretched cardiomyocytes. Additional coimmunoprecipitation assays performed with nuclear extracts confirmed the interaction of FAK with nuclear MEF2 in stretched NVRMs. An unrelated nuclear protein anti-Sm-D1, used as control, did not interact with FAK (Figure 2B). Taken together, these data indicate that FAK accumulates and associates with MEF2 transcription factors in the nucleus of cardiomyocytes stimulated by cyclic stretch. The FAK FAT Domain Interacts Directly with the MADSBox/MEF2 Domain Members of the MEF2 family of transcription factors (MEF2A-D) contain highly conserved N-terminal MADS-box and MEF2 domains, which are responsible for protein dimerization, sequence-specific DNA-binding, and interaction with transcription cofactors (Black and Olson, 1998). In contrast, the C-terminal region (transactivation domain) is highly variable among the MEF2 family members. The full-length structure of FAK contains the FERM (band 4.1, ezrin, radixin, moesin) and the FAT domains situated N- and C-terminal to the kinase domain, respectively (Figure 2C). While FERM regulates activation of the kinase domain (Cooper et al., 2003; Lietha et al., 2007), the four-helix bundle FAT mediates FAK interaction with distinct protein partners as well as its subcellular localization (Hayashi et al., 2002;

Please cite this article in press as: Cardoso et al., FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.06.003

Figure 2. FAK Interacts with MEF2 in Stretched Cardiomyocytes (A) MEF2 immune complex isolated from cytosolic and nuclear extracts of NS and ST NRVMs. Samples were probed using specific antibodies for FAK and MEF2 as indicated. (B) Representative co-immunoprecipitation experiment using anti-FAK, anti-MEF2, and anti-Sm-D1 antibodies and nuclear extracts from NS and ST NRVMs. (C) Schematic representation of mouse FAK and MEF2 domains and the residue numbers of the related amino acids. (D) Pull-down experiments using constructs of FAK domains fused to GST (bottom) and nuclear extracts of ST NRVMs. The upper panel shows the representative MEF2 immunoblot used to detect the association with the constructs of FAK domains. (E) Fluorescence polarization curve of FAT domain binding to FITC-MEF2C_95. Kd is shown as the best-fit value of three repeat experiments, performed in three technical replicates. See also Figure S4. The error bars are SD. The uncropped blots are shown in Figure S6B.

Hildebrand et al., 1993). To narrow down which FAK domains are involved in the association with MEF2, we performed pull-down assays using recombinant glutathione-S-transferase (GST)tagged N-terminal FERM (GST-FERM), kinase (GST-KINASE), and C-terminal (GST-FAT) domains of FAK, and demonstrated that only the GST-FAT construct precipitated MEF2 from nuclear extracts of stretched cardiomyocytes (Figure 2D). The quantification of the interaction was performed by the titration of recombinant FAK FAT domain to fluorescein-labeled MEF2C_95 (MADS-box/MEF2 domain of MEF2C, residues 1–95) in a fluorescence polarization assay yielded a dissociation constant (Kd) of 7 ± 0.8 mM (Figure 2E). In addition, a microscale thermophoresis (MST) assay was performed with an unlabeled FAT domain (0.003–50 mM) titrated into a fixed concentration of labeled MEF2C_95 (100 nM), which yielded an apparent Kd of 10.0 ± 2.0 mM (Figure S4). The data indicate that the FAT domain of FAK and the MADS-box/MEF2 domain of MEF2 are necessary and sufficient to mediate a high-affinity interaction between FAK and MEF2. Structural Basis for the Binding of the FAK FAT Domain to MEF2C To obtain insights on the specificities of this interaction, we determined the crystal structure of the FAT:MEF2C_95 heterocomplex at 2.9 A˚ resolution, solved by molecular replacement using FAT (PDB: 1K40) (Hayashi et al., 2002) and MEF2A (PDB: 3KOV) (Wu et al., 2010) structures as search models. Parameters

