Nuclear Localization of the DOCK180/ELMO Complex

ABB Archives of Biochemistry and Biophysics 429 (2004) 23–29 www.elsevier.com/locate/yabbi Nuclear localization of the DOCK180/ELMO complex Jinhu Yin,a Lisa Haney,b Scott Walk,b Sharleen Zhou,c Kodi S. Ravichandran,b and Weidong Wanga,* a Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MA 21224, USA b Beirne Carter Center for Immunology Research and the Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA c Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA Received 19 March 2004, and in revised form 18 May 2004 Available online 2 July 2004 Abstract DOCK180 protein plays a key role during development, cell motility, and phagocytosis. It forms a complex with another protein ELMO, and this complex acts as a guanine nucleotide exchange factor (GEF) for Rac. However, DOCK180-containing complexes have not been purified by unbiased biochemical approaches, and the nature and subcellular localization of these complexes remain unclear. Here, we show that a large fraction of endogenous DOCK180 is present as a 700 kDa nuclear complex with ELMO proteins. In addition, this nuclear DOCK180/ELMO complex has functional Rac-GEF activity. Furthermore, endogenous DOCK180 could be found in complexes with different ELMO isoforms (ELMO1, 2 or 3) in different cell lines, depending on the ELMO isoforms expressed. These studies suggest that DOCK180 may associate with different ELMO proteins to form cell-type specific complexes and may have functions in both the nucleus and the cytoplasm. Ó 2004 Elsevier Inc. All rights reserved. Keywords: DOCK180; ELMO; Guanine nucleotide exchange factor; Molecular complex; Nuclear localization Mammalian DOCK180 protein and its homologues nucleotide exchange factors (GEFs)1 [16–18], DOCK180 in Drosophila Myoblast City and Caenorhabditis elegans contains a novel domain, termed Docker domain, which (CED-5) represent an evolutionarily conserved family of specifically recognizes nucleotide-free Rac and can proteins involved in multiple biological processes. promote loading of GTP by Rac in vitro [19]. Recent DOCK180 participates in actin cytoskeletal changes evidences suggest that in addition to Dock180, 10 ad- during cell migration and phagocytosis of apoptotic cells ditional proteins in the human genome contain a Docker [1–6]. The Drosophila (Myoblast City) plays a role in domain, and that these constitute a Dock180 super- myoblast fusion, dorsal closure, and cytoskeletal orga- family of proteins [20]. These different DOCK180 family nization during embryonic development [7–9]. The C. members have been linked to various biological pro- elegans CED-5 is implicated in engulfment of cell cesses including cell adhesion, migration, phagocytosis corpses and in distal tip cell migration [5,10–15]. The of apoptotic cells, and tumor cell metastasis. DOCK180 family of proteins has been suggested to Several lines of evidence have suggested that function as an upstream regulator of the small GTPase DOCK180 forms a complex with another protein Rac, which mediates membrane ruffling and other cy- ELMO1 [5,19], which, like DOCK180, has also been toskeletal changes [8,10]. Although DOCK180 does not identified as an evolutionarily conserved upstream contain the conventional Dbl homology (DH) domain found in essentially all known mammalian Rac guanine 1 Abbreviations used: DTT, dithiothreitol; ELMO, engulfment and cell motility; GEF, guanine nucleotide exchange factor; IP, immuno- * Corresponding author. Fax: 1-410-558-8331. precipitation; PMSF, phenylmethylsulfonyl fluoride; SDS–PAGE, E-mail address: [email protected] (W. Wang). sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 0003-9861/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.05.014 24 J. Yin et al. / Archives of Biochemistry and Biophysics 429 (2004) 23–29 regulator of Rac. CED-12, the ELMO1 homologue in (DTT), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), C. elegans, has been shown to function genetically at 2 lg/ml leupeptin, and 2 lg/ml aprotinin). The mixture the same step as DOCK180/CED-5 in engulfment of was incubated with 2 lg of DOCK180 antibodies in the apoptotic cells and cell migration in nematodes presence of 100 ll of protein A-beads (Amersham– [5,10,13,14]. In cell culture studies, DOCK180 itself is Pharmacia) for at least 12 h at 4 °C. The immunopre- insufficient for GTP loading of Rac, and a complex with cipitate was washed three times with the IP buffer. The ELMO is required [19]. Recent studies indicate that the complex on the beads was either used in the GEF assay DOCK180/ELMO complex functions as a bi-partite or was eluted from the beads using 100 mM glycine–HCl GEF for Rac [19,21]. At least three ELMO proteins buffer (pH2.5). The eluted complex was subjected to (ELMO1, 2, and 3) have been reported in mammals, and sodium dodecyl sulfate–polyacrylamide gel electropho- none of them contains any obvious catalytic domains. resis (SDS–PAGE) and immunoblotting analysis. Less Although endogenous DOCK180 and ELMO1 have than 10% of the input was loaded as control for im- been shown to occur as a complex in cells [19], this munoblotting. For mass spectrometric analysis, the complex has not been purified by unbiased biochemical proteins were visualized by Coomassie blue staining, approaches. Thus, the nature of the DOCK180/ELMO excised from the gel, and digested with trypsin. The complex, and its localization remain unclear. Here, we peptides obtained were analyzed by matrix-assisted laser have immunoisolated DOCK180-containing complexes desorption ionization-time of flight mass spectrometry. from several mammalian cell lines. We show that DOCK180 is present in a complex with ELMO in both Nuclear extract fractionation the nucleus and the cytoplasm, and that DOCK180 can form complexes with multiple ELMO isoforms in dif- The HeLa S3 nuclear or cytoplasmic extract was di- ferent cell lines. rectly applied to a Superose 6 column (HR16/50; Amersham) equilibrated with the column running buffer containing 20 mM Hepes (pH 7.9), 200 mM NaCl, 1 mM Experimental procedures DTT, 0.1 mM PMSF, 5 lg/ml leupeptin, and 5% glycerol. Fractions were collected and analyzed by SDS– Cells and antibodies PAGE and immunoblotting. HeLa S3 and CHO-K1 cells were obtained from In vitro GEF assay the National Cell Culture Center. Nuclear and cytoplasmic extracts were prepared as previously described The DOCK180 complex immobilized on the protein [22]. Three DOCK180 antibodies (sc-5635, sc-13163, A beads was analyzed for Rac GEF activity as follows and sc-6167) were obtained from Santa Cruz Bio- [19]: 1 lg of bacterially expressed and purified Rac was technology. Another DOCK180 antibody was kindly incubated with 50 lCi of [a-32P]GTP (3000 Ci/mmol) in provided by Dr. Matsuda (Japan). The ELMO1 an- a loading buffer containing 40 mM Mops (pH 7.1), tibody has been described previously [19], which has 1 mM EDTA, 1 mg/ml BSA, and 0.3 lM unlabeled weak cross-reactivity to ELMO2. The rabbit ELMO2 GTP, for 20 min on ice. Magnesium chloride was then antibody was generated against a recombinant GST– added at a final concentration of 10 mM, and the mix- ELMO2 fusion protein (encompassing amino acid ture was incubated on ice for an additional 10 min. residues 1–551 of ELMO2) expressed in E. coli. The [a-32P]GTP-loaded Rac (50 ng) was added to the specific reactivity of the ELMO2 antibody was con- DOCK180 complex, and the mixture was diluted using firmed using ELMO1, ELMO2, and ELMO3 proteins an exchange buffer containing 25 mM Mops (pH 7.1), expressed in 293T cells, as well as bacterially produced 6.25 mM magnesium chloride, 0.6 mM NaH2PO4, recombinant proteins (data not shown). The ELMO2 0.5 mg/ml BSA, and 1.25 mM GDP (unlabeled), so that antibody was purified on a protein-A column prior to the final volume was 100 ll. After 15-min incubation at use in immunoblotting. 30 °C, 50 ll of the exchange reaction was subjected to filter binding to nitrocellulose and scintillation counting. Immunoisolation of DOCK180 complexes The presence of GEF-activity was detected by loss of radioactivity bound to Rac (as a result of the exchange DOCK180 complexes were isolated from both nu- reaction). [32P]GTP bound to Rac when incubated with clear and cytoplasmic extracts of HeLa and CHO-K1 the exchange buffer alone was set at 100%. The data cells by using an immunoprecipitation protocol previ- presented are representatives of at least three indepen- ously described (Lee et al. unpublished data; [23]). dent experiments. In a time course of this exchange as- Briefly, 1 ml (7 mg/ml) of nuclear extract was diluted 10 say with the DOCK180 complex, the maximal exchange fold with IP buffer (20 mM Hepes [pH 7.9], 200 mM on Rac was detected between 10 and 20 min. Hence, a NaCl, 10% glycerol, 0.1% NP-40, 1 mM dithiothreitol 15-min point was chosen for the assay. J. Yin et al. / Archives of Biochemistry and Biophysics 429 (2004) 23–29 25 Immunofluorescence In contrast, although nucleotide-free Rac, p130Cas and CrkII have been shown to interact with DOCK180 [1– Cell fixation and blocking followed a published pro- 5], we failed to detect these proteins as part of the tocol [24]. Cells were incubated with a DOCK180 anti- DOCK180 complex (data not shown), implying that the body (Santa Cruz Biotechnology) in blocking buffer (5% interactions between these polypeptides and DOCK180 milk in PBS with 0.2% Triton X-100) at 4 °C overnight, are not stable enough to withstand the isolation proce- and then stained with Alexa Fluor 568-conjugated sec- dure used here. ondary antibody (Molecular Probes). DNA was stained Superose 6 gel-filtration fractionation revealed that using the TOTO-3 dye (Molecular Probes).

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