Oncogene (2014) 33, 4021–4035 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc REVIEW Rho guanine nucleotide exchange factors: regulators of Rho GTPase activity in development and disease DR Cook1, KL Rossman2,3 and CJ Der1,2,3 The aberrant activity of Ras homologous (Rho) family small GTPases (20 human members) has been implicated in cancer and other human diseases. However, in contrast to the direct mutational activation of Ras found in cancer and developmental disorders, Rho GTPases are activated most commonly in disease by indirect mechanisms. One prevalent mechanism involves aberrant Rho activation via the deregulated expression and/or activity of Rho family guanine nucleotide exchange factors (RhoGEFs). RhoGEFs promote formation of the active GTP-bound state of Rho GTPases. The largest family of RhoGEFs is comprised of the Dbl family RhoGEFs with 70 human members. The multitude of RhoGEFs that activate a single Rho GTPase reflects the very specific role of each RhoGEF in controlling distinct signaling mechanisms involved in Rho activation. In this review, we summarize the role of Dbl RhoGEFs in development and disease, with a focus on Ect2 (epithelial cell transforming squence 2), Tiam1 (T-cell lymphoma invasion and metastasis 1), Vav and P-Rex1/2 (PtdIns(3,4,5)P3 (phosphatidylinositol (3,4,5)-triphosphate)-dependent Rac exchanger). Oncogene (2014) 33, 4021–4035; doi:10.1038/onc.2013.362; published online 16 September 2013 Keywords: Rac1; RhoA; Cdc42; guanine nucleotide exchange factors; cancer; mouse models INTRODUCTION mouse fibroblast focus formation assay that led to the discovery of 10 Ras homologous (Rho) family proteins (20 human members) mutant Ras in human cancer, analysis of genomic DNA isolated comprise a major branch of the Ras superfamily of small GTPases,1 from a human diffuse B-cell lymphoma resulted in the discovery of with RhoA, Rac1 and Cdc42 the most extensively studied the DBL oncogene that encoded an N-terminally truncated and and characterized.2 Rho GTPases specifically regulate actin activated protein.11 Dbl was also detected earlier as an oncogene organization, cell motility, polarity, growth, survival and gene that caused the tumorigenic growth of NIH 3T3 cells after transcription.3 These proteins act as binary switches that cycle transfection with genomic DNA from the MCF-7 human breast between an active GTP-bound and an inactive GDP-bound state carcinoma cell line (designated mcf2),12 and only later was it (Figure 1).4 Rho guanine nucleotide exchange factors (RhoGEFs) determined to be identical to Dbl.13 Dbl was subsequently shown accelerate the intrinsic exchange activity of Rho GTPases to to catalyze the GTP/GDP exchange activity of Cdc42.14 Additional stimulate formation of Rho-GTP.5 Rho GTPase-activating proteins NIH 3T3 focus formation and related biological assays identified (RhoGAPs) stimulate the intrinsic GTP hydrolysis activity of Rho Dbl-related proteins, in particular Vav, Tiam1 and Ect2 (Table 1 and GTPases, resulting in the inactive GDP-bound state. Rho guanine Figure 2). That RhoGEFs were discovered initially as oncoproteins nucleotide-dissociation inhibitors (GDIs) comprise a third class of provided the first suggestion that Rho GTPases may also have a 6 regulatory proteins. RhoGDIs bind Rho GTPases in a GDP-bound function in oncogenesis. state and also extract and sequester Rho GTPases from the cell Dbl and the Dbl-related proteins share a B200-amino-acid membrane where Rho GTPases can be activated. Once activated, catalytic Dbl homology (DH; also called RhoGEF) domain and an the GTP-bound GTPase then associates with effector targets that B 7–9 immediately adjacent regulatory 100-amino-acid pleckstrin number over 100. In this review, we focus on the classical Dbl homology (PH) domain5 (Figure 3). Additional RhoGEFs with this family RhoGEFs and their role in development and disease. In tandem DH–PH domain structure were identified by genetic and particular, we focus on the RhoGEFs with the best validated roles in biochemical approaches and by in silico database searches. There cancer (Vav1/2/3, Ect2 (epithelial cell transforming sequence 2), are 70 human Dbl family RhoGEFs, many of which have conserved Tiam1/2 (T-cell lymphoma invasion and metastasis 1/2), P-Rex1/2 orthologs found in all vertebrate species and in invertebrates, (PtdIns(3,4,5)P3 (phosphatidylinositol (3,4,5)-triphosphate)-dependent including Drosophila, Caenorhabditis elegans, Saccharomyces Rac exchanger)) and their analyses in cell culture, mouse models cerevisiae and S. pombe. The nomenclature for many Dbl RhoGEFs and human cancers. is complicated by the different names used in independent discoveries, by different names for orthologs of different species, by the existence of different gene products because of alter- THE DISCOVERY OF RHOGEFS: DIVERSE IN NUMBERS AND native RNA splicing and by establishment of the ARHGEF STRUCTURE gene family nomenclature by the HUGO Gene Nomenclature The first RhoGEF was identified initially as an oncogene