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PI3K Links NKG2D Signaling to a Crkl Pathway Involved in Natural Killer Cell Adhesion, Polarity, and Granule Secretion1

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PI3K Links NKG2D Signaling to a Crkl Pathway Involved in Natural Killer Cell Adhesion, Polarity, and Granule Secretion1 The Journal of Immunology PI3K Links NKG2D Signaling to a CrkL Pathway Involved in Natural Killer Cell Adhesion, Polarity, and Granule Secretion1 Colin M. Segovis,* Renee A. Schoon,* Christopher J. Dick,* Lucas P. Nacusi,* Paul J. Leibson,* and Daniel D. Billadeau2*† The NK cell-activating receptor NKG2D plays a critical role in the destruction of malignant cells, but many of the cell-signaling mechanisms governing NKG2D-mediated cellular cytotoxicity are unknown. We have identified an NKG2D-mediated signaling pathway that governs both conjugate formation and cytotoxic granule polarization. We demonstrate that an interaction between the regulatory subunit of PI3K, p85, and the adaptor protein CrkL is required for efficient NKG2D-mediated cellular cytotoxicity. We show decreased NK cell-target cell conjugate formation in NK cells treated with PI3K inhibitors or depleted of CrkL. Independent of adhesion, we find that microtubule organization center polarization toward target cells expressing the NKG2D ligand MICA or toward anti-NKG2D-coated beads is impaired in the absence of CrkL. Ab-stimulated granule release is also impaired in NK cells depleted of CrkL. Furthermore, our data indicate that the small Ras family GTPase Rap1 is activated downstream of NKG2D engagement in a PI3K- and CrkL-dependent manner and is required for conjugate formation, MTOC (microtubule organizing center) polarization, and NKG2D-dependent cellular cytotoxicity. Taken together, our data identify an NKG2D-activated signaling pathway that collectively orchestrates NK cell adhesion, cell polarization, and granule release. The Journal of Immunology, 2009, 182: 6933–6942. ytotoxic lymphocytes, such as NK cells and CD8ϩ T gates down two distinct arms of the NKG2D signaling pathway: lymphocytes, play a critical role in our body’s immune PI3K and Grb2/Vav1 (4, 7, 8). Although a number of proteins have C response to malignant and virally infected cells. A prin- been shown to play a role downstream of these proximal signaling ciple effector mechanism of cytotoxic lymphocytes is the process molecules, questions remain regarding the signaling pathways reg- of cell-mediated cytotoxicity, during which a target cell is de- ulating NKG2D-mediated conjugate formation, granule polariza- stroyed by a cytotoxic lymphocyte. Cell-mediated cytotoxicity oc- tion, and secretion. curs in discrete steps: formation of a cytotoxic lymphocyte-target Integrins have been established as regulators of cell adhesion cell conjugate, polarization of the cytotoxic machinery toward the and are required for proper NK cell-target conjugate formation. In effector-target cell interface known as the cytotoxic synapse (CS),3 ␤ ␤ fact, blocking 1 or 2 integrins inhibits NK cell-mediated cyto- and finally lytic granule secretion (1, 2). Although the steps of toxicity (9). Moreover, patients with leukocyte adhesion deficien- cytotoxicity have been established, many questions remain regard- cies display impaired NK cell function (10–12). ITAM-containing ing the regulation of these steps. receptors have been shown to regulate integrins in T lympho- NKG2D is a non-ITAM containing NK- and T cell-activating cytes, neutrophils, macrophages, and NK cells (13–15). Nolz receptor that recognizes “stress” ligands, such as the tumor-asso- and colleagues established a role for WAVE2 in integrin acti- ciated MICA (in humans) and Rae-1 (in mice), which are up- vation through Abl- and CrkL-C3G-mediated activation of regulated on the surface of cells undergoing genotoxic stress (3–5). Rap1 in T cell signaling (16). Vinculin and talin have also been Upon NKG2D ligation, the transmembrane protein DAP10 is implicated in integrin affinity maturation downstream of the phosphorylated by the Src kinase Lck (6). The signal then propa- TCR (17). However, whether signaling pathways activated by NKG2D signaling affect integrin activation or NK cell adhesion is unknown. In fact, two recent reports did not observe any † *Department of Immunology and Division of Oncology Research, College of Med- increase in conjugate formation upon stimulation of NKG2D, icine, Mayo Clinic, Rochester, MN 55905 suggesting that signaling through NKG2D/DAP10 may not ac- Received for publication November 17, 2008. Accepted for publication March 31, 2009. tivate integrins (8, 18). The costs of publication of this article were defrayed in part by the payment of page The Crk family of adaptor proteins are a widely expressed fam- charges. This article must therefore be hereby marked advertisement in accordance ily of signal transduction mediators involved in normal and ma- with 18 U.S.C. Section 1734 solely to indicate this fact. lignant cell processes (19). This family contains Crk, expressed as 1 Supported by the National Institutes of Health (Grants CA47752 and AI65474 to two isoforms, CrkI and CrkII, and CrkL (Crk-like) (19). CrkI has D.D.B.). D.D.B. is a Leukemia and Lymphoma