and statistics of data collection and model refinement are summarized in Table 1. Overall, the asymmetric unit is composed of one FAT dimer, where each one of the subunits is bound to a MEF2C_95 dimer, and a third FAT monomer (FAT-3) inserted in the middle of the FAT dimer in the asymmetric unit (Figure 3A). The main contact region within the hetero-complex is formed between the helix a2 of each FAT monomer and a solvent-exposed pocket formed by a pair of H2 helices of the MEF2 dimer. Additional contacts are also shared between FAT a3 helices and the MEF2 H2 helix (Figures 3B and 3C). The interface is located on the opposite side of the MEF2 DNA-binding site. The FAT dimer in the asymmetric unit is stabilized by a crosscontact via the swap of helices a1 (Figure 3A; FAT-1 and FAT-2). Such self-arrangement has been previously described in crystal structures of FAT (Arold et al., 2002) with alleged biological implications (Dixon et al., 2004; Kadare et al., 2015; Zhou et al., 2006). However, we observed that the interaction with MEF2 occurs independently of the FAT a1-swapped dimeric conformation, as indicated by the SAXS experiments (described below; Figures 4A and 4B). In addition, a third FAT molecule is also present in the asymmetric unit (Figure 3A; FAT-3) inserted in the middle of the FAT dimer, where the a3 and a4 helices of FAT-3 make contacts with the a1 of FAT-1 and with the a4 of FAT-2. Although not involved in the direct interaction with MEF2, this additional FAT-3 molecule is crucial for mediating extensive contacts with symmetry mates. Intrigued by such an intricate assembly found in the crystallographic asymmetric unit, we sought to determine the minimum biologically relevant organization of the FAT:MEF2C_95 complex. In this regard, we performed SAXS measurement of the FAT:MEF2C_95 complex in solution and compared the results Structure 24, 1–10, August 2, 2016 3

Please cite this article in press as: Cardoso et al., FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.06.003

Table 1. X-Ray Data Collection and Refinement Statistics: Molecular Replacement PDB:

5F28

Data Collection Space group

P 42 21 2

Cell dimensions a, b, c (A˚)

139.21, 139.21, 90.35

a, b, g ( )

90.0, 90.0, 90.0

Resolution (A˚)

49.22–2.90 (3.08–2.90)a

Rpim

0.096 (0.433)

Mn(I) half-set correlation CC(1/2)

0.99 (0.51)

I/sI

8.7 (2.0)

Completeness (%)

100 (100)

Redundancy

13.7 (13.7)

Refinement Resolution (A˚)

43.2–2.9

No. of reflections

20,211

Rwork/Rfree

0.208/0.235

No. of atoms Protein

5,418

Water

58

B factors Protein

48.70

Water

37.80

Rmsd Bond lengths (A˚)

0.004

Bond angles ( )

0.6

All-atom clashscore

2.11

a

Values in parentheses are for highest-resolution shell.

of the predicted scattering to combinations of the complexes found in the asymmetric unit. Collectively, the scattering curve (Figure 4A), Guinier plot, and p(r) (Figure S3) strongly suggest that an arrangement in which a single FAT molecule bound to one MEF2C_95 dimer (calculated Rg of 2.06 nm) is the most probable complex in solution, compared with the full asymmetric unit content (3 FAT monomers and 2 MEF2C_95 dimers, calculated Rg of 2.98 nm). Therefore, we propose that the FAT monomer bound to an MEF2 dimer is the biologically relevant organization (Figure 4B). Interface Analysis of the FAT:MEF2C_95 Complex A PISA server (Protein, Interfaces, Surfaces and Assemblies) analysis showed that each FAT:MEF2C_95 interaction occluded about 660 A˚2 of solvent-accessible area, with a solvation free energy gain of
7 kcal/mol. The FAT a2 helix (residues 949–974) forms a triple-helix bundle with the MEF2 dimer H2 helices, in which residues Pro952, Val954, Lys955, and Arg962 in FAT make extensive contacts with residues Asp63, Leu66, Leu67, Tyr69, Thr70, Tyr72, and Asn73 located on the dimer cleft of MEF2C_95 (Figures 3B and 3C). More specifically, the FAT Leu959 side chain binds to a hydrophobic pocket formed by MEF2C_95 Leu66 and Thr70 residues (Figure 3B). On the other hand, the FAT domain a3 helix (residues 977–1005) engages in4 Structure 24, 1–10, August 2, 2016

teractions with chain B of MEF2 helix H2. Residues Asp991, Leu994, and Ile998 contact residues Leu67 and Asn73 at helix H2 of MEF2C_95 chain B (Figure 3C). In order to validate the proposed interface of FAT:MEF2C_95, we performed several point-mutations and assayed the complex formation by pulling down the recombinant purified proteins. As expected, GST-MEF2C_95 pulled down FAT wild-type construct. While the FAT mutations K955E, L959E, and R962E attenuated the interaction with MEF2C_95, the mutations I998E and K1018 did not affect the interaction (Figure 4C). Far-UV circular dichroism confirmed that such mutations had no disruptive impact on FAT folding (Figure S3). In addition, fluorescence polarization assays were performed by the titration of recombinant FAT mutant K955E to fluorescein-labeled MEF2C_95. As expected, the FAT mutant K955E which interacts poorly with MEF2 shifted the interaction curve to the right (Figure S4). FAK Binds to MEF2 and Forms a Ternary Complex with DNA The crystallographic type of interaction between MEF2C_95 and FAT raises the interesting possibility that FAK may establish a ternary complex with MEF2 and DNA. To test this possibility, we performed an electrophoretic mobility shift assay. While the addition of MEF2C_95 alone causes a specific shift of the responsive element, FAT itself does not. However, addition of FAT displaced even further the MEF2C_95 band, indicating the formation of the ternary complex MEF2:FAK:DNA (Figure S5). Given the observation that FAK:MEF2 can form a ternary complex with DNA, we wanted to devise a model for this association. In this context, we superposed the FAK:MEF2 complex to the already described structures of Cabin-1:MEF2B:DNA (PDB: 1N6J) (Han et al., 2003), HDAC9:MEF2B:DNA (PDB: 1TQE) (Han et al., 2005), and p300 TAZ2:MEF2A:DNA (PDB: 3P57) (He et al., 2011). The Ca backbones of the MEF2 chain A of the four structures were superimposed on one another. These analyses confirmed that FAT:MEF2 association is compatible with MEF2 association to DNA, with some minor adjustments of flexible loops (Ca root-mean-square deviation [rmsd] of 2.3 A˚ for chain A of MEF2). It also shows that the FAT a2 helix binds to MEF2 in a manner similar to the Cabin1, HDAC9, and p300 a helices fragments, with the a-helix long axis lying diagonally on each MEF2 monomer’s helix H2 (Figure 4D). These observations further imply that FAK may have to compete with these proteins for MEF2 interaction. FAK Regulates the Transcriptional Activity of MEF2 To investigate the potential impact of FAK signaling on MEF2 activity, we co-transfected the cardiomyoblast H9c2 cell line with a MEF2-responsive reporter (3xMEF2-LUC) (Naya et al., 1999) and plasmids expressing mutants or wild-type FAK. In these cells, overexpression of wild-type FAK strongly stimulated (5-fold) the reporter expression over baseline (Figure 5A). Remarkably, FAK mutants K955E, L959E, and R962E (shown to disrupt the binding of FAT domain to MEF2C_95) failed to enhance the reporter expression to the same level as the wild-type FAK or I998E and K1018E mutants. These data strongly suggest that the interaction with FAK stimulates MEF2 transcriptional activity.

Please cite this article in press as: Cardoso et al., FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.06.003

Figure 3. Structure of the FAK FAT Domain Bound to MEF2C_95 (A) Overall views of the FAT:MEF2C_95 complex crystal structure in cartoon representation. (B and C) FAT:MEF2C_95 complex interface showing the a2 and a3 helices of FAT (gray) and helices H2 of the MEF2 dimer (green and cyan). Interacting residues are shown in stick model mode, colored by element: carbon (by chain), nitrogen (blue), and oxygen (red). Residues that make hydrophobic interactions are shown as sticks and dots, and labeled with black fonts.

FAK Cooperates with MEF2C to Regulate the Expression of the Proto-Oncogene Jun Prior studies from our laboratory have implicated a co-operation between FAK and MEF2 in the stress-induced transcriptional activation of the MEF2’s target Jun in cardiomyocytes (Nadruz et al., 2005). Jun is an established transcriptional driver of load-induced cardiac hypertrophy (Wang et al., 1998; Windak et al., 2013). Given the physical and functional interactions between FAK and MEF2, we tested whether FAK might play a role on the regulation of Jun transcription. Accordingly, we carried out chromatin immunoprecipitation (ChIP) followed by PCR (ChIP-PCR) of the Jun promoter region in samples of control and overloaded rat left ventricle to ask whether the recruitment of FAK by MEF2 mediates increased MEF2 binding to this region. Both anti-FAK and anti-MEF2 antibodies precipitated the Jun promoter region containing the MEF2-binding site (Figure 5B). While the anti-MEF2 antibody immunoprecipitates the Jun promoter in samples of control and overloaded rat hearts, the anti-FAK antibody immunoprecipitates the Jun promoter region only on samples of overloaded hearts, suggesting that FAK and MEF2 interact and may contribute to regulate the load-induce Jun expression in the heart. To further confirm whether FAK can influence MEF2 transcriptional control of Jun we expressed full-length MEF2C and a myc-

tagged version of FAK in NRVMs. The results show that MEF2C, but not FAK expression alone, could increase the expression of Jun transcripts above the basal level. However, FAK co-expression potentiated the upregulation of Jun transcripts induced by MEF2C in NRVMs (Figure 5C). Notably, co-expression of FAK mutant K955E or the empty vector did not interfere in the activity of MEF2C toward Jun expression. To further strengthen this observation, we transfected NRVMs with the luciferase reporter gene containing MEF2-reponsive Jun promoter (pJTXGL3-LUC) (Clarke et al., 1998). Overexpression of MEF2C resulted in a 2.5-fold induction of luciferase expression, while overexpression of FAK alone did not change the gene reporter expression (Figure 5D). Furthermore, the overexpression of both MEF2C and FAK, but not the FAK mutant K955E or the empty vector, strongly stimulated the expression of the reporter gene. These results show that FAK potentiates the MEF2 induction of Jun promoter. DISCUSSION Better defining the molecular processes underlying changes in gene expression that occur in cardiomyocytes in response to mechanical stress is key to the understanding of pathogenesis of cardiac disease. Here, we have unraveled an unappreciated Structure 24, 1–10, August 2, 2016 5

Please cite this article in press as: Cardoso et al., FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.06.003

Figure 4. SAXS Analysis, Mutagenesis, and Structural Comparison of the FAT: MEF2C_95 Complex with Related Structures (A) Experimental SAXS data of the FAT:MEF2C_95 complex (blue dots). The superposition of the calculated scattering curve of the best model obtained by BUNCH and the crystallographic model are shown in red and green lines, respectively. (B) Ab initio model, calculated from the SAXS data, shown as blue beads and the complex as a cartoon. (C) Pull-down assays performed with GSTMEF2C_95 and FAT-WT or mutants, as indicated. Top panel: localization of the mutant residues showed as spheres. Middle panel: FAK FAT immunoblot used to detect the association with the GST-MEF2C_95 construct. Bottom panel: Coomassie-stained SDS-PAGE of purified FATWT or mutants. MW, molecular weight. (D) Backbone superposition of different structures of protein:MEF2 complexes (FAK FAT, Cabin1, HDAC9, and p300). The structures are superimposed by the Ca backbone of the MEF2 chain A (green) from the four related structures. The DNA from the related structures is shown in orange. See also Figures S3 and S4 and Table S1. The uncropped blot is shown in Figure S6C.

interaction between FAK and MEF2 mediated by the FAK FAT domain that underlies the coupling of mechanotransduction to transcriptional regulation in cardiomyocytes. X-ray crystallography, SAXS, and biochemistry were used to define the structural basis for such interaction and provide insights into how FAK can influence MEF2 transcriptional activity. Importantly, the experiments we report in isolated cardiomyocytes, H9C2 cell lineage, and overloaded left ventricle of rats demonstrate that the interaction with FAK stimulates MEF2 transcriptional activity. We established that the FAK:MEF2 complex associates with chromatin and modulates the load-induced expression of the stress responsive gene Jun, a highly pleiotropic transcription factor implicated in the survival and hypertrophic responses of cardiomyocytes to mechanical stress. Evidence in this study and that reported earlier indicate that mechanical stress increases FAK activity and its nuclear accumulation in cardiomyocytes (Fonseca et al., 2005; Senyo et al., 2007; Torsoni et al., 2003). Studies using endothelial cells, muscle cells, and fibroblasts have reported a substantial increase of nuclear FAK in response to cellular stress (Lim et al., 2012; Luo et al., 2009). In addition, FAK has similarly been found in the nucleus of different cancer cells (Lim et al., 2008; Serrels et al., 2015). Little information is available on the trafficking route that drives FAK to the nucleus, but the FAK N-terminal FERM domain contains a nuclear localization signal that is necessary for FAK nuclear translocation, and the FAK kinase domain contains a functional nuclear export signal (Lim et al., 2008; Ossovskaya et al., 2008). In line with these observations, evidence has accumulated to support newly 6 Structure 24, 1–10, August 2, 2016

emerging duties for FAK in gene transcription and chromatin remodeling. A striking example was the discovery that nuclear FAK drives signals to cell survival in cancer cells by inactivating tumor suppressor protein p53 (Lim et al., 2008). Further, nuclear FAK was shown to interact with the core promoter complex transcription factor IID to stimulate the transcription of chemokines and cytokines, a mechanism that enables tumor immunological tolerance by the local recruitment of regulatory T cells (Serrels et al., 2015). Extending these observations, we now provide multiple levels of evidence that FAK directly activates MEF2 transcriptional activity toward upregulation of Jun in cardiomyocytes. First, we show by immunoblotting of subcellular fractions and immunofluorescence that FAK accumulates in the nucleus of cardiomyocytes undergoing mechanical stress. Second, we show by co-immunoprecipitation, pull-down, and co-crystallization that FAK interacts directly with MEF2 through a high-affinity binding of its FAT domain to MEF2. Third, we show by using reporter gene and gel shift assays that the interaction with FAK activates MEF2 transcriptional activity. Finally, we show by a combination of reporter gene and ChIP assay studies that FAK co-occupies Jun promoter with MEF2 in overloaded neonatal cardiomyocytes and in adult rat heart, promoting Jun upregulation in response to mechanical stress. While these results highlight the important role of the FAK:MEF2 complex in the control of Jun, arguably, this complex might be involved in the control of several genetic programs, as can be anticipated by the extensive array of genes regulated by MEF2 transcription factors in cardiomyocytes (Potthoff and Olson, 2007). A complete understanding of how interplay with FAK results in regulation of MEF2 function requires knowledge of the structural

