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The Journal of Immunology, 2008, 180, 8184 -8191
Copyright © 2008 by The American Association of Immunologists, Inc.
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NK Cell Protease Granzyme M Targets {alpha}-Tubulin and Disorganizes the Microtubule Network1

Niels Bovenschen*, Pieter J. A. de Koning*, Razi Quadir*, Roel Broekhuizen*, J. Mirjam A. Damen{dagger}, Christopher J. Froelich{ddagger}, Monique Slijper{dagger} and J. Alain Kummer2,*

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 {alpha}-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, {alpha}-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct {alpha}-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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cytotoxic lymphocytes, i.e., CTLs and NK cells, are key players in the effector arm of the immune response that eliminates virus-infected cells and tumor cells (1, 2). Cytotoxic lymphocytes predominantly destroy their targets by releasing the content of their cytolytic granules. These granules contain perforin and a family of unique structurally homologous serine proteases known as granzymes (3, 4). Although perforin facilitates the entry of granzymes into the target cell, the latter induce cell death by cleaving critical intracellular substrates (1, 2).

In humans, five different granzymes (GrA, GrB, GrH, GrK, and GrM) are known that differ on the basis of their substrate specificity (3, 4). Over the past few decades, it has been well established that granzyme A (GrA)3 and granzyme B (GrB) serve as important determinants of cellular cytotoxicity. Both granzymes induce nuclear and non-nuclear damage in target cells by cleaving distinct nonoverlapping sets of substrates (1, 2). Two important intracellular substrates of GrB include procaspase 3 (5) and the small Bcl-2 homology domain 3-only protein Bid (6). Cleavage of these proteins leads to DNA fragmentation and mitochondrial damage, respectively. GrA predominantly kills by cleaving nuclear (e.g., Ku70), mitochondrial, and cytoplasmic substrates (e.g., SET complex components) (2, 7, 8, 9). Cleavage of these substrates results in single-stranded nicking of chromosomal DNA.

In contrast to GrA and GrB, far less is known about the other human granzymes. It has been demonstrated that granzyme M (GrM), which is specifically expressed by NK cells, mediates a novel major and perforin-dependent cell death pathway with unique morphological hallmarks that plays a significant role in NK cell-induced death (10). The molecular mechanism by which GrM induces cell death remains unclear. One study has found that GrM-induced cell death occurs independently of caspases, DNA fragmentation, and reactive oxygen species (ROS) generation (10), whereas other recent reports have demonstrated the opposite (11, 12). This suggests that GrM targets multiple independent cell death pathways, which has also been demonstrated for GrA and GrB (1, 2, 5, 6, 7, 8, 9). In the present study, we used a proteomic approach to define potential substrates of GrM. We report that GrM targets the cytoskeleton in tumor cells by cleaving the actin-plasma membrane linker ezrin and the microtubule component {alpha}-tubulin. This likely contributes to the mechanism and the specific morphological changes that coincide with GrM-mediated target cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Abs were anti-{alpha}-tubulin clone B-5-1-2 (Sigma-Aldrich), anti-ezrin clone 3C12 (Zymed Laboratories), anti-β-actin clone 2A2.1 (United States Biological), anti-caspase-3 clone H-277 (Tebu-bio), anti-GST tag (Santa Cruz Biotechnology), and anti-His tag (BD Biosciences). E64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, was from Sigma-Aldrich and benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk) was from Biomol. The chromogenic caspase-3 substrate Ac-Asp-Glu-Val-Asp-p-nitroaniline (Ac-DEVD-pNA) was from Bachem. Purified recombinant human GST-k-{alpha}-1-tubulin was purchased from Cytoskeleton. Human perforin was purified as described (13). Protein was quantified by the Bradford method.

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 µg/ml streptomycin (Invitrogen). Cell-free protein extracts 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 membrane 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 –80°C.

Recombinant proteins

The cDNA encoding mature human GrM (residues Ile26–Ala257) was amplified from IMAGE clone 5222281 and cloned into yeast expression vector pPIC9 (Invitrogen). Catalytically inactive GrM-SA, in which the Ser195 residue in the catalytic center is replaced by Ala (S195A), was generated by site-directed mutagenesis (Stratagene). Plasmids were transformed into the GS115 (his4) strain of Pichia pastoris and granzymes were expressed in conditioned medium for 72 h as described by the manufacturer (Invitrogen). GrM and GrM-SA were purified to homogeneity by cation-exchange chromatography (GE Healthcare) using a linear salt gradient for elution. GrM preparations were dialyzed against 50 mM Tris (pH 7.4) and 150 mM NaCl and stored at –80°C. GrM, but not GrM-SA, was active as determined by a synthetic chromogenic leucine substrate (Bachem) (data not shown). The human k-{alpha}-1-tubulin cDNA was amplified from IMAGE clone 3871729, cloned into the bacterial expression vector pQE80L, and expressed as recommended by the manufacturer (Invitrogen). The L269A, L286A, L286A/L269A, M302A, M302A/L269A, M313A, M313A/L269A, and L317A/L318A tubulin mutants were generated by site-directed mutagenesis. Recombinant His-{alpha}-tubulin protein was purified by metal-chelate chromatography (Clontech), dialyzed against PBS, and stored at –80°C. Ezrin cDNA was from RZPD German Resource Center for Genome Research. The pGEX-GST-ezrin bacterial expression construct, in which the GST tag is fused to the N terminus of human ezrin, was provided by Dr. H. Rehmann (UMC Utrecht, The Netherlands). Recombinant GST-ezrin was expressed and purified as described above for recombinant His-{alpha}-tubulin.

