ARHGAP21 associates with FAK and PKCf and is redistributed after cardiac pressure 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, Brazil b Department of Histology and Embryology, UNICAMP, Brazil c Department of Internal Medicine, Cardiology, UNICAMP, Brazil article info Article history: Received 1 July 2008 Available online 26 July 2008 Keywords: ARHGAP21 Cardiomyocyte Acute mechanical stress Rat Cell signaling abstract ARHGAP21 is highly expressed in the heart, which demonstrates activity over Cdc42 and interacts with proteins of the cytoskeleton and adherent junctions. The main cause of cardiac hypertrophy is mechanical stimulus; therefore we analyzed ARHGAP21 expression after acute mechanical stress in the myocardium and its association with FAK and PKCf. We demonstrated that ARHGAP21 is relocated to Z-lines and costameres 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 and PKCf 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 binds to 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. Ó 2008 Elsevier Inc. All rights reserved. The recently described member of RhoGAPs, ARHGAP21, codes 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 several cell lines, acting preferentially upon Cdc42 and RhoA, which have a 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 signaling cascade 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 overload, which imposes a mechanical stress on cardiac walls affecting pathways such as proliferation, differentiation, growth, and cellular survival. Protein complexes involved in these events engage members of the small GTPases, such as RhoA, Rac1, and Cdc42. 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 after pressure overload [7–9]. PKCf is a regulator of cardiomyocyte myo- filament protein phosphorylation [10], but PKCf participation in cardiac remodeling process is controversial. However, PKCf involvement in MAPK and NFjB pathways [11,12], well-known proteins implicated in cardiac hypertrophy, indicates that PKCf might play an important role during the hypertrophic process. Herein, we demonstrated that ARHGAP21 relocates from the nucleus to cardiomyocyte Z-lines and costameres after pressure overload, implying a possible role of ARHGAP21 in mechanical stress. 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 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.07.085 Abbreviations: LV, left ventricle; NWR, normal Wistar rats; SHR, spontaneously hypertensive rats; RhoGAP, Rho GTPase activating protein; PKCf, zeta isotype of protein kinase C; FAK, focal adhesion kinase; FAT, focal adhesion targeting; FERM, band 4.1, ezrin, radixin, moesin; MAPK, mitogen-activated protein kinase; PIP3, phosphatidylinositol phosphate-3,4,5-triphosphate; NFjB, nuclear factor kappa B. * Corresponding author. Address: Hemocentro—UNICAMP, R. Carlos Chagas, 480, Cidade Universitária ‘‘Zeferino Vaz”, CEP 13083-970, Campinas/SP, Brazil. Fax: +55 19 3289 1089. E-mail address: sara@unicamp.br (S.T.O. Saad). Biochemical and Biophysical Research Communications 374 (2008) 641–646 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc