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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 Kummer^2,*

^* Department of Pathology, University Medical Center, Utrecht; Department of Biomolecular Mass Spectrometry, Utrecht University, Utrecht, The Netherlands; and Department of Medicine, Evanston Northwestern Healthcare Research Institute, Evanston, IL 60201

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 GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymerization 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 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.

¹ This work was supported by the Netherlands Organization for Scientific Research 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.).

² Address correspondence and reprint requests to Dr. J. Alain Kummer, Department of Pathology, University Medical Center, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands. E-mail address: j.a.kummer{at}umcutrecht.nl

³ Abbreviations used in this paper: GrA, granzyme A; GrB, granzyme B; GrM, granzyme M; GrM-SA, GrM with S195A mutation in catalytic center; HSP, heat shock protein; MAP, microtubule-associated protein; MS, mass spectrometry; PI, propidium iodide; ROS, reactive oxygen species; Ac-DEVD-pNA, acetyl-Asp-Glu-Val-Asp-p-nitroaniline; E64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone.