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 PKC zeta. We demonstrated that ARHGAP21 is relocated to Z-lines and costameres after pressure overload, and interacts with PKC zeta and FAK in control rats (sham), rats submitted to aortic clamping and spontaneously hypertensive rats (SHR). Co-transfection using ARHGAP21 and PKC zeta constructions demonstrated that ARHGAP21 associates with PKC zeta-GST and endogenous FAK. Pull-down assay showed that ARHGAP21 binds to the C-terminal region of FAK. Moreover, ARHGAP21 binds to PKC zeta phosphorylated on Thr410 in sham and SHR. However, ARHGAP21 only binds to FAK phosphorylated on Tyr925 of SHR. Additionally, PKC zeta is phosphorylated by mechanical stimuli. These results suggest that ARHGAP21 may act as a signaling or scaffold protein of FAK and PKC zeta signaling pathways, developing an important function during cardiac stress. (c) 2008 Elsevier Inc. All rights reserved.