Identification of Regulatory and Catalytic Domains in the Apoptosis Nuclease DFF40/CAD*

Naohiro Inohara, Takeyoshi KosekiDagger , Shu Chen, Mary A. Benedict§, and Gabriel Núñez

From the Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

The DNA fragmentation factor (DFF) is composed of two subunits, the 40-kDa caspase-3-activated nuclease (DFF40/CAD) and its 45-kDa inhibitor (DFF45/ICAD). During apoptosis, DFF-40/CAD is activated by caspase-3-mediated cleavage of DFF45/ICAD. Mutational analysis of DFF40/CAD revealed that DFF40/CAD is composed of a C-terminal catalytic domain and an N-terminal regulatory domain. Deletion of the catalytic domain (residues 290-345) abrogated the caspase-3-induced nuclease activity of DFF40/CAD but not its ability to interact with DFF45/ICAD. Conversely, removal of the regulatory domain (residues 1-83) yielded a constitutively active DFF40/CAD nuclease that neither bound to its inhibitor nor required caspase-3 for activation. Amino acid alignment revealed that the regulatory domain of DFF40/CAD has homology to the N-terminal region of mammalian and Drosophila DFF45/ICAD and CIDE-N, a regulatory domain previously identified in pro-apoptotic CIDE proteins. Mutational analysis of the N-terminal region revealed mutants with diminished nuclease activity but with intact ability to bind DFF45/ICAD. Thus, CIDE-N represents a new type of domain that is associated with the regulation of the apoptosis/DNA fragmentation pathway.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Apoptosis, a morphologically defined form of programmed cell death plays an essential role in animal development and tissue homeostasis (1). Apoptotic cells undergo multiple changes including membrane blebbing, nuclear condensation, and fragmentation of genomic DNA into nucleosomal fragments. These morphological and biochemical changes are promoted by caspases, a family of cysteine proteases that are activated by apoptotic stimuli. Activated caspases can cleave multiple cytoplasmic and nuclear substrates, a process that appears to play a pivotal role in the execution phase of apoptosis (2).

DNA fragmentation associated with apoptosis is induced by the DNA fragmentation factor (DFF)1, which is activated by caspases, mainly caspase-3 (3-8). DFF is composed of two protein subunits, a 40-kDa caspase-activated nuclease (DFF40/CAD), also called CPAN, and its 45-kDa inhibitor (DFF45/ICAD) (3-8). Cleavage of DFF45/ICAD by caspase-3 releases DFF40/CAD from its inhibitor leading to the induction of nuclease activity, nuclear condensation, and DNA fragmentation in vitro (3-8). However, the molecular basis for DFF40/CAD function and its regulation by DFF45/ICAD remains poorly understood.

In a previous study, we identified a conserved family of proteins named CIDEs, that include CIDE-A, CIDE-B, and Fsp27, based on amino acid homology to the N-terminal region of DFF45/ICAD (9). Unlike DFF45/ICAD, CIDEs are pro-apoptotic proteins that induce DNA fragmentation as well as other apoptotic features such as membrane blebbing and nuclear condensation (9). The activity of CIDEs is inhibited by DFF45/ICAD (9). Mutational analysis revealed that CIDEs contains two domains, CIDE-N and CIDE-C (9). The CIDE-C domain is necessary and sufficient for apoptosis, whereas CIDE-N acts as a regulatory domain required for the inhibitory activity of DFF45/ICAD (9).

Here we report that DFF40/CAD has two domains with distinct biological functions. A catalytic domain located in the C-terminal region mediates nuclease activity, whereas a regulatory domain is located in the N-terminal half of DFF40/CAD. The regulatory domain of DFF40/CAD has homology to a conserved CIDE-N domain that was previously identified in the pro-apoptotic CIDE proteins. These results identify the N-terminal region of DFF40/CAD as a conserved domain that regulates the activation and activity of this apoptosis-associated nuclease.

    MATERIALS AND METHODS

Construction of Expression Plasmids-- The entire cDNA open reading frame of mouse DFF40/CAD was amplified by polymerase chain reaction from cDNA synthesized from total RNA from mouse testis and cloned into the XbaI and EcoRI sites of pcDNA3-HA and pcDNA-3-Myc (9) to produce expression plasmids pcDNA3-HA-CAD and pcDNA34-Myc-CAD encoding HA-tagged and Myc-tagged DFF40/CAD, respectively. pcDNA3-Flag-DFF45, pcDNA3-caspase-9-HA, pcDNA3-TNFR1-Flag (tumor necrosis factor receptor-1), and pcDNA3-beta -gal have been previously described (9-11). The expression plasmids encoding deletion mutants of HA-DFF40/CAD, pcDNA3-HA-CADDelta 290-345, pcDNA3-HA-CADDelta 162-345, and pcDNA3-Myc-CAD-Delta 1-83 were constructed by polymerase chain reaction methods. The authenticity of all constructs was confirmed by dideoxy sequencing.

Transfection, Expression, and Immunodetection of Tagged Proteins-- 5 × 106 human 293T cells were transfected with expression plasmids by a calcium phosphate method as described (10). The total amount of transfected plasmid DNA was adjusted with pcDNA3 plasmid to be the same within individual experiments. 24 h post-transfection, 293T cells were harvested and lysed with 0.2% Nonidet P-40 isotonic lysis buffer (12). Total lysates were subjected to 15% SDS-polyacrylamide electrophoresis and immunoblotted with mAb to Flag (Kodak).

In Vitro Nuclease Assay and in Vivo DNA Fragmentation Assay-- The in vitro nuclease activity was assayed with immunopurified DFF in the presence or absence of recombinant caspase-3. Briefly, 5 × 106 293T cells were transfected with plasmids as indicated in the figure legends. 24 h post-transfection, cells were lysed with Nonidet P-40 lysis buffer, and tagged DFF40/CAD proteins were immunoprecipitated with anti-HA or anti-Myc polyclonal antibody and protein-A-Sepharose, washed five times with Nonidet P-40 lysis buffer, and then with CPAN buffer (10 mM HEPES, 1 mM EGTA, 5 mM MgCl2, 50 mM NaCl, 1 mg/ml bovine serum albumin, pH 7.5) (6). The in vitro nuclease activity of the immunoprecipitated protein was assayed by incubation in CPAN buffer with or without 50 units of recombinant caspase-3 (a gift from Margarita Garcia-Calvo, Merck) and 1 µg of plasmid DNA substrate and subsequent analysis of plasmid DNA degradation by agarose gel electrophoresis, as described (6). For assay of in vivo DNA fragmentation, genomic DNA was extracted from 2 × 104 293T cells transfected with pcDNA3, pcDNA3-caspase-9-HA, pcDNA3-TNFR1-Flag, or DFF40/CAD expression plasmids as indicated in the figure legends, 24 h post-transfection as described (8). The same amount of each DNA was subjected to 1% agarose gel electrophoresis. The amount of DNA loaded was 10-fold lower than previously described (9). The cell death assay of 293T was performed as reported (9).

    RESULTS AND DISCUSSION

The N-terminal Half of DFF40/CAD Mediates Binding to DFF45/ICAD whereas the C-terminal Region Is Required for Nuclease Activity-- To identify regions of DFF40/CAD that are important for nuclease activity, binding, and sensitivity to DFF45/ICAD, we constructed a series of plasmids to express deletion mutants of DFF40/CAD (Fig. 1A). We transfected these expression plasmids encoding HA-tagged or Myc-tagged wild type and mutant DFF40/CAD proteins into 293T embryonic kidney cells and purified the proteins by affinity chromatography.


View larger version (59K):
[in this window]
[in a new window]
 
