Biochemical and Biophysical Research Communications 374 (2008) 641–646

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Biochemical and Biophysical Research Communications

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ARHGAP21 associates with FAK and PKCf and is redistributed after cardiacpressure overload

Luciene Borges a, Carolina Louzão Bigarella a, Mariana Ozello Baratti a, Daniella P. Crosara-Alberto a,Paulo P. Joazeiro b, Kleber G. Franchini c, Fernando F. Costa a, Sara Teresinha Olalla Saad a,*

a Department of Internal Medicine, Hematology and Hemotherapy Center, School of Medical Science, University of Campinas—UNICAMP, Campinas, São Paulo 13083-970, Brazilb Department of Histology and Embryology, UNICAMP, Brazilc Department of Internal Medicine, Cardiology, UNICAMP, Brazil

a r t i c l e i n f o

Article history:Received 1 July 2008Available online 26 July 2008

Keywords:ARHGAP21CardiomyocyteAcute mechanical stressRatCell signaling

0006-291X/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.bbrc.2008.07.085

Abbreviations: LV, left ventricle; NWR, normal Wihypertensive rats; RhoGAP, Rho GTPase activating pprotein kinase C; FAK, focal adhesion kinase; FAT, focband 4.1, ezrin, radixin, moesin; MAPK, mitogen-acphosphatidylinositol phosphate-3,4,5-triphosphate; N

* Corresponding author. Address: Hemocentro—UNCidade Universitária ‘‘Zeferino Vaz”, CEP 13083-970,19 3289 1089.

E-mail address: sara@unicamp.br (S.T.O. Saad).

a b s t r a c t

ARHGAP21 is highly expressed in the heart, which demonstrates activity over Cdc42 and interacts withproteins of the cytoskeleton and adherent junctions. The main cause of cardiac hypertrophy is mechanicalstimulus; therefore we analyzed ARHGAP21 expression after acute mechanical stress in the myocardiumand its association with FAK and PKCf. We demonstrated that ARHGAP21 is relocated to Z-lines andcostameres after pressure overload, and interacts with PKCf and FAK in control rats (sham), rats submit-ted to aortic clamping and spontaneously hypertensive rats (SHR). Co-transfection using ARHGAP21 andPKCf constructions demonstrated that ARHGAP21 associates with PKCf-GST and endogenous FAK. Pull-down assay showed that ARHGAP21 binds to the C-terminal region of FAK. Moreover, ARHGAP21 bindsto PKCf phosphorylated on Thr410 in sham and SHR. However, ARHGAP21 only binds to FAK phosphor-ylated on Tyr925 of SHR. Additionally, PKCf is phosphorylated by mechanical stimuli. These results sug-gest that ARHGAP21 may act as a signaling or scaffold protein of FAK and PKCf signaling pathways,developing an important function during cardiac stress.

� 2008 Elsevier Inc. All rights reserved.

The recently described member of RhoGAPs, ARHGAP21, codes overload, which imposes a mechanical stress on cardiac walls

for 1957aa protein with PDZ, pleckstrin homology (PH) and Rho-GAP domains [1]. Although this gene is highly expressed in brain,skeletal, and cardiac muscles, there are few reports regarding ARH-GAP21, and none regarding function in the heart. ARHGAP21 is lo-cated in nucleus, juxtanuclear, and adherent junctions of severalcell lines, acting preferentially upon Cdc42 and RhoA, which havea fundamental participation during cardiac hypertrophy [2,3].Moreover, ARHGAP21 interacts with ARF1, ARF6, and a-catenin,important proteins in cytoskeleton and adherent junctions [4,5],suggesting that this protein may play a crucial role in the signalingcascade activated by cardiac hypertrophic stimuli.

Cardiac hypertrophy involves different stimuli, e.g., neurohu-moral activation, growth factors, cytokines, and mechanical stress[6]. A primary event that triggers cardiac hypertrophy is pressure

ll rights reserved.

star rats; SHR, spontaneouslyrotein; PKCf, zeta isotype ofal adhesion targeting; FERM,tivated protein kinase; PIP3,FjB, nuclear factor kappa B.

