Vav1 Controls DAP10-Mediated Natural Cytotoxicity by Regulating Actin and Microtubule Dynamics This information is current as Daniel B. Graham, Marina Cella, Emanuele Giurisato, Keiko of October 1, 2021. Fujikawa, Ana V. Miletic, Tracie Kloeppel, Karry Brim, Toshiyuki Takai, Andrey S. Shaw, Marco Colonna and Wojciech Swat J Immunol 2006; 177:2349-2355; ; doi: 10.4049/jimmunol.177.4.2349 http://www.jimmunol.org/content/177/4/2349 Downloaded from References This article cites 35 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/177/4/2349.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Vav1 Controls DAP10-Mediated Natural Cytotoxicity by Regulating Actin and Microtubule Dynamics1 Daniel B. Graham,* Marina Cella,* Emanuele Giurisato,* Keiko Fujikawa,† Ana V. Miletic,* Tracie Kloeppel,* Karry Brim,* Toshiyuki Takai,‡ Andrey S. Shaw,* Marco Colonna,* and Wojciech Swat2* The NK cell-activating receptor NKG2D recognizes several MHC class I-related molecules expressed on virally infected and tumor cells. Human NKG2D transduces activation signals exclusively via an associated DAP10 adaptor containing a YxNM motif, whereas murine NKG2D can signal through either DAP10 or the DAP12 adaptor, which contains an ITAM sequence. DAP10 signaling is thought to be mediated, at least in part, by PI3K and is independent of Syk/Zap-70 kinases; however, the exact mechanism by which DAP10 induces natural cytotoxicity is incompletely understood. Herein, we identify Vav1, a Rho GTPase guanine nucleotide exchange factor, as a critical signaling mediator downstream of DAP10 in NK cells. Specifically, using mice Downloaded from deficient in Vav1 and DAP12, we demonstrate an essential role for Vav1 in DAP10-induced NK cell cytoskeletal polarization involving both actin and microtubule networks, maturation of the cytolytic synapse, and target cell lysis. Mechanistically, we show that Vav1 interacts with DAP10 YxNM motifs through the adaptor protein Grb2 and is required for activation of PI3K-dependent Akt signaling. Based on these findings, we propose a novel model of ITAM-independent signaling by Vav downstream of DAP10 in NK cells. The Journal of Immunology, 2006, 177: 2349–2355. http://www.jimmunol.org/ atural cytotoxicity mediated by NK cells is regulated by Activating NK cell receptors primarily signal through ITAM- multiple activating and inhibitory receptors, which con- containing adaptor molecules such as DAP12, CD3␨, and FcR␥, N fer innate defenses against tumor cells and virus-infected which initiate cellular activation signals by recruiting Syk/Zap-70 cells. The multistep process leading to target cell lysis involves the family kinases (6–9). Additional NK cell activating receptors, formation of a cytolytic synapse and polarized degranulation of the such as NKG2D, trigger cytotoxicity independently of ITAMs by NK cell (1–4). Initial contact between the NK cell and the target associating with DAP10, a unique adaptor containing a YxNM cell is mediated by integrins and facilitates engagement of NK- motif that recruits PI3K (10) and Grb2 (11). Initiation of NKG2D activating receptors by their cognate ligands (2, 4). Postconjuga- signals occurs upon recognition of specific ligands, stress-induced tion events are orchestrated by signals emanating from activating MHC class I-like molecules such as MICA, MICB, and UL-16 by guest on October 1, 2021 receptors and involve F-actin accumulation at the NK-target con- binding protein in humans, as well as Rae-1, H-60, and MULT1 in tact site and microtubule-organizing center (MTOC)3 polarization mice (8, 12). Subsequent to ligand engagement, human NKG2D toward the target cell. In turn, MTOC polarization leads to the signals through DAP10, whereas murine NKG2D signals through establishment of a microtubule network guiding cytolytic granules distinct adaptor molecules. Full-length NKG2D-long (NKG2D-L) to the synapse where they fuse with the plasma membrane and signals through DAP10, whereas a shorter splice variant release perforin and granzymes to lyse target cells (2, 4). Cytoskel- (NKG2D-S) signals through both DAP10 and DAP12 (13–15). In etal remodeling is critical for NK cytotoxicity because pharmaco- this regard, the relative proportion of NKG2D-L and NKG2D-S in logic inhibition of