Two-dimensional gel electrophoresis and spot identification by mass spectrometry

Washed HeLa cells (108 cells/ml) in 25 mM Tris (pH 8), 30 mM NaCl, and 1 mM DTT were subjected to three rounds of freeze/thaw lysis and cell-free extracts (50 µg) were incubated with GrM (1 µM) or GrM-SA (1 µM). After 1 h at 37°C, samples were precipitated using the Plus One two-dimensional Clean-up kit as recommended by the manufacturer (GE Healthcare) and solubilized in 8 M urea, 2 M thiourea, 4% CHAPS, 20 mM DTT, 0.2% Biolyte (pH range 3–10), and 0.2% bromophenol blue (200 µl) for isoelectric focusing. Samples (50 µg) were rehydrated passively into 11-cm pH 3–10 immobilized pH gradient (IPG) strips for 15 h at room temperature before isoelectric focusing in the IPGphor system (GE Healthcare) for 20 kVh. The IPG strips were reduced for 60 min in 2% (w/v) DTT, 6 M urea, 2% (w/v) SDS, 20% (v/v) glycerol, and 0.375 M Tris (pH 8.8) and alkylated for 30 min in the same buffer containing 2% (w/v) iodoacetamide instead of DTT. Strips were mounted on 10% SDS-polyacrylamide gels, proteins were separated, and two-dimensional gels were stained by mass spectrometry (MS)-compatible silver staining. Gel features were evaluated by PDQuest 7.4 software and selected spots were excised robotically with a ProteomeWorks Spot Cutter (Bio-Rad). Gel cores were destained and subjected to in-gel tryptic digestion. Peptide mixtures were applied to liquid chromatography-tandem MS (Finnigan LTQ) and the results were analyzed by MASCOT (www.matrixscience.com).

GrM-mediated cell death

Washed Jurkat cells (1 x 106) were treated with GrM (1 µM) or GrM-SA (1 µM) in the presence or absence of a sublytic dose of perforin (40 ng/ml) in 50 mM HEPES (pH 7.4), 150 mM NaCl, 2.5 mM CaCl2, and 1% (w/v) BSA for 4 or 10 h at 37°C. Cells were washed in the same buffer and used for cytospins, propidium iodide (PI) flow cytometry, or direct lysis with SDS-PAGE loading buffer. Cytospins were fixed with 96% (v/v) ethanol for 10 min and stained with Giemsa or immunostained with an Ab against {alpha}-tubulin (clone B-5-1-2), followed by a tetramethylrhodamine isothiocyanate-conjugated Ab to visualize {alpha}-tubulin by confocal microscopy. For flow cytometry, cells were incubated with PI (46 µg/ml) for 10 min at room temperature. Cell viability after a 24-h incubation period with GrM/perforin or GrM-SA/perforin was measured by trypan blue staining.

Tubulin polymerization assay

A tubulin polymerization assay kit (Cytoskeleton) was used to address the effects of GrM on tubulin polymerization dynamics. A purified bovine {alpha}- and β-tubulin preparation (3 mg/ml) virtually free of microtubule-associated proteins (MAPs) (Cytoskeleton catalog no. HTS02) in 80 mM PIPES (pH 6.9), 0.5 mM EGTA, and 2 mM MgCl2 was incubated with GrM (1 µM), GrM-SA (1 µM), paclitaxel (Taxol) (5 µM), or buffer in 20 mM Tris (pH 7.0). With the exception of Taxol, samples were preincubated for 2 h at 30°C. Microtubule polymerization was initiated by the addition of GTP (1 mM) and 5% (v/v) glycerol. Changes in microtubule turbidity were measured kinetically at 340 nm at 37°C (Anthos Labtec).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
GrM-induced cleavage events in tumor cell lysates

To define potential intracellular substrates of GrM, we used a protease-proteomic approach. Because it is difficult to deliver large amounts of GrM to all target cells via perforin, we used freeze/thaw lysis of HeLa tumor cells to gently disrupt the plasma membrane and minimally alter the native proteome. These protein extracts were incubated with purified recombinant mature human GrM or the catalytically inactive GrM-SA mutant and cleavage events were analyzed by two-dimensional gel electrophoresis (Fig. 1). Spots present in greater abundance in the control sample indicate possible GrM substrates, whereas spots present in greater abundance in the GrM-treated sample reflect potential cleavage products. Of ~1500 proteins that were resolved in this proteomic screen, ~15 spots clearly disappeared (Fig. 1A) and ~22 spots clearly appeared (Fig. 1B) following the incubation with GrM. These changes were highly reproducible and could also be detected when samples were labeled fluorescently and analyzed on the same two-dimensional gel using fluorescence two-dimensional difference gel electrophoresis (fl-2D-DIGE; data not shown). Spots were excised that exhibited high reproducibility and displayed > 3-fold changes in abundance following GrM treatment. We were able to identify 16 of 37 excised protein spots from two-dimensional gels using tandem MS (Table I). Several spots that consistently changed following GrM treatment could not be identified, most likely because protein levels were too low. The identity of potential GrM substrates that could be identified include a group of highly homologous proteins involved in chaperone systems and cellular stress response (i.e., heat shock protein (HSP) 90β, endoplasmin, and protein disulfide isomerase), proteins involved in translational machinery (i.e., heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, U5 small nuclear ribonuclear protein (snRNP) component, EL-Tu, EL-1-{alpha}-1, and hnRNP E1), a protein of less well defined function (i.e., LRP130), some miscellaneous (i.e., GAPDH and carbamoylphosphate synthetase I), and several components that control the integrity of the cytoskeleton (i.e., β-actin, ezrin, and {alpha}-tubulin) (Table I).


Figure 1
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  		FIGURE 1. Identification of GrM-induced cleavage events in tumor cell lysates. HeLa cell freeze/thaw lysates were incubated with GrM-SA (1 µM) (A) or GrM (1 µM) (B) for 60 min at 37°C. Proteins were separated by two-dimensional gel electrophoresis (10%) and visualized by silver staining. Proteins that are reduced in abundance after GrM incubation represent potential GrM substrates (A) and new spots that appear during GrM treatment are cleavage products (B). This experiment was performed three times with similar results and the changed protein spots (n = 37) were excised from two-dimensional gels. Protein spots that could be identified (n = 16) by liquid chromatography-tandem MS are indicated by white circles.

 

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  		Table I. Overview of GrM-induced cleavage events identified by tandem mass spectrometrya

 
GrM cleaves cytoskeleton-related proteins

The group of proteins that control the integrity of the cytoskeleton (i.e., β-actin, ezrin, and {alpha}-tubulin) was selected for further studies because inactivation or down-regulation of these proteins has been causally linked to cell death and they are known to protect tumor cells from apoptosis (15, 16, 17, 18, 19). In addition, these proteins reside in the cytoplasm and as such are accessible for GrM. Finally, {alpha}-tubulin has recently been identified as a physiological substrate of the cytotoxic lymphocyte component GrB (19, 20) and is currently considered one of the most successful targets for anticancer chemotherapy (16, 18). To verify the cleavage of β-actin, ezrin, and {alpha}-tubulin by GrM in protein extracts, HeLa cell lysates were incubated in the presence or absence of GrM or GrM-SA and subjected to immunoblotting by using Abs against these proteins (Fig. 2). Incubation of lysates with GrM resulted in time-dependent cleavage of ezrin and {alpha}-tubulin, but not β-actin. Unlike ezrin and {alpha}-tubulin, the β-actin protein was identified by tandem MS from cleaved β-actin fragments that appeared during GrM cleavage (spot no. 9 in Fig. 1B). This precluded information on the efficiency by which intact β-actin is cleaved by GrM. Western blot analysis, however, now indicates that the bulk of the intact β-actin protein remains uncleaved (Fig. 2). Although the mAbs used could not detect cleavage products, GrM-mediated cleavage of ezrin and {alpha}-tubulin was illustrated by the progressive time-dependent disappearance of both ezrin and {alpha}-tubulin protein bands. The molecular weights of noncleaved ezrin and {alpha}-tubulin matched the molecular weights of the protein spots excised from GrM-SA-treated lysates on two-dimensional gels (Fig. 1A) and that of the theoretical molecular mass of these proteins (Table I). Cleavage of {alpha}-tubulin by GrM in cell lysates was virtually completed after 30–60 min of incubation, whereas complete cleavage of ezrin occurred after 2–4 h. GrM-SA did not show any reactivity with these substrates, indicating that all cleavage events are specific for GrM proteolytic activity. Similar results were obtained when Jurkat cell lysates were used (data not shown). These data indicate that ezrin and {alpha}-tubulin are direct or indirect substrates of human GrM and that {alpha}-tubulin kinetically was more efficiently cleaved in tumor cell lysates as compared with ezrin.


Figure 2
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  		FIGURE 2. Ezrin and {alpha}-tubulin are cleaved by GrM in tumor cell lysates. HeLa cell lysates were incubated with GrM (1 µM), GrM-SA (1 µM), or buffer for the indicated times at 37°C. Samples were immunoblotted using Abs against β-actin, ezrin, and {alpha}-tubulin. These experiments were repeated at least three times with the same results.

 
GrM cleaves {alpha}-tubulin and ezrin in tumor cells undergoing cell death

To examine whether {alpha}-tubulin and ezrin are also cleaved in intact cells undergoing cell death induced by GrM, we used a cell death assay in which perforin was used to deliver GrM into the tumor cell (Fig. 3). It has been established that GrM-induced cell death can be detected by typical morphological changes and by measuring membrane integrity by PI flow cytometry (10). Consistent with this, GrM/perforin-treated cells demonstrated the morphological changes as described (10), such as signs of chromatin condensation and the presence of large cytoplasmic vacuoles (Fig. 3C). As expected (10), GrM/perforin-treated cells showed higher PI staining as compared with controls (Fig. 3, E–H). Lysates of GrM- or GrM-SA-treated cells were subjected to immunoblotting and cleavage of {alpha}-tubulin and ezrin was monitored (Fig. 3I). Cleavage of {alpha}-tubulin or ezrin was not observed in cells treated with buffer, perforin alone, or perforin in combination with GrM-SA, but was detected in cells treated with perforin and GrM. After 4 h of incubation a modest reduction of {alpha}-tubulin could be observed, whereas only a minor reduction of ezrin was found. This is consistent with the observed differential cleavage kinetics of both proteins by GrM (Fig. 2). Cleavage of {alpha}-tubulin and ezrin, however, was nearly complete after a 10-h incubation period. In line with the results obtained in Fig. 2, no discernible hydrolysis of β-actin was observed. GrM-induced cell death was irreversible, as cell viability of GrM/perforin-treated Jurkat cells was 2.1 ± 2.0% (mean ± S.D.) as compared with 100 ± 11.9% for GrM-SA/perforin after a 24 h incubation period (p < 0.001). These results indicate that {alpha}-tubulin and ezrin, but not β-actin, are cleaved in tumor cells that undergo irreversible cell death induced by GrM in combination with perforin.


Figure 3
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  		FIGURE 3. Ezrin and {alpha}-tubulin are cleaved by GrM in tumor cells undergoing cell death. A–D, Jurkat cells (1 x 106) were treated with a sublytic dose of perforin (40 ng/ml) and GrM (1 µM), perforin (40 ng/ml) and GrM-SA (1 µM), perforin (40 ng/ml) alone, or GrM (1 µM) alone for 4 h at 37°C. Cells (1 x 105) were centrifuged on glass slides and visualized by Giemsa staining. Bar, 20 µm. E–H, Cells (1 x 104) were incubated with PI (1 µg/ml) for 10 min at room temperature and analyzed by flow cytometry. I, At indicated time points the cells were lysed and whole cell protein extracts were immunoblotted using Abs against {alpha}-tubulin, ezrin, and β-actin. These experiments were repeated at least three times with the same results.

 
GrM cleaves {alpha}-tubulin and ezrin in a caspase-independent manner

Lu et al. (11) recently demonstrated that one way by which GrM induces cell death involves proteolytic activation of procaspase-3, whereas another study shows that GrM-mediated cell death completely occurs independently of caspases (10). Therefore, we have addressed the role of caspases and other cysteine proteases during GrM-mediated proteolysis of {alpha}-tubulin and ezrin. To this end, we used the pan-caspase inhibitor z-VAD-fmk and the broad spectrum cysteine protease inhibitor E64. Tumor cell lysates were incubated with GrM and proteolysis of {alpha}-tubulin and ezrin was monitored by immunoblotting (Fig. 4A). Neither z-VAD-fmk nor E64 affected GrM-mediated cleavage of {alpha}-tubulin and ezrin, indicating that neither caspases nor other cysteine proteases are involved in this process. E64 and z-VAD-fmk did not affect the activation of procaspase-3 by GrB (Fig. 4A), which is expected because GrB is a serine protease (1, 2, 3, 4). Next, we investigated the capability of GrM to cleave and/or activate procaspase-3. In contrast to Lu et al. (11) but consistent with Kelly et al. (10), GrM did not cleave procaspase-3 (Fig. 4A). Also at higher GrM concentrations and longer incubation times GrM did not cleave this procaspase (Fig. 4B). In agreement with these findings, GrM did not activate procaspase-3 as determined by the small chromogenic caspase-3 substrate Ac-DEVD-pNA (Fig. 4C). GrB was included as positive control for cleavage and activation of procaspase-3 in these experiments. Indeed, the activity of caspase-3 was completely inhibited by the pan-caspase inhibitor z-VAD-fmk. As expected, E64 did not affect caspase-3 activity, given that E64 does not inhibit caspases (21). E64 was functionally active because it efficiently blocked the cathepsin-binding properties of DCG-04, which is an active site-directed probe that is used to specifically label active cathepsin proteases (data not shown). Taken together, these results indicate that GrM neither cleaves nor activates procaspase-3 and that {alpha}-tubulin and ezrin are not cleaved by GrM in a caspase(-3)-dependent or other cysteine protease-dependent manner.


Figure 4
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  		FIGURE 4. GrM cleaves {alpha}-tubulin and ezrin in a caspase-independent manner. A, Jurkat cell lysates were incubated with GrM (1 µM), GrM-SA (1 µM), or GrB (1 µM) for 2 h at 37°C, in the absence (–) or presence (+) of z-VAD-fmk (10 µM) or E64 (10 µM). Samples were immunoblotted, using Abs against {alpha}-tubulin, ezrin, or caspase-3. B, Jurkat cell lysates were incubated with GrM (2 µM), GrM-SA (2 µM), or GrB (0.5 µM) for 0–4 h at 37°C. Samples were immunoblotted, using Abs against caspase-3. These experiments were repeated at least three times with the same results. C, Jurkat cell lysates were incubated with GrM (1 µM), GrM-SA (1 µM), or GrB (1 µM) for 4 h at 37°C in the absence (–) or presence (+) of z-VAD-fmk (10 µM) or E64 (10 µM). Samples were incubated with the chromogenic caspase-3 substrate Ac-DEVD-pNA (0.5 mM) and measured kinetically at 405 nm for 60 min. Data are presented as pmol of p-nitroaniline that is cleaved from the chromogenic substrate per minute as mean ± S.D. of three independent experiments.

 
GrM directly cleaves {alpha}-tubulin and ezrin

We investigated whether {alpha}-tubulin and ezrin constitute direct GrM substrates rather than being the substrates of secondary proteases other than cysteine proteases or caspases present in tumor cells. To this end, GrM or GrM-SA was incubated with purified recombinant GST-{alpha}-tubulin or GST-ezrin. Treatment of purified GST-ezrin with GrM, but not GrM-SA, resulted in the disappearance of the expected ~100 kDa GST-ezrin protein band (Fig. 5). Using anti-GST (Fig. 5A) or anti-C-terminal ezrin Abs (Fig. 5B), cleavage products were detected by immunoblotting. This indicates that ezrin is a direct substrate of GrM. Whereas GrM-SA-treated and untreated GST-{alpha}-tubulin remained intact, treatment of purified {alpha}-tubulin with increasing concentrations of GrM resulted in the progressive disappearance of the ~75-kDa GST-fused {alpha}-tubulin protein and the appearance of two major cleavage products of ~52 and ~23 kDa (Fig. 6A). This cleavage event already occurred at low nanomolar concentrations of GrM (5–20 nM) and relatively high tubulin concentrations (1 µM). Immunoblotting with an Ab against GST revealed that the 52-kDa band represents the N-terminal {alpha}-tubulin moiety fused to the GST tag (data not shown). Higher concentrations of GrM further processed the N-terminal 52-kDa cleavage product, indicating that GrM cleaves {alpha}-tubulin at least at two sites. Taking a closer look at the molecular weights of the cleavage fragments of {alpha}-tubulin and knowing the proposed P1 primary and P2-P4 subsite specificities of GrM (22, 23), we mutated the Leu residue at position 269 into Ala. Although this mutant His-tagged {alpha}-tubulin was also cleaved by GrM, a different amino-terminal cleavage product with increased m.w. was observed (Fig. 6B). Using a highly sensitive fluorescent protein staining on GrM-cleaved wild-type and L269A mutant {alpha}-tubulin, the up-shift of the N-terminal proteolytic fragment of L269A {alpha}-tubulin mutant was again evident (Fig. 6C). Strikingly, however, under these conditions at least seven other {alpha}-tubulin cleavage fragments appeared when {alpha}-tubulin was cleaved by GrM (Fig. 6C). The L269A {alpha}-tubulin mutant kinetically was equally well cleaved by GrM as compared with wild-type {alpha}-tubulin (Fig. 6D), strongly suggesting that other GrM cleavage sites in {alpha}-tubulin are at least equally important. Furthermore, the N-terminal {alpha}-tubulin cleavage fragment appeared at low GrM concentrations with limited proteolysis at multiple sites until it completely disappeared when higher GrM concentrations were used (Fig. 6D). We have attempted to identify additional GrM cleavage sites by site-directed mutagenesis of Leu and Met residues more C-terminal of Leu269. These {alpha}-tubulin mutants include L286A, L286A/L269A, M302A, M302A/L269A, M313A, M313A/L269A, and L317A/L318A. Except for L269A, we were not able to demonstrate a difference in proteolysis of these mutants, either alone nor in combination with L269A (data not shown). This indicates that other Leu or Met residues in {alpha}-tubulin are more important GrM cleavage sites or that GrM cleaves {alpha}-tubulin after other amino acids than the proposed Leu or Met (22, 23). The latter would be consistent with a Ser residue being a GrM cleavage site in (inhibitor of caspase-activated DNase (ICAD; Ref. 11). Cleavage at alternate sites in substrates has also been found for GrA and its substrates Ku70 and SET (9). Cleavage by GrM after Leu269 bisects the MAP-binding domain of {alpha}-tubulin (Fig. 6E) and this cleavage site is conserved in all human {alpha}-tubulin isoforms except {alpha}-tubulin-L3 (Fig. 6F). The latter isoform, however, harbors P1 Met that can also be hydrolyzed by GrM (22, 23). Thus, {alpha}-tubulin proteolysis by GrM is direct, efficient, complex, and occurs at multiple cleavage sites. One cleavage site includes Leu269 and at least one other cleavage site is positioned slightly more C-terminal thereof.


