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

  • Naohiro Inohara
    Affiliations
    From the Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109
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  • Takeyoshi Koseki
    Footnotes
    Affiliations
    From the Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109
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  • Shu Chen
    Affiliations
    From the Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109
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  • Mary A. Benedict
    Footnotes
    Affiliations
    From the Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109
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  • Gabriel Núñez
    Correspondence
    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;
    Affiliations
    From the Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109
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  • Author 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.
    ‡ 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.
Open AccessPublished:January 01, 1999DOI:https://doi.org/10.1074/jbc.274.1.270
      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 andDrosophila 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.
      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.
      Apoptosis, a morphologically defined form of programmed cell death plays an essential role in animal development and tissue homeostasis (
      • Jacobson M.D.
      • Weil M.
      • Raff M.C.
      ). 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 (
      • Cohen G.M.
      ).
      DNA fragmentation associated with apoptosis is induced by the DNA fragmentation factor (DFF)1, which is activated by caspases, mainly caspase-3 (
      • Liu X.
      • Zou H.
      • Slaughter C.
      • Wang X.
      ,
      • Enari M.
      • Sakahira H.
      • Yokoyama H.
      • Okawa K.
      • Iwamatsu A.
      • Nagata S.
      ,
      • Sakahira H.
      • Enari M.
      • Nagata S.
      ,
      • Halenbeck R.
      • MacDonald H.
      • Roulston A.
      • Chen T.T.
      • Conroy L.
      • Williams L.T.
      ,
      • Liu X.
      • Li P.
      • Widlak P.
      • Zou H.
      • Luo X.
      • Garrard W.T.
      • Wang X.
      ,
      • Mukae N.
      • Enari M.
      • Sakahira H.
      • Fukuda Y.
      • Inazawa J.
      • Toh H.
      • Nagata S.
      ). 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) (
      • Liu X.
      • Zou H.
      • Slaughter C.
      • Wang X.
      ,
      • Enari M.
      • Sakahira H.
      • Yokoyama H.
      • Okawa K.
      • Iwamatsu A.
      • Nagata S.
      ,
      • Sakahira H.
      • Enari M.
      • Nagata S.
      ,
      • Halenbeck R.
      • MacDonald H.
      • Roulston A.
      • Chen T.T.
      • Conroy L.
      • Williams L.T.
      ,
      • Liu X.
      • Li P.
      • Widlak P.
      • Zou H.
      • Luo X.
      • Garrard W.T.
      • Wang X.
      ,
      • Mukae N.
      • Enari M.
      • Sakahira H.
      • Fukuda Y.
      • Inazawa J.
      • Toh H.
      • Nagata S.
      ). 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 (
      • Liu X.
      • Zou H.
      • Slaughter C.
      • Wang X.
      ,
      • Enari M.
      • Sakahira H.
      • Yokoyama H.
      • Okawa K.
      • Iwamatsu A.
      • Nagata S.
      ,
      • Sakahira H.
      • Enari M.
      • Nagata S.
      ,
      • Halenbeck R.
      • MacDonald H.
      • Roulston A.
      • Chen T.T.
      • Conroy L.
      • Williams L.T.
      ,
      • Liu X.
      • Li P.
      • Widlak P.
      • Zou H.
      • Luo X.
      • Garrard W.T.
      • Wang X.
      ,
      • Mukae N.
      • Enari M.
      • Sakahira H.
      • Fukuda Y.
      • Inazawa J.
      • Toh H.
      • Nagata S.
      ). 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 (
      • Inohara N.
      • Koseki T.
      • Chen S.
      • Wu X.
      • Nunez G.
      ). 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 (
      • Inohara N.
      • Koseki T.
      • Chen S.
      • Wu X.
      • Nunez G.
      ). The activity of CIDEs is inhibited by DFF45/ICAD (
      • Inohara N.
      • Koseki T.
      • Chen S.
      • Wu X.
      • Nunez G.
      ). Mutational analysis revealed that CIDEs contains two domains, CIDE-N and CIDE-C (
      • Inohara N.
      • Koseki T.
      • Chen S.
      • Wu X.
      • Nunez G.
      ). 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 (
      • Inohara N.
      • Koseki T.
      • Chen S.
      • Wu X.
      • Nunez G.
      ).
      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.

      Acknowledgments

      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.

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