Please cite this article in press as: Cardoso et al., FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.06.003

Figure 5. FAK Activates MEF2 Transcriptional Activity (A) Luciferase reporter assay performed in H9c2 cardiomyoblasts co-transfected with 3xMEF2LUC and full-length myc-tagged FAK (FAK) or mutants, as indicated. The lower panel shows representative Myc-tag and GAPDH immunoblotting used as a loading control. (B) Representative scheme of rat Jun promoter and primers used for ChIP-PCR assay (upper). The lower panel shows the representative PCR of the ChIP assay for the Jun promoter region containing a conserved MEF2-binding site (106 bp upstream from the transcription starting site). Myocardial extracts were isolated from samples of rat left ventricle after sham-operation (SO) or transverse aortic constriction (TAC; 1 hr). ChIP assays were performed with antibody to FAK, MEF2, or no antibody (No-Ab) used as control for immunoprecipitation. Input shows the PCR product of total chromatin without prior immunoprecipitation (n = 4). (C) Real-time PCR of Jun gene in samples of NRVMs transfected with constructions as indicated. (D) Luciferase reporter assay performed in NRVMs co-transfected with a luciferase reporter gene containing MEF2-reponsive Jun promoter (pJTXGL3LUC) and constructs as indicated. Full-length MEF2C (M), myc-FAK (FAK), myc-FAK mutant K955E (M + K955E), empty myc vector (Vector). Data in (A, C, and D) represent the average fold induction from four independent experiments; error bars show SEM. *p < 0.05 vs. CT; #p < 0.05 vs. M by one-way ANOVA followed by Bonferroni’s multiple comparison test. See also Figure S5. The uncropped blots are shown in Figure S6D.

features of the FAK:MEF2 complex. A structural model of MEF2, based on the previously determined crystal structure of the MEF2-DNA binary complex displayed a hydrophobic pocket that sits in the opposite side of the DNA-binding surface at the core of the dimeric arrangement of MADS-box/MEF2 domains (Wu et al., 2010). This pocket is an intrinsic structural property of MEF2 that provides a binding site for the MEF2 transcriptional cofactors (Han et al., 2003, 2005; He et al., 2011). Our current data reveal that this pocket also provides a platform for binding of the amphipathic a2 helix of FAT, while the FAT a3 helix engages additional contacts with the MEF2 groove. Critical for the interaction were the side chains of FAK FAT a2 helix K955 and R962 that establish salt bridges with the MEF2 pocket. Located at the center of the FAT:MEF2 interface is L959 of the FAT a2 helix, the side chain of which inserts deeply into a hydrophobic site formed at the bottom of the MEF2 dimer. Further evidence that these residues are relevant in cells has come from the observation that FAK mutation at these sites attenuates the interaction with MEF2 in vivo, as well as the activation of the Jun reporter gene. These observations suggest that the 3D helix a2 structure, rather than the sequence of the amino acids, is the dominant feature recognized by the MEF2 dimer. In fact, the interaction of the FAT a2 helix with MEF2 is reminiscent of the ones observed for cofactors of MEF2 such as p300, Cabin1, and HDAC9 that interact with the MEF2 dimer through a conserved amphipathic helix (Han et al., 2003, 2005; He et al., 2011). Thus, MEF2 has the potential to recognize substrates with a similar 3D structure, and this function might serve

as a mechanism of signal integration with multiple signaling pathways. That FAK, by binding to MEF2, functions as a co-activator is supported by the observation that addition of FAK enhances the expression of the MEF2 tandem reporter gene in cellular assays. Our current study provides a fundamental framework to aid our understanding of signaling mediated by mechanical stress in cardiomyocytes. The insights derived from the delineation of the structure of the FAK:MEF2 complex and its relevance for transcription in overloaded cardiomyocytes could provide a rationale for developing small molecules as therapeutic agents in heart disease. EXPERIMENTAL PROCEDURES Antibodies The following specific antibodies were used in the study: FAK (C-20; sc-558, Santa Cruz Biotechnology); FAK (A-17 sc-557, Santa Cruz Biotechnology); MEF2 (C-21; sc-313, Santa Cruz Biotechnology); GAPDH (FL-335, sc25778, Santa Cruz Biotechnology); Sm-D1 (C15, sc-20822, Santa Cruz Biotechnology); Phospho-FAK pTyr397 (44625G; Invitrogen); and Myc-tag (mAb9E10, Sigma-Aldrich). Cell Culture Primary cardiomyocytes were prepared from neonatal rat hearts (NRVMs) according to the protocol described previously (Pereira et al., 2014). The cardiomyocytes cultured in Bioflex plates were stretched in a Flexercell FX3000 strain unit to 10% of their resting length at a frequency of 1 Hz (0.5-s stretch/0.5-s relaxation) for 1 hr. At the conclusion of the experimental protocols, cells were scraped from membranes and lysed for immunoblotting or

