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Inhibition of Homodimerization of Poly(ADP-ribose) Polymerase by Its C-terminal Cleavage Products Produced during Apoptosis*

  • Jin Woo Kim
    Affiliations
    Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, 305-701, South Korea
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  • Karam Kim
    Affiliations
    Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, 305-701, South Korea
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  • Kewon Kang
    Affiliations
    Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, 305-701, South Korea
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  • Cheol O. Joe
    Correspondence
    To whom correspondence should be addressed. Tel.: 82-42-869-2627; Fax: 82-42-869-2610
    Affiliations
    Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, 305-701, South Korea
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  • Author Footnotes
    * This work was supported in part by a grant from Korea Science and Engineering Foundation, a grant from the Ministry of Education (Genetic Engineering Program) and a grant from the Ministry of Science and Technology, South Korea.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.
Open AccessPublished:March 17, 2000DOI:https://doi.org/10.1074/jbc.275.11.8121
      The biochemical role of the C-terminal fragment of poly(ADP-ribose) polymerase (PARP) was investigated in HeLa cells undergoing UV-mediated apoptosis. During the course of apoptosis, the C-terminal cleavage product of PARP interacted with intact PARP and down-regulated PARP activity by blocking the homodimerization of PARP. The basic leucine zipper motif in the auto-modification domain of the C-terminal fragment of PARP represented the site of association, and Leu405 was critical to the ability of the basic leucine zipper motif to associate with intact PARP. The expression of the C-terminal fragment of PARP stimulated UV-mediated apoptosis. These results suggest that the C-terminal cleavage product of PARP produced during apoptosis blocks the homodimerization of PARP and inhibits the cellular PARP activity. The inhibition of the cellular PARP activity might prevent cellular NAD+ depletion and stimulate apoptosis by maintaining the basal cellular energy level required for the completion of apoptosis.
      PARP
      poly(ADP-ribose) polymerase
      GST
      glutathione S-transferase
      GFP
      green fluorescence protein
      bZip
      basic leucine zipper
      AMC
      7-amino-4-methylcoumarin
      FITC
      fluorescein isothiocyanate
      HA
      hemaglutinin
      NLS
      nuclear localization signal
      PAGE
      polyacrylamide gel electrophoresis
      GPCR
      G protein-coupled receptor
      Ex
      excitation
      Em
      Emission
      Activation of caspases has been generally accepted as a common cellular event that occurs in cells undergoing apoptosis (
      • Cryns V.
      • Yuan J.
      ). Numerous cellular proteins are cleaved by caspases during the course of apoptosis. However, the biochemical consequence of proteolytic cleavage of the substrate proteins by caspases during the course of apoptosis has been poorly understood. PARP1 has been known to be cleaved by caspases during apoptosis (
      • Lazebnik Y.A.
      • Kaufmann S.H.
      • Desnoyers S.
      • Poirier G.G.
      • Earnshaw W.C.
      ). The cleavage of PARP yields a 25-kDa N-terminal fragment containing two zinc-fingers and an 89-kDa C-terminal fragment containing the auto-modification domain and the NAD+ binding domain (
      • Kaufmann S.H.
      • Desnoyers S.
      • Ottaviano Y.
      • Davidson N.E.
      • Poirier G.G.
      ). The two zinc-finger motifs in the N-terminal DNA-binding domain bind to DNA at the sites of single- or double-stranded breaks, resulting in the catalytic activation of PARP. The DNA-bound, activated PARP utilizes NAD+ to synthesize poly(ADP-ribose) on various nuclear proteins, such as DNA polymerase α (
      • Yoshihara S.
      • Simbulan C.M.G.
      ) and β (
      • Ohashi Y.
      • Itaya A.
      • Tanaka Y.
      • Yoshihara K.
      • Kamiya T.
      • Matsukage A.
      ), topoisomerase I (
      • Ferro A.M.
      • Olivera B.M.
      ) and II (
      • Darby M.K.
      • Schmitt B.
      • Jongstra-Bilen J.
      • Vosberg H.P.
      ), histones (
      • Huletsky A.
      • Niedergang C.
      • Frechette A.
      • Aubin R.
      • Gaudreau A
      • Poirier G.G.
      ), p53 (
      • Wesierska-Gadek J.
      • Schimid G.
      • Cerni C.
      ), DNA-dependent protein kinase (
      • Ruscetti T.
      • Lehnert B.E.
      • Halbrook J.
      • Le Trong H.
      • Hoekstra M.F.
      • Chen D.J.
      • Peterson S.R.
      ), and PARP itself (
      • Lindahl T.
      • Satoh M.S.
      • Poirier G.G.
      • Klungland A.
      ).
      Many studies have described PARP as a positive regulator of apoptosis. The overexpression of PARP in the transfected cells was known to stimulate apoptosis (
      • Van Gool L.
      • Meyer R.
      • Tobiasch E.
      • Cziepluch C.
      • Jauniaux J.C.
      • Mincheva A.
      • Lichter P.
      • Poirier G.G.
      • Burkle A.
      • Kupper J.H.
      ). The pro-apoptotic role of PARP activation was further supported by the finding that a specific chemical inhibitor of PARP, 3-aminobenzamide, suppresses apoptosis (
      • Nosseri C.
      • Coppola S.
      • Ghibelli L.
      ). However, the controversial roles of PARP during apoptosis have been argued in the studies using PARP-deficient mice. Wang et al. (
      • Wang Z.Q.
      • Stingl L.
      • Morrison C.
      • Jantsch M.
      • Los M.
      • Schulze-Osthoff K.
      • Wagner E.F.
      ) proposed a dispensable role of PARP in apoptosis by showing a normal level of apoptosis in PARP-deficient mouse cells treated with various apoptosis inducers. An indispensible role of PARP on apoptosis was presented by Simbulan-Rosenthal et al. (
      • Simbulan-Rosenthal C.M.
      • Rosenthal D.S.
      • Iyer S.
      • Boulares A.H.
      • Smulson M.E.
      ), who reported an early burst of poly(ADP-ribosyl)ation of nuclear proteins during Fas-mediated apoptosis in PARP+/+ cells, whereas no induction of apoptosis was shown in PARP−/− cells by Fas stimulation.
      The importance of PARP cleavage in apoptosis has recently been recognized. For example, it has been suggested that the activity of PARP is stimulated by DNA breaks during the early course of apoptosis, but proteolytic cleavage decreases PARP activity in the late course of apoptosis (
      • Rosenthal D.S.
      • Ding R.
      • Simbulan-Rosenthal C.M.
      • Vaillancourt J.P.
      • Nicholson D.W.
      • Smulson M.
      ). Oliver et al. (
      • Oliver F.J.
      • de la Rubia G.
      • Rolli V.
      • Ruiz-Ruiz M.C.
      • de Murcia G.
      • Menissier-de Murcia J.
      ) observed a delayed apoptosis in PARP−/− cells expressing an uncleavable mutant of PARP. They suggested that PARP cleavage might be a sign that cells should undergo apoptosis because cells were unable to repair the cellular injury triggered by the apoptosis inducers. More recently, the importance of PARP cleavage during apoptosis was emphasized by the finding that the extensive poly(ADP-ribosyl)ation of p53 early during apoptosis decreases, as activated caspase-3 cleaves PARP and the expression of p53-responsive pro-apoptotic genes, bax andFas, is elevated during the late course of apoptosis (
      • Simbulan-Rosenthal C.M.
      • Rosenthal D.S.
      • Luo R.
      • Smulson M.E.
      ). However, the biological relevance of PARP cleavage during apoptosis and the cellular function of the cleavage products have not been yet clarified.
      Recent studies have proposed that an adequate level of intracellular ATP is required for the completion of apoptosis (
      • Leist M.
      • Single B.
      • Castoldi A.F.
      • Kuhnle S.
      • Nicotera P.
      ,
      • Eguchi Y.
      • Srinivasan A.
      • Tomaselli K.J.
      • Shimizu S.
      • Tsujimoto Y.
      ). Because the intracellular ATP level is directly affected by the catalytic activity of PARP, apoptosis, an energy-requiring process, may well be influenced by PARP activity. The N-terminal fragment of PARP containing the DNA-binding domain preferentially binds to DNA breaks and prevents the activation of PARP, whose activity is stimulated by DNA binding (
      • Rhun Y.L.
      • Kirkland J.B.
      • Shah G.M.
      ). The pro-apoptotic role of the N-terminal fragment of PARP on apoptosis has been strongly supported by a recent finding that the N-terminal fragment of PARP irreversibly binds to DNA ends produced during apoptosis (
      • Smulson M.E.
      • Pang D.
      • Jung M.
      • Dimtchev A.
      • Chasovskikh S.
      • Spoonde A.
      • Simbulan-Rosenthal C.
      • Rosenthal D.
      • Yakovlev A.
      • Dritschilo A.
      ). However, the role of the C-terminal fragment of PARP on apoptosis has not been delineated. In the present study, we propose a putative role for the C-terminal cleavage product of PARP on apoptosis based on the following findings: (a) the C-terminal fragment of PARP interacts with intact PARP through an association between the auto-modification domains and (b) the C-terminal fragment suppresses its catalytic activity by blocking homodimerization of PARP. We have found that the disruption of homodimerization of PARP by the cleaved C-terminal fragment stimulates apoptosis in UV-treated HeLa cells.

      DISCUSSION

      Although PARP cleavage has been used as a prominent biochemical hallmark of apoptosis, the physiological relevance and functional consequence of PARP cleavage during apoptosis have not been clarified. A recent report by Oliver et al. (
      • Oliver F.J.
      • de la Rubia G.
      • Rolli V.
      • Ruiz-Ruiz M.C.
      • de Murcia G.
      • Menissier-de Murcia J.
      ) showed that Fas-induced apoptosis was delayed in cells expressing an uncleavable PARP mutant. Furthermore, Herceg and Wang (
      • Herceg Z.
      • Wang Z.-Q.
      ) reported that the failure of PARP cleavage induced necrosis by the depletion of cellular energy in cells treated with various apoptotic inducers. These results suggested that the inactivation of PARP by the caspase-mediated proteolytic cleavage is a necessary requirement for the completion of apoptosis. In the present study, we attempted to interpret the biological meaning of the proteolytic cleavage of PARP during the course of apoptosis. We found that apoptosis requires the self-regulatory function of PARP, whose catalytic activity is regulated by its proteolytic cleavage products. The N-terminal fragment of PARP containing the DNA-binding domain preferentially binds to DNA breaks and thereby prevents the activation of PARP (
      • Molinete M.
      • Vermeulen W.
      • Burkle A.
      • Murcia M.
      • Kupper J.H.
      • Hoeijmakers J.H.J.
      • de Murcia G.
      ,
      • Schreiber V.
      • Hunting D.
      • Trucco C.
      • Gowans B.
      • Grunwald D.
      • de Murcia G.
      • de Murcia J.M.
      ). It has been demonstrated that the N-terminal fragment of PARP irreversibly binds to DNA ends produced during apoptosis (
      • Smulson M.E.
      • Pang D.
      • Jung M.
      • Dimtchev A.
      • Chasovskikh S.
      • Spoonde A.
      • Simbulan-Rosenthal C.
      • Rosenthal D.
      • Yakovlev A.
      • Dritschilo A.
      ). The binding of the N-terminal fragment of PARP to DNA breaks has generally been known to contribute to apoptosis by blocking the access of repair enzymes to the DNA breaks generated during the course of apoptosis.
      The roles of the C-terminal fragment of PARP on apoptosis have not yet been described. The present study proposes that the intermolecular association of the cellular PARP with the C-terminal fragment containing the auto-modification domain contributes to apoptosis by blocking the homodimerization of PARP in cells irradiated with UV. The C-terminal fragments of PARP containing the auto-modification domain interacted with intact PARP, whereas the N-terminal fragment containing the DNA-binding domain interacted with DNA. The association between the N-terminal fragment and intact PARP disappeared after DNase I treatment, suggesting that the association was mediated by DNA (Fig.1). We further demonstrated that the bZip motif in the auto-modification domain is the site of self-association of the cellular PARP (Fig. 2 A). The homodimerization of PARP was blocked by the expression of PARP cleavage products containing the bZip motif in HeLa cells (Fig. 2 B). The hydrophobic amino acid Leu405 in the bZip motif was of critical importance in the structural framework required for the intermolecular association and an efficient dimerization of PARP (Fig. 3). The association of PARP with the basic components of the auto-modification domain in the C-terminal fragment seem to compete with the homodimerization of PARP.
