RhoD is a member of the classical Rho GTPases and it has an essential role in the regulation of actin dynamics. Furthermore, RhoD also localizes to early endosomes and recycling endosomes, indicating additional roles in the regulation of endosome trafficking. A yeast two-hybrid screen identified Zipper-Interacting Protein Kinase (ZIPK) as a RhoD target. We found that RhoD interacts with ZIP kinase in a GTP dependent manner and modulates actin and focal adhesion reorganization. Interestingly, while ectopic expression of ZINC in fibroblasts induces actin reorganization and actomyosin contraction seen as stress fiber bundling and membrane blebbing, the concomitant expression of active RhoD suppressed this phenotype. Previously, RhoD has been associated with focal adhesion regulation, and in line with this notion, we observed that ZIPK resulted in reorganization of focal adhesion and increased adhesion size. Importantly, the RhoD activity suppressed ZIPK-dependent effects on FAR activity, indicating a functional interplay between RhoD and FAK in the focal adhesion dynamics. (C) 2013 Elsevier Inc. All rights reserved.