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Previous Article | Next Article 
Molecular and Cellular Biology, April 2002, p. 2810-2820, Vol. 22, No. 8
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.8.2810-2820.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Center for Blood Research and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115
Received 28 December 2001/ Accepted 22 January 2002
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ABSTRACT |
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INTRODUCTION |
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To understand the GzmA-mediated cell death pathway, affinity chromatography with inactive Ser
Ala mutant GzmA (S-AGzmA) was used to determine potential substrates and other proteins that associate with GzmA (8). A 270- to 420-kDa cytoplasmic complex (SET complex) from K562 cell lysates binds to immobilized S-AGzmA (9). The purified SET complex reconstitutes single-stranded DNA nicking in isolated nuclei in the presence of GzmA (10). The SET complex contains the tumor suppressor protein pp32 (5, 10, 28, 59, 61), the nucleosome assembly protein (NAP) SET (10, 31, 59, 60), and the base excision repair enzyme APE/ref-1 (20; Z. Fan, P. J. Beresford, D. Zhang, Z. Xu, C. D. Novina, A. Yoshida, Y. Pommier, and J. Lieberman, submitted for publication). SET and APE, but not pp32, are substrates of GzmA in CTL attack and after GzmA loading with perforin (8, 10; Fan et al., submitted). pp32 (also known as PHAPI, I1PP2A) is a highly conserved protein phosphatase 2A (PP2A) inhibitor with putative nuclear localization and export signals which shuttles between the nucleus and the cytosol via binding to the nuclear export receptor crm1 (13, 28). SET (also known as PHAPII, TAF-Iß, I2PP2A) was initially identified as a translocated gene in acute undifferentiated leukemia (60). SET can stimulate adenovirus replication of a viral chromatin template (31). Recent reports show that SET and its homologues bind to the transcriptional coactivators CBP/p300 and the core histones and may serve as a link between transcriptional coactivators and chromatin (40, 46). APE is a multifunctional protein with DNA binding and endonuclease activity associated with its C terminus and redox function in the N-terminal domain (reviewed in reference 22). APE, a major member of the base excision repair pathway, recognizes and is the rate-limiting enzyme in the repair of DNA apurinic/apyrimidinic (AP) sites, the most frequent type of DNA damage in cells (7, 34). It also activates by a redox-based mechanism the DNA binding activity of many transcription factors, including AP-1 (Fos/Jun), NF-
B, and Myb (64).
SET and its homologue TAF-I
and pp32 and its homologue APRIL were recently shown to coassociate in a smaller 
150-kDa nuclear complex which has been shown to inhibit histone acetylation and to bind to HuR, a protein that increases the stability of mRNAs with AU-rich regions that are encoded by many early-response genes, protooncogenes, and cytokine genes (13, 40). The size of the nuclear SET complex suggests that SET, pp32, and their homologues may constitute all the components of the nuclear complex, although each of these proteins is so acidic (predicted pIs of 4) that it is hard to see how they can stay together without a balancing charge. However, the endoplasmic reticulum (ER)-associated SET complex is substantially larger and not all of its known components (SET, pp32, and APE) account for its apparent molecular weight as estimated by gel filtration (10; Fan et al., submitted). All of the components of the ER-associated SET complex can also be found in the nucleus, where most of their known functions would require them to be. The functions of the cytoplasmic SET complex are unknown, as are the signals, which regulate its nuclear import and export. In this study we show that, despite its nuclear function, the DNA bending protein HMG2 (high mobility group protein 2; reviewed in reference 14) is a component of the ER-associated SET complex. HMG2 binds directly to SET and colocalizes in part with SET to associate with the ER. Just as GzmA destroys the NAP activity of SET and the DNA repair and redox activities of APE, it also cleaves and functionally inactivates the DNA binding and bending activities of HMG2.
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MATERIALS AND METHODS |
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Recombinant proteins and purified PFP. Recombinant GzmA, inactive S-AGzmA, and GzmB were produced and purified as previously reported (8, 65). PFP was purified from the rat RNK-16 cell line and used at a sublytic concentration, titrated to induce <10% cytolysis, as described previously (44). Recombinant pp32 (rpp32) was expressed in BL21-DE3 cells from pET 30a, recombinant SET (rSET) was expressed from pET 26b, recombinant APE was expressed from pET 14b (a kind gift of I. Hickson, University of Oxford), and recombinant GST (rGST) was expressed from pET 30b (Novagen) as described previously (10; Fan et al., submitted). Recombinant proteins with His6 tags were purified sequentially over Novagen nickel and Bio-Rad anion-exchange columns. HMG2 cDNA, a kind gift of P. Sharp, was expressed from pET15b and purified as described previously (48).
