Identification of a Novel Actin-Binding Domain within the Rho Guanine Nucleotide Exchange Factor TEM4 Natalia Mitin*, Kent L. Rossman, Channing J. Der Lineberger Comprehensive Cancer Center and Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America Abstract Spatio-temporal activation of Rho GTPases is essential for their function in a variety of biological processes and is achieved in part by regulating the localization of their activators, the Rho guanine nucleotide exchange factors (RhoGEFs). In this study, we provide the first characterization of the full-length protein encoded by RhoGEF TEM4 and delineate its domain structure, catalytic activity, and subcellular localization. First, we determined that TEM4 can stimulate guanine nucleotide exchange on RhoA and the related RhoB and RhoC isoforms. Second, we determined that TEM4, like other Dbl RhoGEFs, contains a functional pleckstrin homology (PH) domain immediately C-terminal to the catalytic Dbl homology (DH) domain. Third, using immunofluorescence analysis, we showed that TEM4 localizes to the actin cytoskeleton through sequences in the N-terminus of TEM4 independently of the DH/PH domains. Using site-directed mutagenesis and deletion analysis, we identified a minimal region between residues 81 and 135 that binds directly to F-actin and has an ,90-fold higher affinity for ATP-loaded F-actin. Finally, we demonstrated that a single point mutation (R130D) within full-length TEM4 abolishes actin binding and localization of TEM4 to the actin cytoskeleton, as well as dampens the in vivo activity of TEM4 towards RhoC. Taken together, our data demonstrate that TEM4 contains a novel actin binding domain and binding to actin is essential for TEM4 subcellular localization and activity. The unique subcellular localization of TEM4 suggests a spatially- restricted activity and expands the diversity of mechanisms by which RhoGEF function can be regulated. Citation: Mitin N, Rossman KL, Der CJ (2012) Identification of a Novel Actin-Binding Domain within the Rho Guanine Nucleotide Exchange Factor TEM4. PLoS ONE 7(7): e41876. doi:10.1371/journal.pone.0041876 Editor: Anna Maria Delprato, BioScience Project, United States of America Received May 25, 2012; Accepted June 27, 2012; Published July 24, 2012 Copyright: 2012 Mitin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH GI SPORE Developmental Research grant to N.M. and K.L.R. (CA106991) and NIH grant CA129610 to C.J.D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nmitin@med.unc.edu Introduction Rho family GTPases are molecular switches that cycle between inactive, GDP-bound and active, GTP-bound states [1]. Rho- GEFs catalyze the exchange of bound GDP for GTP on Rho GTPases rendering them biologically active to signal to down- stream effectors. The largest family of RhoGEFs in humans, with 68 human members, is the Dbl family of proteins [2,3], which is characterized by a tandem catalytic Dbl homology (DH) and regulatory pleckstrin homology (PH) domain cassette responsible for accelerating the intrinsic nucleotide exchange activity of one or more members of the Rho family small GTPases. Together with DOCK family RhoGEFs, there are over 80 RhoGEFs that can act on 12 out of 20 Rho GTPases (8 Rho GTPases are predicted or verified to be constitutively activated and regulated by RhoGEF- independent mechanisms) [4]. This apparent signaling redundan- cy is perhaps most striking for activators of RhoA, where at least 24 RhoGEFs have been documented for this single Rho GTPase [2,3]. Beyond the DH-PH cassette, Dbl family proteins are highly divergent and possess other interaction motifs or catalytic domains that account for the distinct mechanisms by which the activity of Dbl family RhoGEFs is regulated [2]. For example, the Dbl family RhoGEF, GEF-H1 is sequestered by microtubules through the C- terminal region of GEF-H1 and its release off the microtubular lattice promotes the spatial activation of RhoA and changes in the actin cytoskeleton essential for cellular migration [5–7]. Another Dbl family RhoGEF, Ect2 is sequestered in the nucleus during interphase and is released during mitosis to bind the centralspin- dlin complex to promote spatio-temporal activation of RhoA and formation of the actin-rich contractile ring that is essential for cytokinesis [8–10]. Thus, each RhoGEF can integrate distinct stimulus-dependent, spatio-temporally restricted Rho GTPase activation which leads to a rearrangement of the actin cytoskeleton essential for a variety of cellular processes. In this report, we show that the RhoGEF, TEM4/ARHGEF17 [11], displays a subcellular localization unique amongst RhoGEF family members as it was associated with actin stress fibers. We found that TEM4 binds actin directly through a novel actin binding domain (ABD) within the N-terminus, and the TEM4 ABD is essential for TEM4 localization to the actin cytoskeleton and the in vivo activity. Materials and Methods Expression Constructs, Cell Culture and Reagents A full-length TEM4/ARHGEF17 (NM_014786) 7.5 kb cDNA sequence was obtained by subcloning the exon (PCR amplified from human genomic DNA) encoding the first 456 N-terminal amino acids into the KIAA0337 clone (Kazusa DNA Research Institute, Japan) in pBS vector (Stratagene). cDNAs encoding full- length or truncation mutants of TEM4 were subsequently subcloned into the pEGFP mammalian expression vector PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e41876