ARHGAP21 associates with FAK and PKCζ and is redistributed after cardiac pressure overload

https://doi.org/10.1016/j.bbrc.2008.07.085Get rights and content

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 PKCζ. We demonstrated that ARHGAP21 is relocated to Z-lines and costameres after pressure overload, and interacts with PKCζ and FAK in control rats (sham), rats submitted to aortic clamping and spontaneously hypertensive rats (SHR). Co-transfection using ARHGAP21 and PKCζ constructions demonstrated that ARHGAP21 associates with PKCζ-GST and endogenous FAK. Pulldown assay showed that ARHGAP21 binds to the C-terminal region of FAK. Moreover, ARHGAP21 binds to PKCζ phosphorylated on Thr410 in sham and SHR. However, ARHGAP21 only binds to FAK phosphorylated on Tyr925 of SHR. Additionally, PKCζ is phosphorylated by mechanical stimuli. These results suggest that ARHGAP21 may act as a signaling or scaffold protein of FAK and PKCζ signaling pathways, developing an important function during cardiac stress.

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Materials and methods

Experimental animal model. Male Wistar rats (160–200 g) underwent acute pressure overload induced by controlled constriction of transversal aorta as previously described [13]. Left ventricle (LV) of male spontaneously hypertensive rats (SHR) at 8 weeks of age were used to extract total protein for co-immunoprecipitation and Pulldown assays. Three independent experiments were carried out for each procedure. The animals were obtained from the Animal Breeding Center of the University of Campinas.

Relocation of ARHGAP21 to Z-lines and costameres after pressure overload

We separated LV into cytoplasmic (CF), membrane (MF), and nuclear (NF) fractions. ARHGAP21 was weakly expressed in CF (Fig. 1A) and expression decreased in cardiomyocyte NF after aortic clamping (AoCo) when compared with control animals (sham). In contrast, a progressive increase of ARHGAP21 occurred in MF.

Confocal microscopy demonstrated that ARHGAP21 is hardly expressed in myocyte nucleus from AoCo hearts (Fig. 1B; lower), whereas sham cardiomyocytes had their nucleus completely labeled (Fig.

Discussion

In the present study, we demonstrated that ARHGAP21 is recruited from the nuclei and myofilaments to costameres, Z-lines, and intercalated disks of cardiomyocytes after AoCo. These data suggest that ARHGAP21 is modulated by mechanical stimuli and can develop an important role during cardiac stress and early hypertrophy.

Moreover, ARHGAP21 associates with PKCζ in cardiac cells of rats submitted to AoCo, sham and SHR. We also demonstrated that ARHGAP21 interacts with PKCζ phosphorylated on Thr410,

Acknowledgments

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We thank Juvani L. Saturno for immunoelectron microscopy and Raquel S. Foglio for English revision. We are also thankful to Dr. Gail Fraser, Dr. Pascale Cossart, Dr. Jun-Lin Guan, and Dr. Michael Schaller for the constructions, and to the students and employees involved in the study.

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