Fig. 1.   C-terminal deletion mutants of DFF40/CAD lose nuclease activity but not the ability to bind DFF45/ICAD. A, schematic structure of WT DFF40/CAD and deletion mutants. Conserved CIDE-N sequence is indicated by a closed box (see Fig. 3) B[en]C, 293T cells were transfected with pcDNA3-HA-CAD, pcDNA3-HA-CADDelta 290-345, or pcDNA3-HA-CADDelta 162-345 in the absence or presence of pcDNA3-Flag-DFF45. 24 h post-transfection, DFF40/CAD proteins were immunoprecipitated by anti-HA mAb. The in vitro nuclease activities of immunoprecipitated DFF40/CAD proteins were assayed with or without caspase-3 treatment, as described under "Materials and Methods." Activity was measured as degradation of added plasmid DNA substrate, visualized by agarose gel electrophoresis (B, top panel). m, DNA size marker fragments. In total lysates (C) and anti-HA immunoprecipitates (B, middle and bottom panels), DFF40/CAD and DFF45/ICAD proteins were detected first with anti-HA polyclonal Ab and then subsequently with anti-Flag polyclonal Ab. The intact 45-kDa DFF45/ICAD (B, middle panel) and 14-kDa fragments of DFF45/ICAD digested by caspase-3 (B, bottom panel) were detected by 5- and 60-s exposures of x-ray film, respectively. D, 293T cells in 4 wells of 12-well plates were co-transfected with pcDNA3-HA-CAD (WT), pcDNA3-HA-CADDelta 290-345 (Delta 290-345), or pcDNA3-HA-CADDelta 162-345 (Delta 162-345) and pcDNA3-TNFR1-Flag (TNFR1) or pcDNA3-caspase-9-HA (Casp. 9) plus pcDNA3-beta -gal. To assay for in vivo DNA fragmentation, genomic DNA was extracted from 1 well of transfected cells and subjected to 1% agarose electrophoresis as described under "Materials and Methods." The percentage of apoptotic cells (apop.) was determined by the beta -galactosidase assay using transfected cells in 3 wells as described under "Materials and Methods." Standard deviation of each percentage was less than 10%.

HA-tagged wild type and C-terminal deletion mutant DFF40/CAD proteins were co-expressed with Flag-tagged DFF45/ICAD and immunoprecipitated by anti-HA antibody. In vitro nuclease activities of these immunoprecipitated proteins were measured by their ability to degrade plasmid DNA substrate in the presence or absence of caspase-3. This in vitro assay revealed that the DFF40/CAD deletion mutants Delta 290-345 and Delta 162-345 lost their caspase-activated nuclease activity when compared with control wild type (WT) DFF40/CAD (Fig. 1B, top panel). In contrast, both DFF40/CAD mutants retained their ability to bind to DFF45/ICAD (Fig. 1B, middle panel). Loss of catalytic activity was not due to differences in protein levels between wild type and mutant proteins because comparable levels of both were detected in total lysates (Fig. 1C) and immunoprecipitates (Fig. 1B, middle panel). In addition, the sensitivity of DFF45/ICAD to caspase-3-mediated cleavage was not affected by these mutations, as DFF45/ICAD bound to either mutant was cleaved by caspase-3 (Fig. 1B, middle and lower panels).

We tested next the ability of WT and mutant DFF40/CAD to promote genomic DNA fragmentation in vivo. In these experiments, we transiently transfected constructs producing WT, Delta 290-345, Delta 162-345, or control plasmid into 293T cells. Because DFF40/CAD requires an apoptotic stimulus to be activated, the cells were also transfected with plasmids producing TNFR1 or caspase-9, both of which are known to activate apoptotic cell death in 293T cells (13). Although both caspase-9 and TNFR1 could cause DNA fragmentation, we loaded a reduced amount of genomic DNA in the in vivo DNA fragmentation assay to visualize the enhancement of DNA fragmentation by DFF40/CAD. In the absence of a death stimulus, neither WT DFF40/CAD nor mutant DFF40/CAD induced DNA fragmentation (Fig. 1D). However, when the cells were co-transfected with plasmids producing TNFR1 or caspase-9, WT DFF40/CAD did promote DNA fragmentation (Fig. 1D). In contrast to WT DFF40/CAD, the Delta 290-345 and Delta 162-345 mutants did not induce DNA fragmentation (Fig. 1D). Thus, the C-terminal region of DFF40/CAD is required for catalytic activity in vitro and in vivo but not for binding to DFF45/ICAD.

The N-terminal Region of DFF40/CAD Has Homology to the N Termini of DFF45/ICAD and CIDEs-- DFF40/CAD exhibits no significant amino acid or structural homology to other known nuclease families (4). Close inspection of the DFF40/CAD amino acid sequence by the BLAST2 program, however, revealed significant amino acid homology between the regulatory domain of DFF40/CAD and the CIDE-N domain previously identified in DFF45/ICAD and CIDE proteins (Fig. 2). The regulatory (CIDE-N) domain of DFF40/CAD showed 49 to 55% similarity with those of CIDE-A, CIDE-B, Fsp27, DFF45/ICAD, and Drosophila DREP-1, respectively (p = 10-2-10-5). Several amino acids in the CIDE-N domain of DFF40/CAD including Lys-35, Asp-54, Gly-55, Thr-56, Phe-63, and Trp-81 are conserved in the CIDE-N domains of CIDE-A, CIDE-B, Fsp27, and DREP-1 (Fig. 2), suggesting that they are important for the function of these proteins.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2.   Homology of CIDE-N domains from DFF40/CAD, CIDE-A, CIDE-B, Fsp27, and DFF45/ICAD. Amino acid sequence and alignments of N-terminal regions of human and mouse DFF40/CAD (4, 6), human and mouse DFF45/ICAD (3, 4), Drosophila melanogaster DREP-1, mouse Fsp27, human and mouse CIDE-A, and mouse CIDE-B (8). The completely conserved residues and partial conserved residues are indicated by black and gray highlights, respectively. Mutations used in the experiments of Fig. 3 are also shown.

Mutant Forms with Substitution of Conserved Residues in the CIDE-N Domain of DFF40/CAD Exhibit Diminished Nuclease Activity-- To determine whether the conserved residues in the CIDE-N domain play a role in the regulation of DFF40/CAD activity, we introduced site-directed mutations at five conserved residues of the CIDE-N domain of DFF40/CAD (Fig. 2). Anti-HA immunoprecipitation showed that all five of these point mutants still bound to DFF45/ICAD (Fig. 3A). However, the G55I, F63S, and W81C mutants of DFF40/CAD exhibited reduced nuclease activity in vitro (Fig. 3B) and in vivo (Fig. 3D), whereas the K35A and T56L mutants did not. In vitro nuclease analysis of 50-fold dilutions of the immunoprecipitated DFF40/CAD proteins from Fig. 3B confirm these differences (Fig. 3C). Immunoblotting revealed similar levels of expression of the mutants, indicating that reduced nuclease activity was not because of diminished expression (Fig. 3A). Under our experimental conditions using the in vitro assay, DFF45/ICAD bound to either WT DFF40/CAD or any of the mutants was completely cleaved by caspase-3.2 In addition, neither WT DFF40/CAD nor any of the mutants bound to DFF45/ICAD protein cleaved by caspase-3.2 These data indicate that reduced nuclease activity of the mutants is not because of caspase-3 insensitivity of DFF45/ICAD bound to mutant proteins or inhibition of DFF40/CAD by an interaction with cleaved DFF45/ICAD fragments.


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 3.   Mutation of conserved residues of CIDE-N domain result in diminished nuclease activity of DFF40/CAD. 293T cells were co-transfected with 4 µg of pcDNA3 (CTRL or -), pcDNA3-HA-CAD (WT), pcDNA3-HA-CAD-K35A, pcDNA3-HA-CAD-G55I, pcDNA3-HA-CAD-T56L, pcDNA3-HA-CAD-F63S, or pcDNA3-HA-CAD-W81C in the presence or absence of pcDNA3-Flag-DFF45. To compare the expression levels and the nuclease activities of WT and mutant DFF40/CAD proteins, 293T cells were also transfected with 0.01 or 0.1 µg of pcDNA3-HA-CAD (WT) in the presence of pcDNA3-Flag-DFF45. A, mutation of the conserved residues does not affect interaction between DFF40/CAD and DFF45/ICAD. HA-DFF40/CAD and Flag-DFF45/ICAD proteins were detected with anti-HA and anti-Flag mAb in total lysate (bottom panel) and anti-HA immunoprecipitates (top panel). B-C, the in vitro nuclease activities of WT and mutant DFF40/CAD immunopurified with anti-HA mAb were measured as described under "Materials and Methods" and Fig. 1. Panel C depicts the relative in vitro nuclease activities obtained using 50-fold dilutions of the immunoprecipitated proteins from panel B. D, to compare in vivo genomic DNA fragmentation activities of the WT and mutant CAD proteins, 293T cells in 4 wells of 12-well plates were co-transfected with plasmid expressing WT or mutant DFF40/CAD and pcDNA3-TNFR1-Flag (TNFR1) or pcDNA3-caspase-9-HA (Casp. 9). Genomic DNA was analyzed as in Fig. 1.

Comparison between wild-type, site-directed mutants of DFF40/CAD and an N-terminal deletion mutant, Delta N, that expresses residues 84-345 of DFF40/CAD (see below) revealed that the G55I, F63S, and W81C mutants and the Delta N mutant exhibit in vitro nuclease activities that are reduced when compared with WT DFF40/CAD (Fig. 3, B and C and Fig. 4A). Thus, amino acids Gly-55, Phe-63, and Trp-81 appear to enhance the nuclease activity of DFF40/CAD after cleavage of DFF45/ICAD by caspase-3 but do not play an essential role in the catalysis reaction. These results suggest that the CIDE-N regulatory domain of CAD/ICAD not only mediates binding to DFF45/ICAD but also promotes the activity of the catalytic nuclease domain located in the C-terminal region of the protein.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 4.   N-terminal deletion mutant of DFF40/CAD retains nuclease activity but not binding to DFF45/ICAD or sensitivity to DFF45/ICAD mediated inhibition. A, 293T cells were transfected with pcDNA3 (-), pcDNA3-Myc-CAD (WT), or pcDNA3-Myc-CADDelta 1-83 (Delta N) in the absence or presence of pcDNA3-Flag-DFF45. 24 h post-transfection, DFF40/CAD proteins were immunoprecipitated by anti-Myc mAb. The in vitro nuclease activities of immunoprecipitated DFF40/CAD proteins with or without caspase-3 treatment were measured as described under "Materials and Methods." To compare in vitro nuclease activity between WT and mutant DFF40/CAD proteins, immunopurified wild type protein was diluted 1-, 6-, 36-, 216-, or 648-fold, and the nuclease activities were measured. m, DNA size marker fragments. B, in total lysates (bottom panel) and anti-Myc immunoprecipitates (top panel), DFF40/CAD and DFF45/ICAD proteins were detected with anti-Myc polyclonal and anti-Flag mAb, respectively. C, 293T were transfected with pcDNA3 (-), 0.01, 0.1, or 1 µg of pcDNA3-Myc-CAD or 4 µg of pcDNA3-Myc-CADDelta 1-83 (Delta N). 24 h post-transfection, transfected cells were harvested and lysed with Nonidet P-40 buffer. Myc-tagged DFF40/CAD and Flag-tagged DFF45/ICAD were subsequently immunodetected with anti-Myc and anti-Flag polyclonal Abs, respectively. Flag-DFF45/ICAD was co-immunoprecipitated with anti-Myc polyclonal Ab and detected with anti-Flag mAb (top panel).

A Regulatory Domain Located in the N-terminal Half of DFF40/CAD-- The results of the experiments shown in Fig. 1 suggested that the nuclease activity of DFF40/CAD resides in the C-terminal half of the protein. To test this hypothesis, we assessed the nuclease activity of the N-terminal deletion mutant, Delta N. The mutant protein retained in vitro nuclease activity, although its activity was reduced compared with that of WT DFF40/CAD (Fig. 4A). In contrast to the WT protein, the Delta N mutant of DFF40/CAD did not bind to DFF45/ICAD (Fig. 4, B and C), indicating that the N-terminal region of DFF40/CAD is required for binding to DFF45/ICAD. Significantly, the Delta N mutant had constitutive nuclease activity in that it did not require caspase-3 for enzymatic activity (Fig. 4A). These results indicate that the N-terminal region of DFF40/CAD, although dispensable for in vitro nuclease activity, has a regulatory role, as it is required both for the interaction of DFF40/CAD with its inhibitor DFF45/ICAD and for caspase-3-dependent activation. In addition, the N-terminal domain of DFF40/CAD is required for optimal nuclease activity, suggesting that this domain regulates the activity of the catalytic C-terminal domain.

