Published May 12, 2014, doi:10.4049/jimmunol.1303348 The Journal of Immunology Essential Role of Elmo1 in Dock2-Dependent Lymphocyte Migration Catherine Stevenson,1 Gonzalo de la Rosa, Christopher S. Anderson, Patrick S. Murphy, Tara Capece, Minsoo Kim, and Michael R. Elliott Elmo1 and Elmo2 are highly homologous cytoplasmic adaptor proteins that interact with Dock family guanine nucleotide exchange factors to promote activation of the small GTPase Rac. In T lymphocytes, Dock2 is essential for CCR7- and CXCR4-dependent Rac activation and chemotaxis, but the role of Elmo proteins in regulating Dock2 function in primary T cells is not known. In this article, we show that endogenous Elmo1, but not Elmo2, interacts constitutively with Dock2 in mouse and human primary T cells. CD4+ T cells from Elmo12/2 mice were profoundly impaired in polarization, Rac activation, and chemotaxis in response to CCR7 and CXCR4 stimulation. Transfection of full-length Elmo1, but not Elmo2 or a Dock2- binding mutant of Elmo1, rescued defective migration of Elmo12/2 T cells. Interestingly, Dock2 protein levels were reduced by 4-fold in Elmo12/2 lymphocytes despite normal levels of Dock2 mRNA. Dock2 polyubiquitination was increased in Elmo12/2 Tcells, and treatment with proteasome inhibitors partially restored Dock2 levels in Elmo12/2 T cells. Finally, we show that Dock2 is directly ubiquitinated in CD4+ T cells and that Elmo1 expression in heterologous cells inhibits ubiquitination of Dock2. Taken together, these findings reveal a previously unknown, nonredundant role for Elmo1 in controlling Dock2 levels and Dock2- dependent T cell migration in primary lymphocytes. Inhibition of Dock2 has therapeutic potential as a means to control recruit- ment of pathogenic lymphocytes in diseased tissues. This work provides valuable insights into the molecular regulation of Dock2 by Elmo1 that can be used to design improved inhibitors that target the Elmo-Dock-Rac signaling complex. The Journal of Immunology, 2014, 192: 000–000. hemokine signaling is an integral component of lym- lymphoid and nonlymphoid tissues. In murine models of cardiac phocyte trafficking, activation, and survival. Rac is a allograft rejection and diabetes, deletion of Dock2 in lymphocytes C member of the Rho family of GTPases that are central was found to be protective, pointing to a role for Dock2 in mi- drivers of actin cytoskeleton dynamics downstream of most che- gration of pathogenic T cells (7, 8). Over the past decade, Dock2 mokine receptors (1–4). Rac cycles between inactive (GDP- has also been found to regulate a range of Rac-dependent functions bound) and active (GTP-bound) states largely because of the ac- in neutrophils, dendritic cells, and NKT cells (9–12). However, the by guest on September 30, 2021. Copyright 2014 Pageant Media Ltd. tion of guanine nucleotide exchange factors (GEFs). Dock2 is a molecular regulation of Dock2 is poorly understood, particularly in ∼200 kDa Rac-GEF restricted to hematopoietic cells in mice and primary cells and in human lymphocytes. 2 2 humans (5). Through the use of Dock2 / mice, it is now well Elmo1 (75 kDa) is a cytoplasmic adaptor protein that physically established that Dock2 is essential for Rac activation and chemo- associates with members of the Dock-A family of Rac-GEFs, of taxis in lymphocytes downstream of multiple chemokine receptors, which Dock1 and Dock2 are the best characterized (5, 13, 14). including CCR7 and CXCR4 (1, 2, 6). Dock2-deficient lympho- Extensive structure–function analyses by a number of groups have cytes show greatly reduced entry, egress, and interstitial motility in shown that Elmo binding enhances Dock1 signaling by increasing its Rac-GEF activity, membrane localization, and protein stability Department of Microbiology and Immunology, University of Rochester Medical Center, (13, 15–21). Studies in invertebrate models and mammalian cell https://www.jimmunol.org Rochester, NY 14642; and David H. Smith Center for Vaccine Biology and Imunology, lines have revealed an evolutionarily conserved role for Elmo1 in University of Rochester Medical Center, Rochester, NY 14642 regulating Dock-Rac signaling in numerous cellular functions, 1Current address: Department of Science and Technology Studies, University College London, London, U.K. including morphology, motility, and phagocytosis (13, 18, 22–25). Received for publication December 17, 2013. Accepted for publication April 10, Elmo1 has also been shown to interact with Dock2 to promote Rac 2014. activation and migration in rodent cell lines (22, 26). More re- 2/2 This work was supported by the Ellison Medical Foundation (to M.R.E.) and Na- cently, studies in Elmo1 mice revealed a critical role for Elmo1 Downloaded from tional Institutes of Health Grants AI027767-24 (to M.R.E, through a Creative and in apoptotic cell clearance during spermatogenesis and hippo- Novel Ideas in HIV Research award from the University of Alabama – Birmingham Center for AIDS Research), HL087088 (to M.K.), and T32AI007285 (to P.S.M. and campal neurogenesis (27, 28). However, as most of our insights T.C.). into Elmo function stem from studies of Elmo1 and Dock1 in Address correspondence and reprint requests to Dr. Michael R. Elliott, Department of nonhematopoietic cells, the mechanisms and outcomes of Elmo1- Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood dependent regulation of Dock proteins in leukocytes remain Avenue, Box 609, Rochester NY 14642. E-mail address: michael_elliott@urmc. rochester.edu largely unknown. The online version of this article contains supplemental material. Elmo1 and Elmo2 are 87% similar at the amino acid level, are Abbreviations used in this article: co-IP, coimmunoprecipitation; GEF, guanine nu- widely expressed, and based on Dock1 studies, have largely been cleotide exchange factor; IB, immunoblotting; IP, immunoprecipitation; pen-strep, considered to be functionally redundant (13). Both proteins contain penicillin-streptomycin; qRT-PCR, quantitative RT-PCR; siRNA, small interfering RNA; pleckstrin homology and proline-rich/PxxP domains located in the S1P, sphingosine 1-phosphate; WT, wild-type. C-terminal 100 aa (15, 29, 30). These C-terminal regions mediate Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 multiple associations with the N termini of Dock1 and Dock2, as www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303348 2 Elmo1 REGULATION OF LYMPHOCYTE MIGRATION revealed through crystallographic and biochemical analyses (30, IP and IB 31). Dock1 and Dock2 contain an N-terminal Src homology 3 Cell lysates were prepared using lysis buffer (20 mM Tris-HCl pH 7.5, 1% domain that mediates interaction with the C-terminal polyproline Triton X-100, 150 mM NaCl, 1 mM EDTA, 13 final protease inhibitor regions of Elmo1 and Elmo2. Interestingly, this PxxP-Src ho- mixture III [Calbiochem]) or Laemmli buffer. Protein quantification of mology 3 association has been reported to be essential for Elmo1 lysates was done using bicinchoninic acid reagent (Pierce). Lysates were interaction with Dock2, but not Dock1 (31). The PxxP motif is boiled for 10 min, and protein separation was carried out on 4–15% SDS- PAGE mini gel (Bio-Rad). After transfer to polyvinylidene difluoride at conserved between mouse and human Elmo1 and Elmo2 (PKEP, 100 V for 1 h, membranes were blocked for 1 h with 5% nonfat dry milk/ 714–717 Elmo1 ), but whether Elmo2 can interact with or regulate TBST before overnight incubation with indicated Abs at 4˚C. SuperSignal Dock2 has not been reported. Pico or Dura ECL reagents were used per manufacturer’s instructions In this study, we used a number of approaches to address the (Pierce). For IP, cell pellets were resuspended in lysis buffer and rotated at 4˚C for 10 min before centrifugation at 12,000 3 g, 5 min, 4˚C. Cleared function of Elmo1 and Elmo2 in regulating Dock2 in primary 2/2 lysates were incubated with anti-Dock2 (1:250), normal rabbit IgG mammalian lymphocytes. Using Elmo1 mice and primary hu- (1:250), or anti-Elmo1 (1:100), in total volume of 500 ml and rotated 18 h man T cells, we demonstrate a previously unknown, nonredundant at 4˚C. A total of 25 ml protein A/G beads was then added to each sample role for Elmo1 in regulation of endogenous Dock2 that may provide and rotated for 2 h at 4˚C. Beads were washed four times in lysis buffer insight toward the development of Dock2-targeting therapeutics. and boiled in Laemmli buffer. For coimmunoprecipitation (co-IP) analysis of endogenous Elmo1 and Elmo2, IPs were split equally after boiling, loaded in duplicate lanes, and blotted with anti-Elmo1 or anti-Elmo2 Abs Materials and Methods separately. For co-IP of Elmo-Flag with Dock2 in 293T cells, all of each Mice anti-Dock2 IP was loaded in a single lane and blotted for Flag using HRP- conjugated anti-DYKDDDDK. For detection of K48-ubiquitinated Dock2 Animal experiments were approved by the University of Rochester Animal under denaturing conditions, anti-Dock2 IPs from wild-type (WT) CD4+ Care and Use Committee. Elmo1-deficient mice have been described T cells were boiled for 5 min in 50 ml denaturing buffer (50 mM Tris pH elsewhere(27).Allmicewere6–12wkofageandonaC57BL/6J 7.5, 70 mM 2-ME, 1% SDS) followed by addition of 350 ml IP lysis buffer background of at least 10 generations. and a second round of IP with anti-Dock2 for 2 h at 4˚C before IB analysis. Reagents qRT-PCR Commercial reagents were purchased as follows: all chemicals unless noted RNA was isolated using DNase I–treated RNeasy columns (Qiagen), and otherwise were purchased from Sigma-Aldrich; chemokines were from cDNA was synthesized from 10–100 ng RNA using iScript (Bio-Rad). PeproTech; ICAM-1 from R&D Systems; Protein A/G agarose beads from qRT-PCR was performed on a 7300 Real Time Thermocycler (Life Santa Cruz; 24-well transwell chambers from Corning; TaqMan quanti- Technologies) using SensiFast Probe Hi-ROX polymerase (Bioline) and tative RT-PCR (qRT-PCR) probes from LifeTech; cell culture media from the following gene-specific TaqMan probes from Life Technologies: Elmo1 Cellgro; and CFSE and TAMRA from Invitrogen.