      There are number of studies suggesting that oligomerization serves as an important biochemical mechanism for the regulation of protein function. Dimerization was known to provide mechanisms for the modulation of catalytic activity of enzymes (
      • Climent I.
      • Sjoberg B.M.
      • Huang C.Y.
      ,
      • Baek K.J.
      • Thiel B.A.
      • Lucas S.
      • Stuehr D.J.
      ) and receptor functions (
      • Zhu X.
      • Wess J.
      ,
      • Heldin C.H.
      ). Several studies have also provided evidence for the inhibition of protein dimerization by the peptides derived from the same protein. For example, the catalytic activity of E. coliribonucleotide reductase was shown to be inhibited by the C-terminal peptide, which inhibits the homodimerization of the enzyme (
      • Climent I.
      • Sjoberg B.M.
      • Huang C.Y.
      ). The truncated G protein-coupled receptors (GPCRs), which inhibit the formation of dimeric arrays of GPCRs, were also identified as negative regulators of GPCR function (
      • Zhu X.
      • Wess J.
      ). It was known that the catalytic function of PARP is maximal when PARP tends to be self-associated to the dimer form (
      • Bauer P.I.
      • Buki K.G.
      • Hakam A.
      • Kun E.
      ,
      • Mendoza-Alvarez H.
      • Alvarez-Gonzalez R.
      ). Data in Fig. 4 implicate that the expression of the C-terminal fragment of PARP in UV-treated HeLa cells suppresses auto-poly(ADP-ribosyl)ation of PARP, probably by blocking the homodimerization of the cellular PARP. The failure of the homodimerization may interfere with the binding of PARP to DNA breaks, resulting in the catalytic inactivation of PARP. However, the expression of a mutated PARP fragment that contains the C-terminal fragment but lacks the auto-modification domain (PARPΔM) also suppressed the cellular PARP activity. The trans-dominant inhibition of PARP activity by the N-terminal fragment might be the case with PARPΔM. Indeed, the cellular PARP activity was inhibited by the expression of the N-terminal fragment of PARP. This result is likely to reflect the fact that the N-terminal fragment of PARP competes with intact PARP for binding at DNA breaks, which activate PARP (Fig.4).
      The pro-apoptotic role of the C-terminal fragment of PARP on apoptosis was examined in Fig. 5. Both annexin-V binding and caspase activation are well defined markers of apoptosis (
      • Toyoshima F.
      • Moriguchi T.
      • Nishida E.
      ). Our results suggest that the pro-apoptotic role of the C-terminal fragment of PARP does not correlate with the enhanced caspase activation in cells undergoing UV-mediated apoptosis. The level of caspase activity in cells expressing the C-terminal fragment that lacks the bZip motif was virtually same as in cells expressing PARP cleavage products containing the bZip motif. However, the intracellular caspase activity was blocked by the expression of a known inhibitor of caspase-3, CrmA (
      • Tewari M.
      • Quan L.T.
      • O'Rourke K.
      • Desnoyers S.
      • Zeng Z.
      • Beidler D.R.
      • Poirier G.G.
      • Salvesen G.S.
      • Dixit V.M.
      ). However, the expression of the C-terminal fragment of PARP stimulated UV-mediated apoptosis as assessed by the appearance of annexin-V positive cells (Fig. 5 B). In addition, the expression of PARP fragments containing the bZip motif (C215 and M) stimulated the induction of morphological changes characteristic to apoptosis in UV-treated HeLa cells (Fig. 5 C). These results suggest that the intermolecular association of PARP at the bZip motif is required for the completion of apoptosis. The homodimerization of PARP might facilitate the progression of apoptotic cell death beyond the caspase activation step.

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