Structure 24, 1–10, August 2, 2016 7

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fixed for analysis by fluorescence microscopy. The H9c2 cell lines were obtained from ATCC (catalog no. CRL-1446; American Type Culture Collection) and maintained according to ATCC instructions. All animals were handled in compliance with the principles of laboratory animal care formulated by the Animal Care and Use Committee of the State University of Campinas (protocol number 3095-1). Confocal Microscopy Fixed NRVMs were blocked with 1% BSA, 0.1% Triton X, and 50 nM glycine in 0.1 M PBS on ice. Then samples were incubated with anti-FAK A-17 (1:100, Santa Cruz Biotechnology) antibody overnight at 4 C. Next, Alexa Fluor 488conjugated goat anti-rabbit (1:200) secondary antibodies were used at room temperature. Slides were then mounted using VECTASHIELD with DAPI. Samples were examined using a Leica TCS SP8 confocal on a Leica DMI6000. Immunoprecipitation NRVMs homogenized in lysis buffer were normalized (500 mg) and incubated with anti-MEF2 antibody (2 mg). The proteins were collected after addition of 30 ml of protein A Sepharose beads (GE Healthcare). Western blots of the immunoprecipitates were performed with anti-FAK c-20 or anti-MEF2 antibodies, used at a dilution of 1:1,000 for immunoblotting. Quantification of mRNAs Total RNA was isolated from extracts of NRVMs as previously described (Santos et al., 2012). For mRNA quantification, target genes expression was analyzed by SYBR Green qRT-PCR (Stratagene). The reactions were performed using the SYBR Green (with Dissociation Curve) program on the Mx3000TM comparative qPCR system (Stratagene). The oligonucleotides used were: Jun sense, 50 -ATT GCT TCT GTA GTG CTC CG-30 ; Jun antisense, 50 -GTT CAT CCG CAA TCT AGC CT-30 ; GAPDH sense, 50 -GGC ATT GCT CTC ATG ACA A-30 ; GAPDH antisense, 50 -ATG TAG GCC ATG AGG TCC AC-30 . All reactions were performed with reference dye normalization. The median cycle threshold value was used for analysis, and all cycle threshold values were normalized to the GAPDH mRNA expression level. Pull-Down Assay The pGEX2T constructs encoding the GST-FERM, GST-KINASE, and GSTFAT fusion proteins were described previously (Santos et al., 2012). GSTFERM, GST-KINASE, or GST-FAT were incubated with the nuclear extracts from NRVMs. Nuclear protein extract was obtained with the NE-PER Nuclear Protein Extraction Kit (Thermo Fisher Scientific). The precipitated pellets were resolved on SDS-PAGE and the membranes immunoblotted with MEF2 antibody. Fluorescence Polarization Assay Purified MEF2C_95 was labeled with the fluorescein isothiocyanate (FITC) fluorophore (Thermo Fisher Scientific) by mixing 50 mM of protein with 10-fold molar excess of FITC in 50 mM borate buffer (pH 8.5) for 1 hr at room temperature in the dark. The excess FITC was removed by desalting (HiTrap Desalting column, GE Healthcare). In order to explore the binding affinity between the recombinant FAT constructions and MEF2C_95, we performed an assay by incubating 100 nM of fluorescein-labeled MEF2C_95 with unlabeled FAT constructions at concentrations ranging from 0.05 to 100 mM. The assays were performed at 20 C in 50 mM Tris buffer (pH 7.5), 150 mM NaCl, and 1 mM DTT in a CLARIOstar microplate reader (BMG Labtech) with excitation and emission wavelengths of 480 and 520 nm, respectively. Experimental values were output as mean ± SD and logistic sigmoidal curves fitted to determine the Kd for each protein:protein complex interaction, according to the equation described previously (Santos et al., 2012). Microscale Thermophoresis The affinity of the FAK FAT domain and MEF2C_95 was also measured by MST. MST measurements were performed with a Monolith NT.115 Instrument (NanoTemper Technologies). Assays were performed by incubating 100 nM of fluorescein-labeled MEF2C_95 (as described above) with unlabeled FAT domain at concentrations ranging from 0.003 to 50 mM. The samples were loaded into premium type glass capillaries (NanoTemper Technologies). Measurements were performed in 50 mM Tris buffer (pH 7.5), 150 mM NaCl, and

8 Structure 24, 1–10, August 2, 2016

0.05% Tween 20. Measurements were performed at 20% LED power and 20% IR-laser power (MST power) with a laser-on time of 30 s and a laser-off time of 5 s. Data were analyzed using NanoTemper MO. Affinity Analysis software, v2.1.2 to determine the Kd. Protein Purification The mouse FAK FAT domain (residues 904–1052) and MEF2C_95 (residues 1–95) were cloned in pET-28a with tobacco etch virus (TEV) protease site and small ubiquitin-related modifier (SUMO) fusion, respectively. The proteins were expressed in Escherichia coli BL21(C41) pRARE2 and lysed on ice with lysis buffer (50 mM Tris-HCl [pH 7.5], 300 mM NaCl, 5 mM imidazole, 1 mM 2-mercaptoethanol, and 5% glycerol) containing a protease inhibitors cocktail. The soluble extract was purified by affinity chromatography using Ni-NTA Superflow resin (Qiagen). Next, ion-exchange chromatography was performed using buffer A (50 mM Tris [pH 7.5], 50 mM NaCl, 1 mM DTT) and buffer B (50 mM Tris [pH 7.5], 1 M NaCl, 1 mM DTT) using HiTrap Q HP and HiTrap SP HP (GE Healthcare). The purified fusion proteins were cleaved with the specific proteases (TEV and SUMO proteases for FAT and MEF2C_95 constructions, respectively), followed by size-exclusion chromatography (HiLoad 16/ 60 Superdex 75 pg, GE Healthcare) in buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM DTT). The FAT:MEF2C_95 complex was obtained by incubation of the previously purified FAT and MEF2C_95 (1:2 M; overnight; 4 C). Crystallization and X-Ray Crystallography The complex was concentrated to 10 mg/ml for crystallization. Crystals were grown at 18 C by the hanging-drop method with a reservoir buffer of 0.1 M magnesium acetate, 0.1 M 2-(N-morpholino)ethanesulfonic acid (pH 6.5), and 12% PEG 8000. On average, the crystals grew to 50 mm in length over the course of 3 weeks. The X-ray diffraction dataset was obtained at beamline ID29 (ESRF), using a wavelength of 0.8729 A˚. Data were processed using Mosflm (Leslie, 1992) and merged and scaled with Aimless (Evans and Murshudov, 2013). The first set of phases was obtained by the molecular replacement technique as implemented in Phaser (McCoy et al., 2007), using the structures of FAT (PDB: 1K40) (Hayashi et al., 2002) and MEF2A (PDB: 3KOV) (Wu et al., 2010) as search models. The region comprising residues 2–91 in the 3KOV dimer (MEF2A) was used as a search model for molecular replacement. However, during refinement of the MEF2C structure herein described, the absence of well-structured atoms for the stretch between residues 2–19 led to the removal of the respective constituent atoms from the final model. Positional and B factor refinement cycles were carried out with Phenix (Adams et al., 2010). Manual building of the extra portions and real space refinement, including Fourier electron density map inspection, were performed with COOT (Emsley et al., 2010). Solvent water molecules, treated as oxygen atoms, were added using the appropriate COOT routine. The overall stereochemical quality of the final models and the agreements between them and experimental data were assessed using Molprobit (Chen et al., 2010) and the appropriate Phaser and COOT routines. Ramachandran plot analysis indicates that 97.6% (or 646) of the modeled residues in the asymmetric unit fall on the preferred regions, while 2.1% (or 14) are found in the allowed regions. Two residues (or 0.3%) are considered outliers. In the final model, the calculated rmsd between the all-matching components (main and side-chain atoms) defining the two equivalent MEF2:FAT interfacing interactions in the asymmetric unit are 0.95 A˚ (between MEF residues 61–75 in chains A and C, 128 atoms each), 0.8 A˚ (between MEF residues 61–75 in chains B and D, 128 atoms each), and 1 A˚ (between FAT residues 948–973 in chains E and F, 201 atoms each). SAXS Analyses and Model Building Scattering data were collected at the D02A-SAXS2 beamline of the Brazilian Synchrotron Light Laboratory (LNLS), Campinas, Brazil, at a wavelength of l = 1.488 A˚, and covered the momentum transfer range 0.33 < s < 3.99 nm
1 (s = 4p sin q/l, where 2q is the scattering angle). The data were normalized to the incident beam intensity and corrected for the detector response using an in-house program. The data were analyzed and processed using the programs contained in the ATSAS package (Petoukhov et al., 2007). In order to check for radiation damage in the sample, three frames of 60 s were collected and compared using PRIMUS (Konarev et al., 2003). The same program was used to average the frames and subtract the buffer.