Society Scholar. an N-terminal SH2 domain followed by a SH3 domain, whereas 2 Address correspondence and reprint requests to Dr. Daniel D. Billadeau, De- partment of Immunology and Division of Oncology Research, Mayo Clinic Col- both CrkII and CrkL contain an additional C-terminal SH3 do- lege of Medicine, 200 First Street SW, Rochester, MN 55905. E-mail address: main. These domains serve as binding sites for a number of pro- [email protected] teins, allowing Crk proteins to participate in multiple signaling 3 Abbreviations used in this paper: CS, cytotoxic synapse; CMAC, 7-amino-4-chlo- pathways (19). Recent work has identified CrkL as an important romethylcoumarin; IP, immunoprecipitation; KIR, killer Ig-related receptor; MICA, MICA expressing BaF3 cells; MTOC, microtubule organizing center; SFDA, 5-sul- mediator of integrin regulation in T lymphocytes (16). Although fofluorescein diacetate; siCrkL, siRNA directed against CrkL; siNeg, negative control no functional role for CrkL has been demonstrated in NK cells, siRNA; siRNA, small interfering RNA; WR, Western Reserve. Crk has been identified as a target following NK inhibitory recep- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 tor signaling (20). www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803840 6934 CrkL REGULATION OF NKG2D-MEDIATED KILLING In this study, we demonstrate that NKG2D-mediated conjugate Conjugate assays formation is PI3K-dependent and involves the adaptor protein Human NK clones were stained with PKH26 per included protocol and CrkL and the small Ras family GTPase Rap1. Significantly, CrkL resuspended at 0.5 ϫ 106 cells/ml in RPMI 1640. Target cells were stained suppression results in diminished cytotoxicity, decreased conju- with either 50 ␮g/ml 5-sulfofluorescein diacetate (SFDA) (Invitrogen) in gate formation, impaired microtubule organizing center (MTOC) PBSfor1hina37°C water bath or 50 ␮M 7-amino-4-chloromethylcou- polarization, and decreased granule secretion by NK cells. We also marin (CMAC) (Invitrogen) for 30 min in serum-free media and then quenched for 15 min in RPMI 1640 plus 10% CS. Target cells were washed show that Rap1 is activated by NKG2D stimulation in a PI3K- and twice in PBS and resuspended at 0.5 ϫ 106 cells/ml in RPMI 1640. NK CrkL-dependent manner and is required for NKG2D-mediated cells (0.25 ϫ 106) were mixed with 0.25 ϫ 106 target cells and spun at 500 conjugate formation, MTOC polarization, and cytotoxicity. rpm for 5 min. The resulting pellet was placed in a 37°C water bath for the desired time. Medium (0.5 ml) was removed from each sample after all samples had been heated for the desired amount of time. Samples were then Materials and Methods vortexed at maximum speed for5stodisrupt nonspecific conjugates and Reagents, cells, and Abs then fixed with 0.5 ml of 4% paraformaldehyde. Conjugate formation was analyzed using a LSRII flow cytotometer (BD Immunocytometry Sys- Reagents were purchased from Sigma-Aldrich unless otherwise stated. Re- tems). The SFDA was excited with a 488-nm laser and the emission was combinant ICAM-1 and VCAM-1, as well as mouse monoclonal IgG1 measured through a 530/30-nm bandpass (BP) filter. CMAC was excited anti-NKG2D, were purchased from R&D Systems. Human NK cells were with a 355-nm laser and the emission was measured through a 450/65-nm cloned and passed as previously described (21). Both the wild-type BaF3 BP filter. The PKH26 was excited with a 532-nm laser and the emission myeloid cell line and the MICA-expressing variant were a gift from Dr. was measured through a 575/26-nm BP filter. Data were analyzed with the L. L. Lanier (University of California at San Francisco, San Francisco, FlowJo software package (Tree Star). CA). The anti-LFA-1 Ab, mHm23, was a gift from T. A. Springer (CBR Institute for Biomedical Research, Boston, MA). Mouse monoclonal IgG1 anti-FcR (CD16) was a gift from Dr. D. Segal (Immunology Branch, Na- Cytotoxicity assays tional Cancer Institute, National Institutes of Health, Bethesda, MD). ␤ 51Cr-release assays were performed as previously described (21). NK cell Mouse monoclonal anti- 1 integrin (Chemicon International), mouse monoclonal CrkL (Abnova), and mouse monoclonal p85, clone AB6 (Up- cytotoxicity was triggered either by coating P815 tumor targets with a state Biotechnology) were also used. Rabbit polyclonal anti-Rap1 was pur- “triggering” Ab, such as anti-NKG2D, or by the BaF3 myeloid cell line chased from Cell Signaling Technology. Rabbit polyclonal anti-SLP76 was expressing the NKG2D receptor ligand MICA. generated as previously described (22). Adhesion assays GST protein precipitation GST-CrkL SH2, GST-CrkL SH3-N, and GST-CrkL SH3-C were con- Adhesions assays were performed as previously described (16). Briefly, a ϫ 6 96-well flat bottom plate was coated overnight with ICAM-1 or VCAM-1 structed as previously described (26). Briefly, 10 10 human NK clones per sample were stimulated with anti-NKG2D and cross-linked, and then and then blocked with 2.5% BSA in PBS. Human NK cells were stained lysed on ice for 10 min with buffer containing 50 mM Tris base, 20 mM with 5 ␮M calcein-AM (Invitrogen) for 30 min in serum-free media and EDTA, and 1% Igepal CA-630.
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