Cytoplasmic SET complex isolation and HMG2 identification. K562 cell lysates (1010 cell equivalents in NP-40 lysis buffer [50 mM Tris HCl, pH 7.5; 0.5% NP-40; 25 mM KCl; 5 mM MgCl2]) were loaded onto an immobilized S-AGzmA column and eluted with 500 mM NaCl in 50 mM Tris-HCl, pH 7.5, as described previously (8). The concentrated S-AGzmA column eluate was applied in Tris-buffered saline to an S400 gel filtration column (Pharmacia). Eluted fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting and compared to the elution profile of Pharmacia gel filtration standards. The S-AGzmA column eluate was also desalted and applied to a Bio-Rad Q column and eluted with an NaCl gradient in 50 mM Tris-HCl, pH 7.5. The dominant 28-kDa band in the flowthrough visualized after Coomassie blue staining was subjected to N-terminal sequencing performed by M. Berne of the Tufts Core facility. A 28-kDa band directly eluted from the S400 column was also analyzed by tryptic digestion and sequencing performed by the Harvard Microchemistry Facility by microcapillary reverse-phase high-pressure liquid chromatography nanoelectrospray tandem mass spectroscopy.
Coimmunoprecipitation and immunoblotting. Antibodies were preincubated with protein A-Sepharose (Pharmacia) for 1 h at 4°C. Antibody-coated beads were washed twice in phosphate-buffered saline (PBS) before being added to recombinant proteins (50 µg/ml) or postnuclear cytosolic lysates (5 x 106 cell equivalents in 20 µl of NP-40 lysis buffer) that had been preincubated at 4°C for 2 h. After overnight incubation with shaking at 4°C, the beads were washed extensively in 1% NP-40, 0.1% SDS in PBS and boiled in 2x SDS loading buffer before electrophoresis through SDS-PAGE gels. Nuclear lysates were prepared by washing the nuclear pellet, obtained after centrifugation at 1,000 x g of NP-40-lysed K562 cells, twice in NP-40 lysis buffer followed by lysis in NP-40 lysis buffer with 500 mM NaCl. S400 column fractions or nuclear and cytoplasmic fractions were similarly analyzed by SDS-PAGE and then transferred to nitrocellulose before probing with indicated antibodies as described.
Laser-scanning confocal microscopy. HeLa cells, grown overnight to subconfluency at 37°C in eight-well chamber slides coated with rat collagen I (Becton Dickinson Labware, Bedford, Mass.), were fixed and permeabilized using the Fix-and-Perm kit (Caltag Laboratories, Burlingame, Calif.) according to the manufacturer's instructions and then blocked with permeabilization buffer with 10% goat serum. All antibodies were diluted in permeabilization buffer with 10% goat serum and incubated with the samples at room temperature; washes between steps were with PBS. Primary antibodies were incubated for 2 h, followed by incubation with species-specific FITC- or TRITC-conjugated secondary antibodies for 1 h. Samples were mounted with ProLong Antifade mounting medium (Molecular Probes, Eugene, Oreg.) and dried overnight. Images were acquired with a Bio-Rad Radiance 2000 laser-scanning confocal microscope by focusing on the central plane of each cell.
Granzyme in vitro and in situ cleavage assay. rHMG2 (1 µM) or postnuclear K562 cell lysates (2 x 105 cell equivalents) were incubated for the indicated times at 37°C with indicated concentrations of GzmA, S-AGzmA, or GzmB in 20 µl of 50 mM Tris-HCl (pH 7.5)-1 mM CaCl2-1 mM MgCl2. Nuclei were pelleted from K562 or HeLa cells lysed in NP-40 lysis buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF) and washed twice in NP-40 lysis buffer and once in NP-40 lysis buffer without NP-40. Nuclei (106 in 50 µl of 50 mM NaCl-0.25 M sucrose-2 mM CaCl2-20 mM Tris-HCl [pH 7.2]) were incubated with the indicated amounts of GzmA or 0.5 µM S-AGzmA at 37°C for 1.5 h. Reaction products were boiled in 5x SDS loading buffer, separated on SDS-12% PAGE gels, and transferred to nitrocellulose for immunoblotting.