ICAMP, R. Carlos Chagas, 480,Campinas/SP, Brazil. Fax: +55

affecting pathways such as proliferation, differentiation, growth,and cellular survival. Protein complexes involved in these eventsengage members of the small GTPases, such as RhoA, Rac1, andCdc42. Focal adhesion kinase (FAK) controls the cardiac hypertro-phic genetic program, growth and cardiac cell survival, with phos-phorylation state and activity tightly linked to cardiac stress afterpressure overload [7–9]. PKCf is a regulator of cardiomyocyte myo-filament protein phosphorylation [10], but PKCf participation incardiac remodeling process is controversial. However, PKCfinvolvement in MAPK and NFjB pathways [11,12], well-knownproteins implicated in cardiac hypertrophy, indicates that PKCfmight play an important role during the hypertrophic process.

Herein, we demonstrated that ARHGAP21 relocates from thenucleus to cardiomyocyte Z-lines and costameres after pressureoverload, implying a possible role of ARHGAP21 in mechanicalstress. Additionally, ARHGAP21 interacts with PKCf and the car-boxyl-terminal portion of FAK in NWR, SHR, and animals submit-ted to aortic clamping (AoCo).

Materials and methods

Experimental animal model. Male Wistar rats (160–200 g) under-went acute pressure overload induced by controlled constriction of

642 L. Borges et al. / Biochemical and Biophysical Research Communications 374 (2008) 641–646

transversal aorta as previously described [13]. Left ventricle (LV) ofmale spontaneously hypertensive rats (SHR) at 8 weeks of agewere used to extract total protein for co-immunoprecipitationand Pulldown assays. Three independent experiments were carriedout for each procedure. The animals were obtained from the Ani-mal Breeding Center of the University of Campinas. The investiga-tion conforms to the Guide for the Care and Use of LaboratoryAnimals published by the US National Institutes of Health (NIHPublication No. 85-23, revised 1996).

Subcellular fractionation. Performed as previously described[14]. Nuclear proteins were obtained from the pellet of LV extractscentrifuged at 1000g. Supernatant was centrifuged at 100,000gseparating cytoplasm (supernatant) from membrane fractions(pellet).

Cell culture and transfection. Myoblast cell line C2C12 (ATCC)was cultivated in DMEM (Gibco) supplemented with 10% fetal bo-vine serum (Gibco) at 37 �C in a 5% CO2 atmosphere. GST-taggedpEBG2T-PKCf-WT was a kind gift from Dr. Gail Fraser (School ofLife Sciences, University of Dundee) [15], and V5-taggedpcDNA3.1-N-termARHGAP and -pcDNA3.1C-termARHGAP were akind gift from Dr. Pascale Cossart (Unité des Interactions bacté-ries-Cellules, Institut Pasteur, INSERM U604) [5]. Briefly, we trans-fected C2C12 cells using Lipofectamine 2000 (Invitrogen) followingthe manufacturer’s protocol. Forty-eight hours afterwards, cellswere selected using 1200 lg/mL of G418 during 3 weeks.

Immunoprecipitation and Western blot. Tissue preparation wasperformed as previously described [16]. Total extracts or immuno-precipitated proteins containing an equal amount of protein wereloaded to each lane, resolved on SDS–PAGE and transferred tomembranes, which were incubated with specific antibodies.

DNA constructs and pulldown assay. pGEX2T, carrying the FAK N-terminal domain (GST-FERM; residues 1–400), pGEX2TK, encodingC-terminal of FAK (GST-C-term; residues 765–1052), and pGEX-KG, containing the catalytic domain sequence (GST-CAT; residues390–696), were given to us by Jun-Lin Guan (FERM domain; Molec-ular Medicine, Cornell University, Ithaca, NY), and Michael Schaller(CAT and C-terminal domains; Microbiology and Cancer Center,University of Virginia School of Medicine). All constructions wereinserted into Escherichia coli BL21 (RIL) Codon Plus and performedas previously described [17–19]. Glutathione beads, conjugatedwith recombinant proteins, were used for the pulldown assayusing NWR and SHR LV extracts. Pulled down pellets were resolvedon SDS–PAGE gel and Western blot was performed as above.

Myocyte isolation and confocal analysis. Cardiomyocytes wereisolated through retrograde perfusion of collagenase buffer as pre-viously described [16]. Cells were fixed in 4% paraformaldehydeand incubated with anti-ARHGAP21 antibody. Slides were labeledwith AlexaFluor�488-conjugated secondary antibody and TRITC-conjugated phalloidin. Positive immunoreactivity was visualizedby laser confocal scanning (Zeiss LM510).

Immunoelectron microscopy. Performed as previously described[8]. LV fragments were fixed in paraformaldehyde/glutaraldehydeand embedded in LR White resin. Thin-sections were stained withanti-ARHGAP21 antibody and then incubated with secondary anti-body, and with gold-conjugated streptavidin (SAV-G/10 nm Sig-ma). The grids were stained with 5% uranyl acetate and 0.5% leadcitrate, and photographed in a transmission electron microscope(LEO 906).

Results

Relocation of ARHGAP21 to Z-lines and costameres after pressureoverload

We separated LV into cytoplasmic (CF), membrane (MF), andnuclear (NF) fractions. ARHGAP21 was weakly expressed in CF

(Fig. 1A) and expression decreased in cardiomyocyte NF afteraortic clamping (AoCo) when compared with control animals(sham). In contrast, a progressive increase of ARHGAP21 oc-curred in MF.

Confocal microscopy demonstrated that ARHGAP21 is hardlyexpressed in myocyte nucleus from AoCo hearts (Fig. 1B; lower),whereas sham cardiomyocytes had their nucleus completely la-beled (Fig. 1B; upper). Contrary to the weak signals in the interca-lated disks of sham rats, we observed a strong ARHGAP21 stainingin those structures after AoCo. A similar pattern was observed inthe plasma membrane labeling of ARHGAP21, which was faint insham rats and strong in AoCo cardiomyocytes.

Immunoelectron microscopy revealed ARHGAP21 located incardiomyocyte costameres and Z-lines from AoCo hearts (Fig. 2C–E). However, ARHGAP21 is located throughout the myocyte myofil-aments of sham animals (Fig. 2A and B). Confocal and immunoelec-tron microscopy negative controls were obtained by incubationwith the primary antibody omitted (Supplemental).

ARHGAP21 associates with PKCf and carboxyl-terminal of FAK andtheir active forms

ARHGAP21 interactions with FAK and PKCf were examined innormal Wistar rat (NWR), rats submitted to AoCo, and spontane-ously hypertensive rats (SHR). We used SHR animal due to in-creased LV weight observed in SHR from the age of two months,corresponding to early hypertrophy [20]. Corresponding LV ex-tracts were incubated with anti-ARHGAP21, -FAK or -PKCf anti-body. Immunoprecipitation assay revealed ARHGAP21 associatedwith FAK and PKCf (Fig. 3A and B). All interactions were observedin NWR, AoCo animals and SHR. We also transfected C2C12 cellline with constructions expressing GST-tagged PKCf-WT, V5-tagged C-terminal and N-terminal portion of ARHGAP21 (Fig. 4Aand B). We observed that anti-V5 antibody co-immunoprecipitatedboth PKCf-GST and endogenous FAK. Moreover, anti-GST antibodypulled ARHGAP21 down but not FAK. Transfections were per-formed twice.

Three different regions of FAK were incubated with NWR andSHR LV extracts. We demonstrated that the GST-C-terminal ofFAK pulled endogenous ARHGAP21 down but neither GST-FERMnor GST-CAT precipitated ARHGAP21 in either NWR or SHR(Fig. 3C).

The ability of ARHGAP21 to associate with active forms of FAKand PKCf was also examined (Fig. 3D). ARHGAP21 associated withPKCf phosphorylated on Thr410 in both NWR and SHR. Further-more, ARHGAP21 interacted with FAK phosphorylated on Tyr925only in SHR.

Effect of pressure overload on PKCf, a-catenin and Cdc42

We evaluated Cdc42 and a-catenin distribution, previously de-scribed as interacting with ARHGAP21. AoCo modulated neither a-catenin nor Cdc42 contents (Fig. 4D). Moreover, PKCf phosphoryla-tion on Thr410 increased in cytoplasm and membrane fractions ofAoCo hearts, when compared with total PKCf content (Fig. 4C). P-FAK Tyr925 increase was also verified, according to a previousstudy (data not shown) [8].

