A Novel Family of DOCK180-Related Proteins 4903
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Research Article 4901 Identification of an evolutionarily conserved superfamily of DOCK180-related proteins with guanine nucleotide exchange activity Jean-François Côté and Kristiina Vuori* The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA *Author for correspondence (e-mail: [email protected]) Accepted 11 October 2002 Journal of Cell Science 115, 4901-4913 © 2002 The Company of Biologists Ltd doi:10.1242/jcs.00219 Summary Mammalian DOCK180 protein and its orthologues DOCK180-mediated Rac activation in vivo. Importantly, Myoblast City (MBC) and CED-5 in Drosophila and we have identified several novel homologues of DOCK180 Caenorhabditis elegans, respectively, function as critical that possess this domain and found that many of them regulators of the small GTPase Rac during several directly bind to and exchange GDP for GTP both in vitro fundamentally important biological processes, such as and in vivo on either Rac or another Rho-family member, cell motility and phagocytosis. The mechanism by which Cdc42. Our studies therefore identify a novel protein DOCK180 and its orthologues regulate Rac has remained domain that interacts with and activates GTPases and elusive. We report here the identification of a domain suggest the presence of an evolutionarily conserved within DOCK180 named DHR-2 (Dock Homology Region- DOCK180-related superfamily of exchange factors. 2) that specifically binds to nucleotide-free Rac and activates Rac in vitro. Our studies further demonstrate that the DHR-2 domain is both necessary and sufficient for Key words: Cytoskeleton, GTPase, Rac, Signaling Introduction (Albert et al., 2000; Cheresh et al., 1999; Dolfi et al., 1998; Mammalian DOCK180 was originally identified as a 180 kDa Gumienny et al., 2001; Kiyokawa et al., 1998a). Additionally, protein that interacts with the proto-oncogene product c-Crk mice lacking DOCK2, which is a DOCK180 homologue (Hasegawa et al., 1996). Recent evidence have demonstrated exclusively expressed in hematopoietic cells (Nishihara, 1999; that DOCK180 and its orthologues in Drosophila and Nishihara et al., 1999), are deficient in lymphocyte migration Caenorbaditis elegans form an evolutionarily conserved and Rac activation in response to chemokines (Fukui et al., protein family [denoted the CDM family (Wu and Horvitz, 2001). 1998)] that regulates several important biological processes. The mechanism by which DOCK180 regulates Rac has The Drosophila orthologue of DOCK180, which is known as remained elusive. Overexpression of DOCK180 or DOCK2 in Myoblast City (MBC), has an essential role in myoblast fusion, 293 cells leads to GTP loading of Rac, and these proteins also dorsal closure and cytoskeletal organization during embryonic associate with nucleotide-free Rac in cell lysates (Kiyokawa et development (Erickson et al., 1997; Rushton et al., 1995). al., 1998a; Nishihara et al., 1999; Nolan et al., 1998). It is not CED-5, which is the C. elegans orthologue of DOCK180, has known, however, whether DOCK180 itself, or one of its in turn been implicated in phagocytosis of apoptotic cell associated protein, interacts with Rac and catalyzes its corpses and in distal tip cell migration (Wu and Horvitz, conversion to GTP-Rac. Like other GTPases, Rac is active 1998). Human DOCK180 is also involved in cytoskeletal when bound to GTP and inactive when bound to GDP. reorganization. Expression of a membrane-targeted form Conversion of the GDP-bound proteins to the active state is of DOCK180 induces spreading of NIH 3T3 cells, and catalyzed by guanine nucleotide exchange factors (GEFs) (Van coexpression of DOCK180 with Crk and its binding partner Aelst and D’Souza-Schorey, 1997). Of note, DOCK180, p130Cas results in accumulation of DOCK180 in focal DOCK2, MBC and CED-5 lack the typical tandem Dbl- complexes (Hasegawa et al., 1996; Kiyokawa et al., 1998b). homology and Pleckstrin-homology (DH-PH) domains found Genetic data in both Drosophila and C. elegans strongly in most GEFs that are involved in activation of Rho-family suggest that DOCK180 mediates its effects by functioning as GTPases, such as Rho, Rac and Cdc42 (Schmidt and Hall, an upstream activator of the small GTPase Rac, which is a 2002). Recently, ELMO1/CED-12 was identified as an regulator of actin-based cytoskeleton (Nolan et al., 1998; upstream regulator of Rac that functions genetically at the Reddien and Horvitz, 2000). In support of the genetic data, same step as DOCK180/CED-5 in engulfment of apoptotic overexpression of DOCK180 in mammalian cells has been cells and cell migration in C. elegans (Gumienny et al., 2001; reported to lead to JNK activation, phagocytosis of apoptotic Reddien and Horvitz, 2000; Wu et al., 2001; Zhou et al., 2001). cells and enhanced cell migration, and all these events can be Mammalian ELMO1 was subsequently shown to directly inhibited by coexpression of a dominant-negative form of Rac interact and functionally cooperate with DOCK180 in Rac- 4902 Journal of Cell Science 115 (24) dependent phagocytosis of carboxylate-modified beads in Matsuda. The DHR-2 domain (aas 1111-1636) of DOCK180, the CHO LR73 cells. ELMO1 lacks any obvious catalytic subdomains of DHR-2 (aas 1111-1515, 1111-1395, 1111-1335, and domains, and when expressed alone in mammalian cells, it fails 1335-1515) and the DOCKER domain [aas 1111-1657, as described to have a notable effect on Rac GTP loading in vivo (Gumienny by Brugnera et al. (Brugnera et al., 2002)] were amplified by using Flag-DOCK180 as a template and ligated into pGEX4T-1 (Amersham et al., 2001). It is thus plausible that formation of a multiprotein ∆ complex around DOCK180 is required for DOCK180 (or Biosciences) or pcDNA3-Myc. The DOCK180 DHR-2 mutant, which contains a deletion of amino acids 1111-1636, was generated another component in the complex) to interact with and/or to by PCR and ligated into pcDNA3.1Zeo. The cDNA for CED-5 was activate Rac. obtained from R. Horvitz, and the cDNAs for KIAA0299, KIAA1058 We report the identification of a region within DOCK180 and KIAA1771 were from the Kazuza DNA Research Institute. The named the DHR-2 (DOCK Homology Region-2) domain, DHR-2 domains of CED-5, DOCK2, DOCK3 (KIAA0299) and which directly interacts with nucleotide-free Rac in vitro and DOCK9 (KIAA1058) were amplified from their respective cDNAs induces the GTP loading of Rac both in vitro and in vivo. and ligated into pGEX4T-1. The DHR-2 domain of DOCK7 Furthermore, we have identified several novel homologues of (KIAA1771) was amplified from reverse-transcribed RNA isolated DOCK180 that possess the DHR-2 domain and found that from 293-T cells (owing to sequence rearrangements in the original many of them bind to and exchange GDP for GTP on either KIAA1771 clone obtained from the Kazuza DNA Research Institute) Rac or Cdc42. Thus, our studies identify a conserved protein and subsequently subcloned into pGEX4T-1. The DHR-2 domains of DOCK180, DOCK2 and DOCK9 were also subcloned into the domain that directly interacts with and activates GTPases mammalian expression vector pcDNA3-Myc. The full-length cDNA and suggest the presence of a previously unidentified, of mouse ELMO1 was obtained from the EST database (GenBank evolutionarily conserved DOCK180-related superfamily of Acc. #AI574349) and subcloned into the pcDNA3-Myc vector. GEFs. pEBB-ELMO1-GFP vector coding for mouse ELMO1 with a C- terminal GFP-tag was obtained from K. Ravichandran and has been described previously (Gumienny et al., 2001). The pET28 Vav2 DPC Materials and Methods (DH-PH-Cysteine Rich) construct has been described previously (Abe Computer analysis et al., 2000) and was a generous gift from C. J. Der. The pRK5-Myc- BLAST searches were performed by using the National Center Rac1, -Cdc42 and -RhoA plasmids, as well as the plasmids encoding for Biotechnology Information (NCBI) Standard BLAST server GST-Rac1 and GST-RhoA were obtained from A. Hall. The pGEX- (http://www.ncbi.nlm.nih.gov/blast). The sequences coding for the Cdc42 construct was from J. Sondek. The pGEX PAK-BD construct various DOCK180-related proteins identified in the BLAST searches has been described previously (Abassi and Vuori, 2002). Anti- were acquired from the NCBI Center and the Kazuza DNA Research DOCK180, anti-Myc, anti-Cdc42 and anti-RhoA antibodies were Institute (Japan). Full-length sequence of MOCA (termed here obtained from Santa Cruz Biotechnologies. The pan-Rac antibody DOCK3) was obtained from D. Schubert and Q. Chen (Kashiwa et was from Upstate Biotechnology. The anti-Flag and anti-GFP al., 2000). The sequences were aligned using ClustalW. With two antibodies were purchased from Sigma and Chemicon, respectively. exceptions (DOCK5 and DOCK10), all available human, Drosophila and C. elegans DNA clones, although not necessarily full-length, covered sequences that coded for the DHR-2 domain and adjacent Cell culture and transfections amino acids. Equal length amino-acid sequences of these clones were COS-1 and HEK 293-T cells were cultured in DMEM supplemented subsequently used for phylogenetic analysis. The amino-acid with 10% fetal bovine serum, penicillin and streptomycin (Gibco- sequence distances were determined with the PHYLIP 3.5 package. BRL). The CHO cell line, subclone LR73, was obtained from P. Gros The phylogenetic tree was derived by neighbor-joining analysis and grown in Alpha MEM Earle’s salts supplemented with 10% fetal applied to pairwise sequence distances by using the Kimura two- bovine serum, penicillin and streptomycin. For transfections, cells parameter method to generate unrooted trees. The final output was were grown to 80-90% confluency in six-well plates. Unless otherwise generated with TREEVIEW. The individual nodes, or branching indicated in the figure legends, each well was routinely transfected points, in the generated tree were examined by bootstrap analysis with with 2 µg of plasmids using the transfection reagents NovaFECTOR 10,000 pseudoreplicates of the data and found to be reliable.