Direct Tumor Lysis by NK Cells Uses a Ras-Independent Mitogen-Activated Protein Kinase Signal Pathway This information is current as Sheng Wei, Danielle L. Gilvary, Brian C. Corliss, Said Sebti, of September 28, 2021. Jiazhi Sun, David B. Straus, Paul J. Leibson, Joseph A. Trapani, Andrew D. Hamilton, Michael J. Weber and Julie Y. Djeu J Immunol 2000; 165:3811-3819; ; doi: 10.4049/jimmunol.165.7.3811 Downloaded from http://www.jimmunol.org/content/165/7/3811 References This article cites 55 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/165/7/3811.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 by guest on September 28, 2021 • Fast Publication! 4 weeks from acceptance to publication *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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Direct Tumor Lysis by NK Cells Uses a Ras-Independent Mitogen-Activated Protein Kinase Signal Pathway 1 Sheng Wei,*‡ Danielle L. Gilvary,*‡ Brian C. Corliss,* Said Sebti,†‡ Jiazhi Sun,†‡ David B. Straus,§ Paul J. Leibson,¶ Joseph A. Trapani,ʈ Andrew D. Hamilton,** Michael J. Weber,†† and Julie Y. Djeu2*‡ Destruction of tumor cells is a key function of lymphocytes, but the molecular processes driving it are unclear. Analysis of signal molecules indicated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase 2 critically controlled lytic function in human NK cells. We now have evidence to indicate that target ligation triggers a Ras-independent MAPK pathway that is required for lysis of the ligated tumor cell. Target engagement caused NK cells to rapidly activate MAPK within 5 min, and PD098059 effectively blocked both MAPK activation and tumoricidal function in NK cells. Target engagement also rapidly activated Ras, detected as active Ras-GTP bound to GST-Raf-RBD, a GST fusion protein linked to the Raf protein fragment Downloaded from containing the Ras-GTP binding domain. However, Ras inactivation by pharmacological disruption with the farnesyl transferase inhibitor, FTI-277, had no adverse effect on the ability of NK cells to lyse tumor cells or to express MAPK activation upon target conjugation. Notably, MAPK inactivation with PD098059, but not Ras inactivation with FTI-277, could interfere with perforin and granzyme B polarization within NK cells toward the contacted target cell. Using vaccinia delivery of N17 Ras into NK cells, we demonstrated that IL-2 activated a Ras-dependent MAPK pathway, while target ligation used a Ras-independent MAPK pathway http://www.jimmunol.org/ to trigger lysis in NK cells. The Journal of Immunology, 2000, 165: 3811–3819. he mitogen-activated protein kinases (MAPK)3 integrate (8–10). Activation of ERK by growth factor receptor triggering multiple intracellular signals within a cell responding to follows a highly conserved set of molecular events, connected to T different external stimuli. Of the MAPK kinase family, upstream signals associated with the receptor. Association of the which includes c-Jun N-terminal kinase/stress-activated protein ki- GRB-2/SOS complex to the receptor recruits Ras to the plasma nase, p38/MAPKAP kinase 2 reactivity kinase (RK), and MAPK/ membrane, resulting in Raf-1 binding to activated GTP-bound extracellular regulated kinase (ERK), the last subgroup is the most Ras. Subsequent activation of Raf-1 leads to its phosphorylation of extensively investigated, particularly in events associated with MAPK/ERK kinase 1 (MEK-1), which, in turn, becomes activated as by guest on September 28, 2021 growth factor receptors and G protein-coupled receptors (1, 2). a dual kinase, with the ability to phosphorylate the TEY motif ERKs are differentiated from the other MAPKs by their conserved in ERK. TEY motif, which becomes dually phosphorylated at the Tyr and ERK has largely been considered a growth- and differentiation- Thr sites upon activation. They play a critical role in gene expres- related signal molecule, involved in transcriptional control of crit- sion related to cell growth and differentiation. They have the abil- ical genes essential for cell proliferation/maturation, but it is likely ity to activate nuclear transcription factors, including Elk-1, c-Jun, that it plays a crucial role in other cellular events. For example, c-Myc, NF-IL-6, and TAL1 (3–7), as well as cytoplasmic sub- ERK has recently been reported to activate superoxide release (11) rsk strates, such as phospholipase A2, pp90 , and RNA polymerase and modulate cell-cell or cell-matrix interactions (12, 13). Cell secretion may also be driven by ERK (14). We therefore undertook the task of investigating whether ERK was important in lympho- *Immunology Program and †Drug Discovery Program, and ‡H. Lee Moffitt Cancer Center, Department of Biochemistry and Molecular Biology, University of South cyte function against tumor cells. Target ligation can direct two Florida College of Medicine, Tampa, FL 33612; §Department of Pathology, Uni- separate and distinct functions in lymphocytes, dictated by the in- versity of Chicago, Chicago, IL 60637; ¶Department of Immunology, Mayo Clinic, Rochester, MN 55905; ʈCellular Cytotoxicity Laboratory, Austin Research Institute, duction and effector phases. In the induction phase, the event trig- Heidelberg, Victoria, Australia; **Department of Chemistry, Yale University, New gered by TCR coupling to its Ag on quiescent T cells leads to †† Haven, CT 06511; and Department of Microbiology, University of Virginia, Char- signals that up-regulate genes associated with cytokine induction lottesville, VA 22908 and cell proliferation, resulting in clonal expansion and functional Received for publication May 30, 2000. Accepted for publication July 14, 2000. activation (15). Upon re-encounter of the Ag, activated T cells The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance express lytic function in the effector phase. The signal pathways with 18 U.S.C. Section 1734 solely to indicate this fact. coupled to the TCR during the initial activation phase must differ 1 This work was supported by a grant from the U.S. Public Health Service (CA83146) from those during the effector phase, because different functions and by American Heart Association Grant AHA 970175. prevail. Research to date has focused on the induction phase, and 2 Address correspondence and reprint requests to Dr. Julie Y. Djeu, Immunology it is established that the TCR is coupled to nonreceptor tyrosine Program, H. Lee Moffitt Cancer Center, University of South Florida, 12902 Magnolia Drive, Tampa, FL 33612. E-mail address: djeu@moffitt.usf.edu kinases, Lck and Zap70 (15). These kinases, in turn, trigger down- stream events via calcium- and Ras-dependent pathways. Zap70 3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; FTI, farnesyl transferase inhibitor; ERK, extracellular regulated kinase 2; LGL, large gran- can phosphorylate a membrane-bound adaptor protein, linker for ular lymphocyte; PLC, phospholipase C; RBD, Ras-GTP binding domain; PI 3-ki- activation of T cells (LAT), which subsequently binds phospho- nase, phosphoinositide 3-kinase; MEK-1, MAPK/ERK kinase 1; TRITC, tetrameth- ␥ ␥ ylrhodamine isothiocyanate; RK, MAPKAP kinase 2 reactivity kinase; GRB2, growth lipase C 1 (PLC 1) and growth factor receptor binding protein 2 factor receptor binding protein 2. (GRB2) (16, 17). Activation of PLC␥1 leads to inositol phosphate Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 3812 RAS-INDEPENDENT MAPK REQUIRED FOR NK LYSIS and intracellular calcium mobilization, while formation of the containing 10% FCS with 2 mM L-glutamine, 100 U/ml penicillin, and 100 GRB2-SOS complex recruits Ras to the membrane, turning on the ␮g/ml streptomycin (30). Raf/MEK/MAPK pathway (18, 19). MAPK is known to activate Isolation of large granular lymphocytes (LGL) from peripheral several important transcription factors required for IL-2 and other blood cytokine gene induction (20). The involvement of Vav and 76-kDa SH2 domain leukocyte protein in T cell signal pathways has also NK cells were derived from LGL isolated from PBMC of normal volun- teers as previously described (4). After plastic adherence for1hat37°C been recently described, and the ability of 76-kDa SH2 domain and passage through nylon wool, the nonadherent mononuclear cells were leukocyte protein to bind Grb2 could provide another link to the placed on a four-step discontinuous Percoll gradient. The cells recovered Ras-dependent MAPK pathway (21, 22). from 42.5–45.0% Percoll usually contained 75–90% LGL, as assessed by Studies to date have thus documented that the conserved Ras/ Giemsa-stained morphology, and were used to test NK function. Raf/MEK/MAPK pathway is invoked by ligands binding to their Pharmacological inhibition of MAPK and Ras Ag receptors. This finding is expected, given that Ag recognition ϫ 6 leads to lymphocyte activation, resulting in gene expression related For MAPK inhibition, NK92 cells or fresh LGL (2.5 10 cells/ml) were incubated for2hat37°C with medium or serial dilutions of PD098059 to clonal expansion, a noted example of which is IL-2. However, (New England Biolabs, Beverly MA) or an equal amount of DMSO to once activated, T cells must express a separate and distinct func- dilute the highest concentration of inhibitor (31).