Figure 5
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  		FIGURE 5. GrM directly cleaves ezrin. Purified recombinant GST-ezrin (75 nM) was treated with indicated concentrations of GrM (0–600 nM) or GrM-SA (600 nM) for 2 h at 37°C. GrM:GST-ezrin stoichiometries were 1.5:1, 2.6:1, 5.3:1, and 7.7:1. Proteins were separated by SDS-PAGE (10%) and subjected to immunoblotting using Abs against the N-terminal GST tag (A) or the C-terminal part of ezrin (B). Full-length GST-ezrin (solid arrow) and cleavage products (dotted arrows) are indicated. ns, Nonspecific.

 

Figure 6
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  		FIGURE 6. GrM directly cleaves {alpha}-tubulin. A, Purified recombinant GST-{alpha}-tubulin (1 µM) was treated with indicated concentrations of GrM (0–500 nM) or GrM-SA (500 nM) for 2 h at 37°C. GrM:GST-{alpha}-tubulin stoichiometries were 1:1000, 1:200, 1:50, 1:10, and 1:2. Proteins were separated by SDS-PAGE (10%) and stained with Coomassie Brilliant Blue. Full-length GST-{alpha}-tubulin (solid arrow) and cleavage products (dotted arrows) are indicated. B, Purified recombinant wild-type His-{alpha}-tubulin (WT) (1 µM) and the His-{alpha}-tubulin mutant in which Leu269 has been replaced by Ala (L269A) were treated with GrM (50 nM) or GrM-SA (50 nM) for 2 h at 37°C. Samples were subjected to Western blot analysis, using an anti-His tag Ab. C, Purified recombinant wild-type (WT) and mutant L269A His-{alpha}-tubulin (1 µM) were treated with (+) or without (–) GrM (50 nM) for 2 h at 37°C. Proteins were separated by SDS-PAGE (10%) and stained with fluorescent Flamingo staining. D, Wild-type and mutant L269A His-{alpha}-tubulin were treated with GrM (0–500 nM) or GrM-SA (500 nM) for 2 h at 37°C. Proteins were separated by SDS-PAGE (10%) and stained by immunoblotting, using an anti-His tag Ab. Full-length His-{alpha}-tubulin (solid arrow), and N-terminal (N1 and N2) cleavage products and other cleavage fragments (dotted arrows) are indicated (B–D). E, Schematic representation of {alpha}-tubulin domain structure, including the GrM cleavage site. F, Sequence alignment of amino acid region 246–289 of human {alpha}-tubulin isoforms. Amino acid identity is indicated in black, except that the GrM cleavage site is depicted in gray. A–D represent P1'–P4' and 1–4 represent P1–P4, respectively.

 
GrM de-regulates tubulin polymerization dynamics

To investigate the effect of GrM on tubulin polymerization rates, we preincubated GrM with a mixture of purified, MAP-depleted bovine {alpha}- and β-tubulin. Following the addition of GTP, microtubule formation was measured kinetically at an absorbance of 340 nm. The kinetics of tubulin polymerization was markedly enhanced in the presence of GrM as compared with GrM-SA or buffer (Fig. 7A). This effect of GrM was comparable to the effect induced by the well-established anti-microtubule, anti-cancer drug paclitaxel (Taxol) (Fig. 7A) (16, 18). GrM cleaved bovine {alpha}-tubulin during the time course of tubulin polymerization (Fig. 7B). This is consistent with the GrM cleavage site (at least P4-P4') being completely conserved in bovine {alpha}-tubulin and multiple other mammalian species. Thus, GrM deregulates the polymerization dynamics of the microtubule network.