Granzyme loading with PFP. K562 cells (2 x 105) in 100 µl of loading buffer (Hanks' balanced salt solution with 1 mg of bovine serum albumin per ml, 1 mM CaCl2, and 1 mM MgCl2) were incubated for indicated times at 37°C with 1 µM GzmA or 1 µM GzmB and sublytic concentrations of PFP. Cells were then incubated for an additional 15 min in 1 mM PMSF before lysing in 20 µl of 0.5% NP-40 lysis buffer containing PMSF. Nuclear pellets were washed twice in NP-40 lysis buffer and extracted in 20 µl of 1% NP-40 lysis buffer containing PMSF. Samples were boiled in 2x SDS loading buffer before SDS-PAGE and immunoblotting.
DNA binding assay. pBR322 DNA (0.3 µg) was incubated with indicated amounts of rHMG2, purified SET complex (2 µg/µl), or 1 µM rGST in 10 mM Tris-HCl (pH 7.8)-100 mM NaCl-1 mM EDTA-10 mM MgCl2-1 mM dithiothreitol-10% glycerol-5 µg of bovine serum albumin per ml at 25°C for 1 h. For GzmA pretreatment of HMG2, 2 µM rHMG2 or 2 µl of pooled SET complex (2 µg/µl) was preincubated with 100 or 500 nM GzmA or 500 nM S-AGzmA at 37°C for 1 h. The reaction mixtures were electrophoresed through 1% agarose gels in 40 mM Tris-acetate, pH 7.8, containing 1 mM EDTA, and analyzed after ethidium bromide staining.
DNA bending assay.
DNA fragments (123 bp), prepared with cohesive ends by AvaI digestion of the 123-bp ladder from Life Technologies, Inc., were 5' end labeled with [
-32P]ATP using T4 polynucleotide ligase (New England Biolabs) and purified through Sephadex G-50 minicolumns (Pharmacia). Radiolabeled fragments (1 nM) in 10 µl of 30 mM Tris-HCl (pH 7.8)-10 mM MgCl2-10 mM dithiothreitol-0.5 mM ATP were ligated with T4 DNA ligase (0.1 U/reaction) (New England Biolabs) at 30°C for 30 min, followed by termination at 65°C for 15 min. rHMG2 (1 µM) or SET complex (2 µg/µl), pretreated with indicated amounts of GzmA or 1 µM S-AGzmA as above, was then added to the 32P-labeled 123-bp DNA fragments for 30 min at 4°C. Deproteinized samples were electrophoresed through 6% nondenaturing gels in 0.5x Tris-borate-EDTA buffer and visualized by autoradiography after drying.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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70). This, taken together with the breadth of the SET complex peak from the sizing column, may indicate some heterogeneity in the complex.
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Experiments analogous to those for the interaction of HMG2 with SET were performed for the interaction with pp32. However, neither rHMG2 nor native HMG2 in the SET complex or in cytoplasmic lysates coprecipitates with pp32, except in the presence of S-AGzmA. This is similar to what we found for the interaction of SET and pp32. The recombinant and native proteins interact only weakly in the absence of S-AGzmA, but they strongly associate in the presence of S-AGzmA. rHMG2 also does not coprecipitate with rAPE (Fig. 2e).
HMG2 distributes both in the cytoplasm and the nucleus and colocalizes with SET in a perinuclear rim. HMG2 is an abundant nonhistone chromosomal protein that has been implicated in the maintenance and establishment of chromatin structure. A major role of HMG box DNA-binding proteins is to bend DNA to facilitate the formation of complex nucleoprotein assemblies. The known functions of HMG2 suggest that it should localize to the nucleus. However, we isolated it from cytoplasmic cell lysates in association with SET, which we previously found to stain in a perinuclear rim with the ER. When postnuclear cytoplasmic supernatants and washed nuclear pellets obtained after NP-40 lysis are analyzed by SDS-PAGE and immunoblotting for SET, most of the protein is found in the cytoplasmic fraction (Fig. 3a). When the same cell fractions are analyzed for HMG2, HMG2 is equally distributed between the nucleus and cytoplasm. These results were confirmed by laser-scanning confocal microscopy using the Caltag Fix-and-Perm kit (Fig. 3b). In the cytoplasm of HeLa cells, HMG2 and SET concentrate in perinuclear regions and colocalize with the ER marker calreticulin and BiP (not shown). HMG2 cytoplasmic staining also coincides with pp32 and APE (10; Fan et al., submitted; and data not shown). However, a signal for HMG2 is clearly detected in the nucleus. This agrees well with the cell fractionation data. Similar results are found when 4% paraformaldehyde is used for fixation in place of the Caltag reagent (not shown). However, if harsher fixation conditions, such as methanol or 1% SDS, are used, the perinuclear staining for SET, pp32, and HMG2 is no longer visible and the signal is limited to the nucleus (reference 10 and data not shown). Differences in staining procedure may explain why some published immunohistochemical studies identify all of these as exclusively nuclear proteins.