Discussion

In the present study, we demonstrated that ARHGAP21 is re-cruited from the nuclei and myofilaments to costameres, Z-lines,and intercalated disks of cardiomyocytes after AoCo. These datasuggest that ARHGAP21 is modulated by mechanical stimuli andcan develop an important role during cardiac stress and earlyhypertrophy.

Fig. 1. ARHGAP21 distribution in controls (sham) and in adult rats submitted to pressure overload (AoCo). (A) Subcellular fractionation of two LV submitted to AoCo atdifferent times (sham, 30 min, 1 and 2 h). Antibodies anti-actin and anti-histone H1 were used as control. (B) Confocal analysis. LV cardiomyocytes were labeled with bothphalloidin-TRITC (red) and anti-ARHGAP21 antibody recognized by AlexaFluor�488-conjugated antibody (green). (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of this paper.)

Fig. 2. Immunoelectron microscopy of cardiomyocytes from rats submitted or notto AoCo. (A) ARHGAP21 in sham animals; (B) details of (A), demonstratingARHGAP21 located throughout myofilaments fibers; (C) ARHGAP21 in ratssubmitted to AoCo for 2 h; (D,E) details of (C), showing ARHGAP21 located in thecostameres (D) and Z-lines (E) after AoCo.

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Moreover, ARHGAP21 associates with PKCf in cardiac cells ofrats submitted to AoCo, sham and SHR. We also demonstrated thatARHGAP21 interacts with PKCf phosphorylated on Thr410, a keyresidue for PKCf activation [21], and PKCf was phosphorylatedon Thr410 in cytoplasm and membrane fractions by AoCo. The roleof PKCf during cardiac hypertrophy is controversial. Changes inPKCf contents in particulate fractions were not detectable in LVof rats submitted to AoCo [22,23]. However, a study demonstratedthat PKCf contents increased in cardiomyocytes submitted to vol-ume overload-induced hypertrophy [24]. These two types ofhemodynamic stimuli activated distinct patterns of signal trans-duction [25], possibly explaining the discrepancy in the PKCfexpression found in previously studies [22–24]. However, noneof these studies reported the PKCf phosphorylation state; thereforethis is the first study to assess PKCf activation during pressureoverload-induced hypertrophy.

PKCf is activated by PI-3,4,5-triphosphate (PIP3) or ligands, e.g.,TNFa [26]. TNFa is enhanced during hemodynamic overloading ofheart [27,28] and induces PKCf phosphorylation in cardiomyocytes[29]. Recently, Wu et al. showed that the constitutive active formof PKCf, mutating in the phosphorylation site T410E, inducedtranslocation of PKCf from cardiomyocyte myofilaments to Z-discs,concluding that PKCf is a regulator of sarcomeric protein phos-phorylation [10]. Interestingly, after AoCo, ARHGAP21 repeatedthe behavior of PKCf phosphorylated on Thr410, suggesting thatPKCf also relocates to Z-discs by pressure overload. Many studiesdemonstrated the involvement of PKCf in MAPK and NFjB activa-tion, important proteins in proliferation and survival pathways andon gene expression of cardiac hypertrophic processes [30,31]. Thus,PKCf may play a role upon MAPK and NFjB signaling and act as aregulator in sarcomeric protein phosphorylation during cardiachypertrophy. As ARHGAP21 interacts with PKCf, the role of

Fig. 3. ARHGAP21 associated with FAK and PKCf. Heart protein extracts, from: (A) sham and rats submitted to AoCo for 2 h, and (B) NWR and SHR; were immunoprecipitated(IP) and immunoblotted with anti-ARHGAP21, anti-FAK, and anti-PKCf antibodies. (C) ARHGAP21 binds to the C-terminal region of FAK. Pulldown assay using GST-taggedFAK constructions: N-terminal FERM domain (FERM-GST), catalytic domain (CAT-GST) and C-terminal region (C-term). Anti-c-Src and anti-GST antibodies used as control. (D)NWR and SHR heart protein extracts were IP with anti-ARHGAP21 and immunoblotted with anti-P-PKCf Thr410, and anti-P-FAK Tyr925 antibodies.

Fig. 4. Co-transfection of C2C12 cells with V5-tagged C- and N-terminal portions of ARHGAP21, and GST-tagged PKCf-WT. (A) Whole-cell extracts from C2C12 transfectedwith C-termARHGAP21-V5, C-termARHGAP21-V5 plus PKCf-WT-GST, N-termARHGAP21-V5, and N-termARHGAP21-V5 plus PKCf-WT-GST. (B) Total extract from transfectedC2C12 with N-termARHGAP21-V5 plus PKCf-WT-GST (lane 1). Respective extracts of transfected C2C12 submitted to IP using anti-V5 (lanes 4–7) or anti-GST (lanes 2 and 3).Normal IgG used as negative control (lane 8). (C) Subcellular fractionation of LV from sham and AoCo rats (sham, 30 min, 1 and 2 h). The phosphorylation of PKCf on Thr410increased in cytoplasm (CF) and membrane (MF) fractions of cardiomyocytes after AoCo. (D) Cdc42 is expressed in CF and MF but not in the nuclei (NF) fraction, whereas a-catenin is more expressed in the NF and MF.

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ARHGAP21 could well be to control mechanisms of cardiomyocytecontraction, survival and gene expression.

We also showed that ARHGAP21 binds to the C-terminal por-tion of FAK in normal rats and SHR. FAK plays crucial role in avariety of intracellular signaling, such as migration, proliferation,differentiation, and adhesion [32]. Initial FAK activation is pro-moted by autophosphorylation at Tyr397 [33]. Src further phos-phorylates FAK at several tyrosine sites, enhancing the catalyticactivity of FAK [34]. FAK is essential for myofibrillogenesis andcostamerogenesis in skeletal muscle, and the expression of C-ter-minal FAT domain of FAK displaces endogenous FAK from focal

adhesions [35]. Moreover, FAK is a well-described protein in-volved in cardiac hypertrophy [7,8,13], and is located at cardio-myocyte focal adhesions and costameres after heart pressureoverload [33].

As FAK recruits other proteins to the sub-membranous region ofthe cell, and as both FAK [8] and ARHGAP21 are located in costa-meres and Z-lines after AoCo, this re-localization could be medi-ated through the interaction of ARHGAP21 with the C-terminalregion of FAK. Additionally, ARHGAP21 possibly participates inthe focal adhesion turnover and cytoskeleton assembly, regulatingFAK activity.

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This evidence is reinforced by the interaction found betweenARHGAP21 and FAK phosphorylated on Tyr925 of SHR. Src-medi-ated phosphorylation of FAK on Tyr925 could displace paxillin,promoting FAK dissociation from focal contacts [36]. This eventis essential for focal adhesion turnover, a process enhanced bymechanical stimuli [37,38]. Recent studies demonstrated thatFAK phosphorylated on Tyr397 accumulates in membrane andintercalated disks [39] Thus, FAK phosphorylated on Tyr397 couldactivate PI3K [40], providing a local source of PIP3, a protein up-stream from PKCf, responsible for direct or indirect activation.

We hypothesized that ARHGAP21 interaction with FAK pro-motes relocation to Z-lines, intercalated disks, and costameres.These regions are composed by transmembrane proteins, integrins,N-cadherin, and associated proteins, such as b-catenin and a-cate-nin, developing a regulatory function in cardiomyocyte communi-cation [41]. In fact, a study showed that a-catenin is necessary forintercalated disk formation and vinculin recruitment, and the cad-herin/catenin/vinculin complex plays a critical role in intercalateddisk structure after stress [42]. As ARHGAP21 has been described tobe a partner of a-catenin, we believe that ARHGAP21 acts as a sig-naling molecule or scaffold protein, orchestrating the catenin/FAK/PKCf signaling pathway.

Taken together, our results suggest that ARHGAP21 might reg-ulate cytoskeleton assembly and control FAK and PKCf activities,orchestrating these two important signaling pathways, and mayhave a crucial role during mechanical stress in the heart.

Acknowledgments

This work was supported by Fundação de Amparo à Pesquisa doEstado de São Paulo (FAPESP) and Conselho Nacional de Desen-volvimento Científico e Tecnológico (CNPq). We thank JuvaniL. Saturno for immunoelectron microscopy and Raquel S. Fogliofor English revision. We are also thankful to Dr. Gail Fraser,Dr. Pascale Cossart, Dr. Jun-Lin Guan, and Dr. Michael Schaller forthe constructions, and to the students and employees involved inthe study.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbrc.2008.07.085.

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