F-actin or microtubule dynamics blocks granule NK cells varies upon in vitro activation with IL-2. Thus, freshly polarization and target cell lysis (1). Moreover, NK cells from isolated (ex vivo) NK cells predominantly express NKG2D-L, patients bearing mutations in WASp that disrupt actin dynamics whereas in vitro activation leads to an increase in NKG2D-S ex- fail to initiate synapse formation and lyse target cells (1, 5). pression (13). Nevertheless, experiments with DAP12-deficient murine NK cells demonstrate that DAP10 is sufficient to mediate NKG2D-dependent cytotoxicity (16). Furthermore, human *Department of Pathology and Immunology, Washington University School of Med- NKG2D promotes NK cytotoxicity despite its inability to interact icine and Siteman Cancer Center, St. Louis, MO 63110; †Department of Pathology and Immunology, Hokkaido University School of Medicine, Sapporo, Japan; and with DAP12 (17). ‡Department of Experimental Immunology, Institute of Development, Aging and Previous studies have implicated the Vav family of Rho guanine Cancer, Tohoku University, Sendai, Japan nucleotide exchange factors in the regulation of several distinct Received for publication March 22, 2006. Accepted for publication May 25, 2006. pathways controlling natural cytotoxicity (18–22). NK cells lack- The costs of publication of this article were defrayed in part by the payment of page ing all three Vav proteins show severely compromised cytotoxicity charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. triggered by both ITAM- and DAP10-associated activating recep- 1 This work is supported by National Institutes of Health Grants R21 AI063024 (to tors (18). However, while deficiency in Vav1 alone primarily im- W.S.) and T32 CA009547 (to D.B.G.). paired the NKG2D-DAP10 cytolytic pathway, lack of Vav2 and 2 Address correspondence and reprint requests to Dr. Wojciech Swat, Department of Vav3 reduced cytotoxicity triggered by receptors that signal Pathology and Immunology, Washington University School of Medicine, 660 South through ITAM-containing adaptors (18). These observations indi- Euclid Avenue, St. Louis, MO 63110. E-mail address: [email protected] cated an unexpected specialization of Vav proteins in regulating 3 Abbreviations used in this paper: MTOC, microtubule-organizing center; NKG2D-L, NKG2D long; NKG2D-S, NKG2D short; SH, Src homology; WT, wild distinct cytotoxic pathways and implicated Vav1 in control of sig- type. nals emanating from DAP10-coupled receptors. However the exact Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 2350 Vav IN CYTOSKELETAL REMODELING BY NKG2D-DAP10 mechanism of Vav1 coupling to DAP10 remains elusive. In addi- Biochemistry tion, a particular issue is how DAP10 controls cytoskeletal remod- Purified splenic NK cells were cultured in IL-2 (1000 U/ml) for 7 days and eling events during the cytolytic response. then starved in serum-free medium for 6 h. NK cells (1.25 ϫ 106/sample) were Herein, we sought to elucidate the mechanism of Vav1 function resuspended in HBSS and incubated on ice with biotinylated anti-NKG2D in DAP10-mediated signaling events that control natural cytotox- (Biolegend) at 1 ␮g/1 ϫ 106 cells. After 15 min, streptavidin (Pierce) was ␮ ϫ 6 icity. Using mice deficient in Vav1 and DAP12, we demonstrate a added at 2 g/1 10 cells, and cells were incubated at 37°C for the indicated time points. Cells were then lysed in radioimmunoprecipitation assay buffer critical function for Vav1 in DAP10-induced PI3K activation, F- and analyzed by Western blotting for phospho-serine 473 Akt (Cell Signaling actin polymerization, and MTOC polarization and provide evi- Technology) or total Akt (Cell Signaling Technology). dence that Vav1 is recruited to DAP10 via Grb2. Conjugate formation Materials and Methods Target cells were stained with CFSE, and NK cells were stained with Mice and NK cell purification hydroethidine. NK cells and targets were pelleted together, gently dis- rupted, and incubated at 37°C for 15 min. The percentage of cells forming Vav1Ϫ/Ϫ and DAP12Ϫ/Ϫ mice have been described elsewhere and were conjugates was determined by FACS. bred to generate Vav1Ϫ/ϪDAP12Ϫ/Ϫ double knockout mice (F6 on C57BL/6 background) (23, 24). Splenic NK cells were purified by pos- NK