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GzmA, but not GzmB, degrades HMG2 in cell cytosol and isolated nuclei. Because GzmA directly cleaves rHMG2 in vitro and HMG2 in cell lysates, we next compared cleavage of HMG2 with cleavage of SET and determined whether GzmB might also cleave HMG2. K562 cell lysates were incubated with various amounts of GzmA, S-AGzmA, or GzmB and analyzed by immunoblotting. HMG2 was degraded after treatment of GzmA beginning at nanomolar concentrations (Fig. 5a). The same blots were stripped and reprobed for SET and pp32. SET is processed by GzmA to produce the previously reported 25-kDa fragment (9, 10) (Fig. 5), while pp32 is unchanged. Cleavage of HMG2 is detected at 10 nM GzmA, but clear cleavage of SET is not seen until a concentration of 50 nM is reached. GzmB and inactive mutant GzmA do not cleave HMG2 in cell lysates.
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HMG2 is cleaved in cells after GzmA loading with PFP. To investigate whether HMG2 cleavage is physiologically relevant, we looked at HMG2 degradation in the cytosol and nuclei of K562 cells loaded with GzmA and PFP (Fig. 6). Treating cells with either GzmA or PFP alone (not shown) or with mutant GzmA with PFP does not result in changes in cytosolic SET or HMG2 (Fig. 6a). Cytosolic HMG2 cleavage in loaded cells occurs within 1 h, and neither uncleaved HMG2 nor the C-terminal cleavage product can be detected within 2 h. SET cleavage, however, is evident within 20 min of loading. pp32, which is not a GzmA substrate, is unchanged and provides a good control for loading. Nuclear fractions were also analyzed 4 h after PFP-mediated GzmA loading (Fig. 6b). Nuclear HMG2 and SET were completely proteolyzed in cells loaded with active GzmA, but not in cells loaded with inactive S-AGzmA or treated with either PFP or GzmA alone. The GzmA concentration required to cleave HMG2 and SET in vivo is comparable to that required to induce cell death and DNA damage (9). Therefore, HMG2 is a direct physiological substrate of GzmA in vivo.
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DISCUSSION |
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HMG1 and HMG2 are highly homologous with a similar structure consisting of two homologous DNA-binding domains (HMG boxes) A and B, each of 70 amino acid residues, and a long acidic C-terminal tail of 30 (HMG1) or 20 (HMG2) acidic residues (14) (Fig. 4b). The acidic tail may be involved in interactions with other basic proteins such as histones or in regulating DNA-binding affinity (27, 37, 51). SET and pp32 in the same complex have even longer C-terminal acidic regions (55 and 82 aa, respectively), which make them both highly acidic (calculated pIs, 4). These acidic regions may bind to as-yet-unidentified basic proteins in the SET complex, which would be required to balance the charge of the complex. Despite their high homology and ability to substitute for one another in most in vitro assays, HMG1 and HMG2 may not be completely redundant. For example, antisense suppression of HMG2 blocks cell cycle progression, which is not compensated for by HMG1 (27). Similarly, mice genetically deficient only in HMG1 suffer from lethal hypoglycemia (17). Many of the amino acid differences between the two proteins are in potential Ser or Thr phosphorylation sites, which suggests that the two proteins may be differentially regulated and have distinct functions. We did not identify a clear 29-kDa HMG1 band in the SET complex, and unlike HMG2, HMG1 does not bind to SET. Therefore HMG2 is preferentially involved in the functions of the SET complex.
Although HMG1 differs by only one amino acid residue at the P1' site from HMG2 (Ala66 in HMG1 versus Ser66 in HMG2), HMG1 is not a substrate of GzmA. This difference in susceptibility to GzmA demonstrates the enzyme's exquisite specificity. At present eight distinct physiologically validated cleavage sites have been identified (Table 1). No obvious similarities among the substrates N terminal to the cleavage site are evident, and cleavage after either Lys or Arg appears to be equally favored. Specificity may come from tertiary structure or from P' sites as has been previously noted (10); P' sites are also important for GzmB substrates (53). Common (but not universal) features in the P' region are Ser at P1' or P2' and a basic residue at P5'. Lack of cleavage of HMG1 supports the importance of the Ser residue in the P' motif.