Fig. 4D is a model depicting the mechanism by which DFF40/CAD nuclease activity may be regulated during apoptosis. When DFF40/CAD is bound to DFF45/ICAD via its N-terminal region, its nuclease activity is suppressed by this interaction. Once DFF45/ICAD is cleaved by caspase-3, the N-terminal region of DFF40/CAD is released, a process that enables the N-terminal region of DFF40/CAD to then activate the C-terminal nuclease domain. Cleavage of DFF45/ICAD by caspase-3 downstream of Asp-117, which is adjacent to its CIDE-N domain, results in the release of DFF45/ICAD fragments from DFF40/CAD (3, 5), supporting an important role for the CIDE-N domain in DFF45/ICAD function. The regulation of the CIDE-N domain of DFF40/CAD is reminiscent of that of the recently described CIDE-N domain of CIDE-A (8). In this protein, the CIDE-N domain is required for DFF45/ICAD inhibition of CIDE-A-mediated apoptosis (8). In addition, deletion of the CIDE-N domain results in enhanced apoptosis, supporting its role as a regulatory domain. Thus the CIDE-N domain represents a novel class of regulatory domains that can control the function of adjacent domains. In the case of DFF40/CAD, its N-CIDE domain appears to play an important role in the activation and regulation of nuclease activity.

    ACKNOWLEDGEMENTS

We are grateful to M. Garcia-Calvo and N. Thorberry of Merck for recombinant caspase-3; V. M. Dixit for pcDNA3-TNFR1-Flag; and L. del Peso, V. Gonzalez, D. Ekhterae, and Y. Hu for critical review of the manuscript.

    FOOTNOTES

* This research was supported in part by Grant CA-64556 from the National Institutes of Health (to G. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a Fellowship from the Japan Science and Technology Corporation.

§ Supported by a Fellowship from the United States Army Medical Research and Material Command.

Recipient of Research Career Development Award CA-64421 from the National Institutes of Health. To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109. Tel.: 734-764-8514; Fax: 313-647-9654; E-mail: Gabriel.Nunez{at}umich.edu.

2 N.Inohara, and G. Núñez, unpublished results.

    ABBREVIATIONS

The abbreviations used are: DFF, DNA fragmentation factor; HA, hemaglutinin; CAD, caspase-3-activated DNase; CIDE, cell death-inducing DFF45-like effector; ICAD, inhibitor of caspase-3-activated DNase; mAb, monoclonal antibody; WT, wild type; TNFR1, tumor necrosis factor receptor-1..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
REFERENCES
  1. Jacobson, M. D., Weil, M., and Raff, M. C. (1997) Cell 88, 347-354[Medline] [Order article via Infotrieve]
  2. Cohen, G. M. (1997) Biochem. J. 326, 1-16[Medline] [Order article via Infotrieve]
  3. Liu, X., Zou, H., Slaughter, C., and Wang, X. (1997) Cell 89, 175-184[Medline] [Order article via Infotrieve]
  4. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S. (1998) Nature 391, 43-50[CrossRef][Medline] [Order article via Infotrieve]
  5. Sakahira, H., Enari, M., and Nagata, S. (1998) Nature 391, 96-99[CrossRef][Medline] [Order article via Infotrieve]
  6. Halenbeck, R., MacDonald, H., Roulston, A., Chen, T. T., Conroy, L., and Williams, L. T. (1998) Curr. Biol. 8, 537-540[Medline] [Order article via Infotrieve]
  7. Liu, X., Li, P., Widlak, P., Zou, H., Luo, X., Garrard, W. T., and Wang, X. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8461-8466[Abstract/Free Full Text]
  8. Mukae, N., Enari, M., Sakahira, H., Fukuda, Y., Inazawa, J., Toh, H., and Nagata, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9123-9128[Abstract/Free Full Text]
  9. Inohara, N., Koseki, T., Chen, S., Wu, X., and Nunez, G. (1998) EMBO J. 17, 2526-2533[Abstract/Free Full Text]
  10. Inohara, N., Koseki, T., Hu, Y., Chen, S., and Nunez, G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10717-10722[Abstract/Free Full Text]
  11. Hu, Y., Benedict, M. A., Wu, D., Inohara, N., and Nunez, G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4386-4391[Abstract/Free Full Text]
  12. Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. (1993) Cell 74, 609-619[Medline] [Order article via Infotrieve]
  13. Duan, H., Orth, K., Chinnaiyan, A. M., Poirier, G. G., Froelich, C. J., He, W.-W., and Dixit, V. M. (1998) J. Biol. Chem. 271, 16720-16724[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.