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Network -Tubulin and Disorganizes the Microtubule Α NK Cell Protease NK Cell Protease Granzyme M Targets α -Tubulin and Disorganizes the Microtubule Network This information is current as Niels Bovenschen, Pieter J. A. de Koning, Razi Quadir, Roel of September 24, 2021. Broekhuizen, J. Mirjam A. Damen, Christopher J. Froelich, Monique Slijper and J. Alain Kummer J Immunol 2008; 180:8184-8191; ; doi: 10.4049/jimmunol.180.12.8184 http://www.jimmunol.org/content/180/12/8184 Downloaded from References This article cites 34 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/180/12/8184.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 September 24, 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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology NK Cell Protease Granzyme M Targets ␣-Tubulin and Disorganizes the Microtubule Network1 Niels Bovenschen,* Pieter J. A. de Koning,* Razi Quadir,* Roel Broekhuizen,* J. Mirjam A. Damen,† Christopher J. Froelich,‡ Monique Slijper,† and J. Alain Kummer2* Serine protease granzyme M (GrM) is highly expressed in the cytolytic granules of NK cells, which eliminate virus-infected cells and tumor cells. The molecular mechanisms by which GrM induces cell death, however, remain poorly understood. In this study we used a proteomic approach to scan the native proteome of human tumor cells for intracellular substrates of GrM. Among other findings, this approach revealed several components of the cytoskeleton. GrM directly and efficiently cleaved the actin-plasma membrane linker ezrin and the microtubule component ␣-tubulin by using purified proteins, tumor cell lysates, and tumor cells undergoing cell death induced by perforin and GrM. These cleavage events occurred independently of caspases or other cysteine proteases. Kinetically, ␣-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct ␣-tubulin proteolysis by Downloaded from GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymer- ization dynamics in vitro and induced microtubule network disorganization in tumor cells in vivo. We conclude that GrM targets major components of the cytoskeleton that likely contribute to NK cell-induced cell death. The Journal of Immunology, 2008, 180: 8184–8191. ytotoxic lymphocytes, i.e., CTLs and NK cells, are key proteins leads to DNA fragmentation and mitochondrial damage, http://www.jimmunol.org/ players in the effector arm of the immune response that respectively. GrA predominantly kills by cleaving nuclear (e.g., C eliminates virus-infected cells and tumor cells (1, 2). Cy- Ku70), mitochondrial, and cytoplasmic substrates (e.g., SET com- totoxic lymphocytes predominantly destroy their targets by releas- plex components) (2, 7–9). Cleavage of these substrates results in ing the content of their cytolytic granules. These granules contain single-stranded nicking of chromosomal DNA. perforin and a family of unique structurally homologous serine In contrast to GrA and GrB, far less is known about the other proteases known as granzymes (3, 4). Although perforin facilitates human granzymes. It has been demonstrated that granzyme M the entry of granzymes into the target cell, the latter induce cell (GrM), which is specifically expressed by NK cells, mediates a death by cleaving critical intracellular substrates (1, 2). novel major and perforin-dependent cell death pathway with by guest on September 24, 2021 In humans, five different granzymes (GrA, GrB, GrH, GrK, and unique morphological hallmarks that plays a significant role in NK GrM) are known that differ on the basis of their substrate speci- cell-induced death (10). The molecular mechanism by which GrM ficity (3, 4). Over the past few decades, it has been well established 3 induces cell death remains unclear. One study has found that GrM- that granzyme A (GrA) and granzyme B (GrB) serve as important induced cell death occurs independently of caspases, DNA frag- determinants of cellular cytotoxicity. Both granzymes induce nu- mentation, and reactive oxygen species (ROS) generation (10), clear and non-nuclear damage in target cells by cleaving distinct whereas other recent reports have demonstrated the opposite (11, nonoverlapping sets of substrates (1, 2). Two important intracel- 12). This suggests that GrM targets multiple independent cell death lular substrates of GrB include procaspase 3 (5) and the small pathways, which has also been demonstrated for GrA and GrB (1, Bcl-2 homology domain 3-only protein Bid (6). Cleavage of these 2, 5–9). In the present study, we used a proteomic approach to define potential substrates of GrM. We report that GrM targets the *Department of Pathology, University Medical Center, Utrecht; †Department of Bio- cytoskeleton in tumor cells by cleaving the actin-plasma mem- molecular Mass Spectrometry, Utrecht University, Utrecht, The Netherlands; and brane linker ezrin and the microtubule component ␣-tubulin. This ‡Department of Medicine, Evanston Northwestern Healthcare Research Institute, Evanston, IL 60201 likely contributes to the mechanism and the specific morphological Received for publication July 25, 2007. Accepted for publication April 14, 2008. changes that coincide with GrM-mediated target cell death. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 This work was supported by the Netherlands Organization for Scientific Research Reagents Grant 916.66.044 (to N.B.), Dutch Cancer Society Grant UMCU-2004-3047 (to J.A.K.), and National Institutes of Health Grant R01 AI044941-07 (to C.J.F.). Abs were anti-␣-tubulin clone B-5-1-2 (Sigma-Aldrich), anti-ezrin clone 2 Address correspondence and reprint requests to Dr. J. Alain Kummer, Department 3C12 (Zymed Laboratories), anti-␤-actin clone 2A2.1 (United States Bio- of Pathology, University Medical Center, Heidelberglaan 100, 3584 CX, Utrecht, The logical), anti-caspase-3 clone H-277 (Tebu-bio), anti-GST tag (Santa Cruz Netherlands. E-mail address: [email protected] Biotechnology), and anti-His tag (BD Biosciences). E64, trans-epoxysuc- 3 Abbreviations used in this paper: GrA, granzyme A; GrB, granzyme B; GrM, gran- cinyl-L-leucylamido-(4-guanidino)butane, was from Sigma-Aldrich and zyme M; GrM-SA, GrM with S195A mutation in catalytic center; HSP, heat shock benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk) protein; MAP, microtubule-associated protein; MS, mass spectrometry; PI, propidium was from Biomol. The chromogenic caspase-3 substrate Ac-Asp-Glu-Val- iodide; ROS, reactive oxygen species; Ac-DEVD-pNA, acetyl-Asp-Glu-Val-Asp-p- Asp-p-nitroaniline (Ac-DEVD-pNA) was from Bachem. Purified recom- nitroaniline; E64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; z-VAD- fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone. binant human GST-k-␣-1-tubulin was purchased from Cytoskeleton. Hu- man perforin was purified as described (13). Protein was quantified by the Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 Bradford method. www.jimmunol.org The Journal of Immunology 8185 Cell lines and cell-free protein extracts HeLa and Jurkat cells were grown in DMEM and RPMI 1640 medium, respectively, supplemented with 10% FCS, 0.002 M glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin (Invitrogen). Cell-free protein ex- tracts were generated from exponentially growing HeLa and Jurkat cells. Cells (108 cells/ml) were washed two times in a buffer containing 50 mM Tris (pH 7.4) and 150 mM NaCl, and lysed in the same buffer by three cycles of freezing/thawing. This method gently disrupts the plasma mem- brane and minimally affects cell compartment integrity (14). Samples were centrifuged for 10 min at 14,000 rpm at 4°C and cell-free protein extracts were stored at Ϫ80°C. Recombinant proteins The cDNA encoding mature human GrM (residues Ile26–Ala257) was am- FIGURE 1. Identification of GrM-induced cleavage events in tumor cell plified from IMAGE clone 5222281 and cloned into yeast expression vec- lysates. HeLa cell freeze/thaw lysates were incubated with GrM-SA (1 tor pPIC9 (Invitrogen). Catalytically inactive GrM-SA, in which the Ser195 ␮M) (A) or GrM (1 ␮M) (B) for 60 min at 37°C. Proteins were separated residue in the catalytic center is replaced by Ala (S195A), was generated by two-dimensional gel electrophoresis (10%) and visualized by silver by site-directed mutagenesis (Stratagene). Plasmids were transformed into staining. Proteins that are reduced in abundance after GrM incubation rep- the GS115 (his4) strain of Pichia pastoris and granzymes were expressed resent potential GrM substrates (A) and new spots that appear during GrM in conditioned medium for 72 h as described by the manufacturer (Invitro- treatment are cleavage products (B). This experiment was performed three gen). GrM and GrM-SA were purified to homogeneity by cation-exchange times with similar results and the changed protein spots (n ϭ 37) were chromatography (GE Healthcare) using a linear salt gradient for elution. Downloaded from excised from two-dimensional gels. Protein spots that could be identified GrM preparations were dialyzed against 50 mM Tris (pH 7.4) and 150 mM ϭ NaCl and stored at Ϫ80°C.
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