Please cite this article in press as: Cardoso et al., FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.06.003

Two different protein concentrations (3 and 10 mg/ml on 50 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1 mM DTT) were evaluated for aggregation by following increases in the measured Rg (radius of gyration) as calculated by autoRg. The Rg was confirmed by using the indirect Fourier transform program GNOM (Semenyuk and Svergun, 1991; Svergun, 1992), which was also used to calculate the distribution function p(r) and Dmax. Crysol was used to calculate the fitting of the experimental data (at 10 mg/ml) to the 3-molecule model (1FAT:2MEF2) and 7-molecule model (3 FAT:4 MEF2, found within the crystal asymmetric unit). The ab initio dummy residue modeling was performed as implemented by Gasbor (Svergun et al., 2001). The results of ten independent runs were averaged by DAMAVER (Volkov and Svergun, 2003) and the obtained damfilt model was superposed to the crystal structure using SUPCOMB (Kozin and Svergun, 2001). Statistical Analyses Data are presented as means ± SEM or means ± SD and analyzed by one-way ANOVA with Bonferroni multiple comparisons test for post hoc comparisons or by Student’s t test. No statistical methods were used to predetermine sample size. Statistical significance of categorical values between groups was designated p < 0.05. ACCESSION NUMBERS The atomic coordinates and structure factors have been deposited in the PDB under accession code PDB: 5F28. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, six figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.str.2016.06.003.

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AUTHOR CONTRIBUTIONS

Evans, P.R., and Murshudov, G.N. (2013). How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214.

A.C.C. and K.G.F. designed the research; A.C.C. and A.L.B.A. performed the protein crystallization, A.C.C., A.H.M.P., and S.R.C., performed the biological imaging, A.C.C. and A.H.M.P. performed the cell-based assays, A.C.C. and R.R.O. performed the mutagenesis and protein purification, S.R.C. and M.C.B. performed the lentiviral production, A.C.C., A.L.B.A., and S.M.G.D. performed the SAXS experiments. All authors discussed and interpreted results. A.C.C. and K.G.F. wrote the paper. All authors contributed to writing and providing feedback.

Fonseca, P.M., Inoue, R.Y., Kobarg, C.B., Crosara-Alberto, D.P., Kobarg, J., and Franchini, K.G. (2005). Targeting to C-terminal myosin heavy chain may explain mechanotransduction involving focal adhesion kinase in cardiac myocytes. Circ. Res. 96, 73–81.

ACKNOWLEDGMENTS This work was funded by the Sa˜o Paulo Research Foundation (FAPESP; Grants 2008/53519-5, 2008/53583-5) and the Brazilian National Research Council (CNPq; Grants 304366/2009-9, 312203/2012-8). We thank the Biosciences National Laboratory (LNBio) and the Synchrotron Light Laboratory (LNLS) at the Brazilian Center for Research in Energy and Materials (CNPEM), for financial support and access to all facilities (LPP, LVV, LEC, LBE, LIB, Robolab, and D02A-SAXS2 beamline). We thank Andrea Dessen (LNBio, Campinas, Brazil, and IBS, Grenoble, France) and Carlos Contreras-Martel (IBS, Grenoble, France) for data collection, and the European Synchrotron Radiation Facility (ESRF) ID29 beamline. Received: January 15, 2016 Revised: May 6, 2016 Accepted: June 4, 2016 Published: July 14, 2016

Franchini, K.G. (2012). Focal adhesion kinase – the basis of local hypertrophic signaling domains. J. Mol. Cell Cardiol. 52, 485–492. Hall, J.E., Fu, W., and Schaller, M.D. (2011). Focal adhesion kinase: exploring Fak structure to gain insight into function. Int. Rev. Cell Mol. Biol. 288, 185–225. Han, A., Pan, F., Stroud, J.C., Youn, H.D., Liu, J.O., and Chen, L. (2003). Sequence-specific recruitment of transcriptional co-repressor Cabin1 by myocyte enhancer factor-2. Nature 422, 730–734. Han, A., He, J., Wu, Y., Liu, J.O., and Chen, L. (2005). Mechanism of recruitment of class II histone deacetylases by myocyte enhancer factor-2. J. Mol. Biol. 345, 91–102. Hayashi, I., Vuori, K., and Liddington, R.C. (2002). The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin. Nat. Struct. Biol. 9, 101–106. He, J., Ye, J., Cai, Y., Riquelme, C., Liu, J.O., Liu, X., Han, A., and Chen, L. (2011). Structure of p300 bound to MEF2 on DNA reveals a mechanism of enhanceosome assembly. Nucleic Acids Res. 39, 4464–4474. Hildebrand, J.D., Schaller, M.D., and Parsons, J.T. (1993). Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions. J. Cell Biol. 123, 993–1005.

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