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Since HMG2 binds preferentially to distorted DNA, it may help target SET and APE to sites of DNA damage where SET unwinds the damaged DNA from the nucleosome and APE participates in its repair. Little is known about the effect of chromatin structure on DNA repair. However, damaged DNA in actively transcribed genes (with more open chromatin) is preferentially repaired (12). Not surprisingly, a recent study showed that a DNA oligonucleotide containing a T(6-4)T photoproduct was repaired 10 times less efficiently in nucleosomal DNA than in naked DNA (24). Therefore, it makes sense for the rate-limiting base excision repair enzyme to have a mechanism for detecting damage within packed chromatin and for unwinding the damaged DNA for easy access for repair. Since SET and APE both facilitate transcription, it is also possible that much of the APE base excision repair occurs during transcription as the integrity of DNA is examined for bends secondary to ongoing oxidative DNA damage. The SET complex also contains an as-yet-unidentified Mg2+-dependent DNase, which induces single-stranded DNA nicks when the SET complex is incubated with GzmA and isolated nuclei (10). This DNase might also be involved in repair.
Because of the nuclear functions of the SET complex proteins, it is perhaps surprising to find that a large proportion of these proteins are in the cytoplasm associated with the ER. They may be stored there for regulated nuclear entry. In fact pp32 has both a canonical nuclear localization signal and leucine-rich regions for binding to crm1 for nuclear export. pp32 binds to crm1 and its nuclear export is inhibited by leptomycin B (13). SET and pp32 also associate in a smaller, 150-kDa complex in the nucleus that binds to HuR, a protein that stabilizes mRNAs with AU-rich regions (13). SET and pp32 might be exported from the nucleus with HuR-bound mRNAs. It is possible that the ER-associated SET complex might also have a posttranscriptional function.
Although most studies have identified HMG2 in the nucleus, in some cells a substantial fraction of HMG2 is cytoplasmic (16). In fact, staining for HMG-2 is highly dependent on the fixation protocol (data not shown). Interestingly, a perinuclear staining pattern of HMG1/HMG2 autoantibodies that develop in patients with refractory ulcerative colitis has been previously reported (50). Autoantibodies to HMG1 and HMG2 have also been found in patients with systemic lupus erythematosis and juvenile rheumatoid arthritis (15, 63). One theory is that autoantibodies arise to proteins and other compounds modified during apoptosis. Since HMG2 is cleaved by GzmA in T-cell-mediated cytolysis, the finding of autoantibodies to HMG2 is consistent with this theory about the origins of autoantibodies. However, HMG2 is not cleaved by GzmB or in GzmB-loaded cells, so it is unlikely to be cleaved during caspase activation.
Most GzmA substrates so far identified (histone H1, core histones, SET, APE, and HMG2) modify chromatin structure. We previously found that GzmA loading of isolated nuclei enhances the access and DNA cleavage by exogenous DNases (68). We have now described several mechanisms used by GzmA which open up chromatin for digestion by apoptotic nucleases. GzmA degrades lamins A, B, and C, disrupting the integrity of the nuclear envelope and possibly interfering with chromatin attachment at matrix attachment regions (67). GzmA also completely degrades the linker histone H1 and cuts the tails from the core histones (68). Chromatin stripped of linker histones is released from a compacted configuration to an extended state (3, 62). Trypsinization of polynucleosomal DNA, which also cuts the tails from core histones (but at somewhat different sites), further opens up compacted chromatin (2). HMG2 is a nonhistone protein that also binds to the internucleosomal linker region of DNA and to core histones (39). The interaction of HMG2 with H2AH2B histone dimers is via the acidic C terminus, whereas the N terminus is involved in the interactions with (H3H4)2 tetramers (11). GzmA cleavage of HMG2 is likely to open up chromatin and contribute to blocking de novo transcription required for the cellular repair response.
Opening up chromatin may also contribute to the observed synergy of GzmA and GzmB in inducing oligonucleosomal DNA fragmentation during CTL cytolysis (9, 33, 45). HMG2, however, has been shown to enhance the nuclease activity of the GzmB- and caspase-activated DNase DFF40 or CAD (57), and HMG2 cleavage would likely counter that effect. However, perhaps one of the homologous HMG proteins such as uncleaved HMG1 could also perform the same function of maintaining the high activity of CAD/DFF40 during CTL-mediated apoptosis.
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ACKNOWLEDGMENTS |
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We thank Zhan Xu and David Oh for technical support. We also thank K. Nagata for supernatant from hybridoma KM1720, Z. Damuni for pp32 plasmid, I. Hickson for APE plasmid, and P. Sharp for HMG2 plasmid.
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FOOTNOTES |
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