Figure 7
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  		FIGURE 7. GrM disturbs tubulin polymerization dynamics. A, Purified bovine tubulin (40 µM) was incubated with GrM (1 µM) (open circles), GrM-SA (1 µM) (open diamonds), Paclitaxel (Taxol) (5 µM) (closed triangles), or buffer alone (closed circles). Except for Taxol, treated samples were preincubated for 2 h at 30°C. Microtubule polymerization was then initiated by the addition of GTP (1 mM) and 5% glycerol and measured kinetically at 340 nm at 37°C. Data represent the mean of three to four independent experiments. B, After 60 min of measurement, cleavage of bovine {alpha}-tubulin by GrM was verified by immunoblotting using an anti-{alpha}-tubulin Ab.

 
GrM disorganizes the microtubule network during killing of tumor cells

To evaluate the physiological effects of GrM-mediated cleavage of {alpha}-tubulin, perforin was used to load Jurkat cells with GrM or GrM-SA for 4 h. GrM-induced cell dead was verified by PI flow cytometry (Fig. 3, E–H) and visual morphological inspection (Fig. 3, A–D) (10). To visualize microtubules, cells were stained with an Ab against {alpha}-tubulin and analyzed by confocal microscopy (Fig. 8). The microtubule network of control perforin/GrM-SA-treated cells appeared as a normal fine structured filamentous tubule network (Fig. 8, A and B) similar to that of untreated cells and cells that were incubated with perforin alone (data not shown). In contrast, many cells that received perforin in combination with GrM displayed an aberrant microtubule network in that {alpha}-tubulin structures were less organized and appeared more diffuse (Fig. 8, C and D). Interestingly, perforin/GrM-treated cells were more flattened and displayed an aberrant shape as compared with controls. The thickness of representative cells (mean ± S.D.) was 6.6 ± 0.7 µm and 3.6 ± 0.3 µm (p < 0.001, n = 7) and the diameter was 12.3 (± 1.1) µm and 17.7 (± 1.4) µm (p < 0.001, n = 7) for GrM-SA- and GrM-treated cells, respectively. These data indicate that GrM disorganizes the microtubule network and cell shape in tumor cells that are attacked by perforin and GrM.


Figure 8
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  		FIGURE 8. GrM disorganizes the microtubule network in tumor cells undergoing cell death. A–D, Jurkat cells (1 x 106) were treated with a sublytic dose of perforin (40 ng/ml) and GrM-SA (1 µM) (A and B) or GrM (1 µM) (C and D) for 4 h at 37°C. {alpha}-Tubulin is visualized by x100 (original magnification) fluorescent immunostaining and confocal microscopy. Bar, 5 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Little is known about the molecular mechanisms by which GrM kills its target cells. It has been postulated that GrM uses multiple pathways to kill, either via caspase-dependent routes that lead to DNA fragmentation and ROS production or via caspase-independent pathways that do not result in fragmentation of DNA and production of ROS (10, 11, 12). Targeting of multiple independent cell death pathways has also been demonstrated for GrA and GrB (1, 2, 5, 6, 7, 8, 9). In the present study, we have demonstrated that GrM neither cleaves nor activates procaspase-3 (Fig. 4), which is in contrast to Lu et al. (11) but consistent with Kelly et al. (10). We have identified several novel potential substrates of GrM. We have shown that GrM directly cleaves the actin-plasma membrane linker ezrin and the microtubule network protein {alpha}-tubulin in tumor cells that are attacked by perforin and GrM (Figs. 3, 5, 6). Ezrin and {alpha}-tubulin are not cleaved by GrM in a caspase(-3)- or cysteine protease-dependent manner (Fig. 4). Cleavage of {alpha}-tubulin by GrM deregulates {alpha}-tubulin function and leads to disorganization of the microtubule network (Figs. 7 and 8). Therefore, tubulin proteolysis by GrM is likely to be a critical event during NK cell-mediated killing.

Microtubules are responsible for cell survival, mitosis, motility, maintenance of cell shape, cell signaling, and intracellular trafficking of macromolecules, vesicles, and organelles (16, 18). The highly dynamic behavior of microtubules is greatly affected by well-known anti-cancer drugs, like vinblastine, vincristine, and Taxol, which all induce abnormal mitosis and cell death (16, 18). Furthermore, down-regulation of {alpha}-tubulin by RNA interference results in the death of tumor cells and limits their mitotic potential (19). We have found that GrM cleaves off the C-terminal part of the {alpha}-tubulin MAP-binding domain (Fig. 6), which regulates microtubule polymerization dynamics and microtubule motor activity (16, 18). Indeed, GrM, like Taxol, shifted the balance of microtubule dynamics toward polymerization (Fig. 7), which has also been demonstrated for GrB (19). In this context, however, we cannot fully exclude the following: 1) that GrM affects in vitro tubulin polymerization by cleaving β-tubulin or trace amounts of MAPs that may accompany tubulins in the tubulin polymerization assay; and/or 2) that GrM-cleaved {alpha}-tubulin is irrelevant to microtubule polymerization in that it is more prone to aggregation as compared with uncleaved {alpha}-tubulin. Nevertheless, GrM disorganizes the microtubule network and cell shape in tumor cells that are attacked by perforin and GrM (Fig. 8). Therefore, we hypothesize that GrM-induced cleavage of {alpha}-tubulin in tumor cells contributes to cell death induced by NK cells and/or that it enhances the NK cell function to kill. The latter possibility would be compatible with the finding that Taxol pretreatment amplifies NK cell-mediated lysis of tumor targets (24). Alternatively, it has been well established that host cell microtubules are indispensable for viral entry, replication, and exit (25). GrM plays a significant role in the elimination of virus-infected cells in vivo (26). This opens the possibility that GrM-mediated disruption of microtubule function terminates viral production in infected cells during NK cell attack. GrM may act in concert with GrB, because the latter also cleaves {alpha}-tubulin and resides in the same NK cell granules (19, 20).

GrM cleaved the actin-plasma membrane linker ezrin by using purified proteins (Fig. 5), tumor cell lysates (Fig. 2), and tumor cells undergoing cell death induced by GrM and perforin (Fig. 3). Ezrin is (over)expressed in a variety of cancers, some of which are associated with poor clinical outcome (27). Linkage of the plasma membrane to the actin cytoskeleton by ezrin allows a cell to interact directly with its microenvironment (27). Ezrin also facilitates several signal transduction pathways, like that of the protein kinases AKT and MAPK (MEK/ERK) that protect cells against apoptosis (15). Therefore, GrM-dependent cleavage of ezrin may impair activation of AKT and MAPK survival pathways and thus may contribute to target cell death. Another possibility may be that GrM inhibits tumor metastatic progression by inactivation of ezrin. This would be consistent with the findings that ezrin is necessary for metastatic progression and that it is required for early metastatic survival of tumor cells in vivo (27). Further studies are required to distinguish between these possibilities.

Fourteen novel potential substrates of GrM were identified (Table I). Apart from {alpha}-tubulin and ezrin, however, direct processing by GrM of these proteins and the precise role thereof remains to be investigated. Some potential GrM substrates may play a role in the mechanism by which GrM induces cell death, for instance proteins involved in chaperone and cell stress response (Table I). HSP90β plays an essential role in maintaining stability and activity of its client proteins, including a set of signaling proteins that regulate key pathways in cell survival and oncogenesis (28). HSP90β is frequently overexpressed in cancer cells and, more importantly, synthetic HSP90β inhibitors have successfully been evaluated in multiple phase II anticancer clinical trials (27). If GrM indeed inactivates HSP90β, this may represent a novel mechanism by which GrM induces target cell death. We are currently addressing this possibility. Interestingly, the HSP90 cochaperones Hop and Hip have recently been identified as novel substrates of GrB (29, 30). Whether or not GrM also plays a role in other cellular processes than cell death remains an intriguing question that deserves further study.

Our proteomic approach has identified a limited set of potential GrM substrates (Fig. 1, Table I). The relatively small number of cleavage events detected suggests that GrM substrate specificity depends on extended binding site(s) on folded proteins rather than short linear peptides that represent the cleavage site (P1), i.e., methionine or leucine (22, 23). This is consistent with the high specificities of GrA and GrB, which also fully depend on secondary structures of folded substrates (31, 32). Remarkably, our proteomic screen did not detect one of the known GrM substrates, i.e., inhibitor of caspase-dependent DNase (ICAD), poly(ADP-ribose) polymerase (PARP), HSP75 (TRAP1), or procaspase-3 (11, 12). Absence of the latter is consistent with our finding that GrM neither cleaves nor activates procaspase-3 in tumor cells (Fig. 4). Although two-dimensional gel electrophoresis is capable of resolving >1000 individual protein spots on a single gel, not all proteins could be visualized because of low abundance or extremes of m.w. or charge. In addition, some of the known GrM substrates may have been detected on the gels but could not be identified by tandem MS. We were able to definitively identify 16 of 37 excised protein spots. Because of the nonquantitative nature of silver staining, it remains difficult to address the cellular abundance of our identified potential GrM substrates.

GrM is highly expressed by NK cells but not in CTLs (33). NK cells play a major role in the innate immune response that forms the first line of defense against tumor cells and virus-infected cells, and they have broad applications in immunotherapy of cancer (34). Knowledge of the precise mechanisms by which NK cells kill tumor cells may lead to further optimization of immunotherapy and/or other proapoptotic anticancer therapies.


   Acknowledgments
 
We thank Dr. M. A. G. G. Vooijs for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.). Back

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 Back

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. Back

Received for publication July 25, 2007. Accepted for publication April 14, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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