in Committee (http://www.genenames.org/genefamilies/ARHGEF). mammalian cells (Table 1 and Figure 2). Using the same NIH 3T3 We have compiled a summary of Dbl RhoGEF nomenclature, 1Division of Chemical Biology and Medicinal Chemistry, University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Chapel Hill, NC, USA; 2Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA and 3Lineberger Comprehensive Cancer Center, Chapel Hill, NC, USA. Correspondence: Dr CJ Der, Department of Pharmacology, University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27599, USA. E-mail: [email protected] Received 20 May 2013; revised 25 June 2013; accepted 26 June 2013; published online 16 September 2013 Aberrant activity of Rho GTPase in development and disease DR Cook et al 4022 Table 1. Discovery of Dbl RhoGEFs as oncogenes Gene Description References Dbl/Mcf2 Identified as an oncogene that caused NIH 3T3 11,12,83 nude mouse tumorigenic growth after transfection of genomic DNA from the MCF-7 human breast carcinoma cell line (designated mcf2); oncogene in an NIH 3T3 focus formation assay using genomic DNA from a primary human diffuse B-cell leukemia; identified in an NIH 3T3 focus formation screen using genomic DNA from a human nodular poorly differentiated lymphoma Vav Identified as an oncogene that caused NIH 3T3 63 nude mouse tumorigenic growth after transfection by genomic DNA from a human esophageal carcinoma. As this represented the sixth oncogene identified from this group, it was given the name of the sixth letter of the Hebrew alphabet. Ect2 Identified as an oncogene in an NIH 3T3 focus 39,176 formation assay using a cDNA expression library from the Balb/MK mouse keratinocyte cell line; designated epithelial cell transforming cDNA 2 Tim Identified as an oncogene in an NIH 3T3 focus 177 formation assay using a cDNA expression library from a human mammary epithelial cell line; designated transforming immortalized mammary oncogene Net1 Identified as an oncogene in an NIH 3T3 focus 178 formation assay using a cDNA expression library from a human neuroepithelioma cell line; designed neuroepithelial cell transforming gene 1 Lbc Identified as an oncogene that caused NIH 3T3 179 nude mouse tumorigenic growth after transfection by genomic DNA from an acute phase CML; designated lymphoid blast crisis oncogene Figure 1. Rho GTPase regulation. The human Rho GTPase family is Lfc Identified as a transforming gene in an NIH 3T3 180 comprised of 20 members. The majority cycle between GDP-bound focus formation assay in a cDNA expression inactive and GTP-bound active states. Unlike RhoGEFs and RhoGAPs library from the 32D mouse hematopoietic cell that possess shared domains and sequence identity, allowing line; designated Lbc’s first cousin Tiam1 Identified by retrovirus insertion for genes that 51 precise determination of the number encoded in the human induced T-cell lymphoma in vitro; designated genome, the majority of Rho GTPase effectors lack a common T-cell lymphoma invasion and metastasis 1 well-defined recognition domain/motif. The information here Ost/Dbs Identified as a transforming gene in an NIH 3T3 181,182 applies mainly to RhoA, Rac1, Cdc42 and related isoforms. Not all focus formation assay using a cDNA expression Rho GTPases are regulated by GEFs, GAPs and/or GDIs, not all are library generated from the rat osteosarcoma post-translationally modified by a geranylgeranyl isoprenoid lipid, cell line ROS; identified as a transforming gene and some do not have known membrane targeting elements. in an NIH 3T3 focus formation assay in cDNA expression library from the mouse hematopoietic cell line 32D; designated Dbl’s based on the names most commonly used in the literature big sister Lsc/Lip Identified as a transforming gene in an NIH 3T3 177,183 together with the additional names used for each RhoGEF focus formation assay using a cDNA expression (Supplementary Table 1). library generated from the murine myeloid In addition to their common structural elements that define progenitor cell line B6SUtA1; designated Lbc’s them as RhoGEFs, there are some distinctive features that mark second cousin; identified as a transforming gene in an NIH 3T3 focus formation assay in individual RhoGEF proteins. Two RhoGEFs possess tandem sets of genomic DNA from a pleiomorphic DH–PH domains (Trio and Kalirin) and four RhoGEFs (Tuba1, 2, 3 liposarcoma and ARHGEF33) lack an apparent PH domain (Figure 3).5 Dbl Clg Identified as a common-site lymphoma/ 184 RhoGEFs also diverge significantly in the N- and C-terminal leukemia guanine nucleotide exchange factor proviral integration site in mouse leukemias sequences flanking the DH/PH domains. These flanking sequences commonly contain a diversity of protein–protein or protein–lipid interaction domains and motifs whose functions are to regulate intrinsic RhoGEF catalytic activity, determine subcellular localiza- tion and/or facilitate complex formation with other proteins subcellular localization and activation of spatially distinct cellular (Figure 3).