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The 89-kDa PARP1 cleavage fragment serves as a cytoplasmic PAR carrier to induce AIF-mediated apoptosis

2020; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1074/jbc.ra120.014479

ISSN

1083-351X

Autores

Masato Mashimo, Mayu Onishi, Arina Uno, Akari Tanimichi, Akari Nobeyama, Mana Mori, Sayaka Yamada, Shigeru Negi, Xiangning Bu, Jiro Kato, Joel Moss, Noriko Sanada, Ryoichi Kizu, Takeshi Fujii,

Tópico(s)

Toxin Mechanisms and Immunotoxins

Resumo

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear protein that is activated by binding to DNA lesions and catalyzes poly(ADP-ribosyl)ation of nuclear acceptor proteins, including PARP1 itself, to recruit DNA repair machinery to DNA lesions. When excessive DNA damage occurs, poly(ADP-ribose) (PAR) produced by PARP1 is translocated to the cytoplasm, changing the activity and localization of cytoplasmic proteins, e.g., apoptosis-inducing factor (AIF), hexokinase, and resulting in cell death. This cascade, termed parthanatos, is a caspase-independent programmed cell death distinct from necrosis and apoptosis. In contrast, PARP1 is a substrate of activated caspases 3 and 7 in caspase-dependent apoptosis. Once cleaved, PARP1 loses its activity, thereby suppressing DNA repair. Caspase cleavage of PARP1 occurs within a nuclear localization signal near the DNA-binding domain, resulting in the formation of 24-kDa and 89-kDa fragments. In the present study, we found that caspase activation by staurosporine- and actinomycin D-induced PARP1 autopoly(ADP-ribosyl)ation and fragmentation, generating poly(ADP-ribosyl)ated 89-kDa and 24-kDa PARP1 fragments. The 89-kDa PARP1 fragments with covalently attached PAR polymers were translocated to the cytoplasm, whereas 24-kDa fragments remained associated with DNA lesions. In the cytoplasm, AIF binding to PAR attached to the 89-kDa PARP1 fragment facilitated its translocation to the nucleus. Thus, the 89-kDa PARP1 fragment is a PAR carrier to the cytoplasm, inducing AIF release from mitochondria. Elucidation of the caspase-mediated interaction between apoptosis and parthanatos pathways extend the current knowledge on mechanisms underlying programmed cell death and may lead to new therapeutic targets. Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear protein that is activated by binding to DNA lesions and catalyzes poly(ADP-ribosyl)ation of nuclear acceptor proteins, including PARP1 itself, to recruit DNA repair machinery to DNA lesions. When excessive DNA damage occurs, poly(ADP-ribose) (PAR) produced by PARP1 is translocated to the cytoplasm, changing the activity and localization of cytoplasmic proteins, e.g., apoptosis-inducing factor (AIF), hexokinase, and resulting in cell death. This cascade, termed parthanatos, is a caspase-independent programmed cell death distinct from necrosis and apoptosis. In contrast, PARP1 is a substrate of activated caspases 3 and 7 in caspase-dependent apoptosis. Once cleaved, PARP1 loses its activity, thereby suppressing DNA repair. Caspase cleavage of PARP1 occurs within a nuclear localization signal near the DNA-binding domain, resulting in the formation of 24-kDa and 89-kDa fragments. In the present study, we found that caspase activation by staurosporine- and actinomycin D-induced PARP1 autopoly(ADP-ribosyl)ation and fragmentation, generating poly(ADP-ribosyl)ated 89-kDa and 24-kDa PARP1 fragments. The 89-kDa PARP1 fragments with covalently attached PAR polymers were translocated to the cytoplasm, whereas 24-kDa fragments remained associated with DNA lesions. In the cytoplasm, AIF binding to PAR attached to the 89-kDa PARP1 fragment facilitated its translocation to the nucleus. Thus, the 89-kDa PARP1 fragment is a PAR carrier to the cytoplasm, inducing AIF release from mitochondria. Elucidation of the caspase-mediated interaction between apoptosis and parthanatos pathways extend the current knowledge on mechanisms underlying programmed cell death and may lead to new therapeutic targets. Poly(ADP-ribose) polymerase 1 (PARP1), in the presence of NAD+, catalyzes poly(ADP-ribosyl)ation, generating poly(ADP-ribose) (PAR) chains attached to acceptor proteins (1Ame J.C. Spenlehauer C. de Murcia G. The PARP superfamily.Bioessays. 2004; 26: 882-893Crossref PubMed Scopus (1113) Google Scholar, 2Gupte R. Liu Z. Kraus W.L. PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes.Genes Dev. 2017; 31: 101-126Crossref PubMed Scopus (261) Google Scholar). Activation of PARP1 catalytic activity occurs in response to DNA damage. Poly(ADP-ribosyl)ation of acceptor proteins modifies their activities, structure, and/or location, which affects diverse cellular functions, including DNA repair, gene expression, and cell death (1Ame J.C. Spenlehauer C. de Murcia G. The PARP superfamily.Bioessays. 2004; 26: 882-893Crossref PubMed Scopus (1113) Google Scholar, 2Gupte R. Liu Z. Kraus W.L. PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes.Genes Dev. 2017; 31: 101-126Crossref PubMed Scopus (261) Google Scholar, 3D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (1481) Google Scholar, 4Gibson B.A. Kraus W.L. New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs.Nat. Rev. Mol. Cell Biol. 2012; 13: 411-424Crossref PubMed Scopus (719) Google Scholar). PARP1 is a 116-kDa protein, consisting of three domains: DNA-binding domain (N terminal), automodification domain (central), and catalytic domain (C terminal) (1Ame J.C. Spenlehauer C. de Murcia G. The PARP superfamily.Bioessays. 2004; 26: 882-893Crossref PubMed Scopus (1113) Google Scholar). The DNA-binding domain recognizes DNA strand breaks, resulting in its dimerization and catalysis by the catalytic domain of transautomodification with PAR of the automodification domain (2Gupte R. Liu Z. Kraus W.L. PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes.Genes Dev. 2017; 31: 101-126Crossref PubMed Scopus (261) Google Scholar, 5Verheugd P. Butepage M. Eckei L. Luscher B. Players in ADP-ribosylation: readers and erasers.Curr. Protein Pept. Sci. 2016; 17: 654-667Crossref PubMed Scopus (23) Google Scholar). PARP1 possesses a nuclear localization sequence (NLS) near the DNA-binding domain and a caspase-cleavage site between the DNA-binding domain and the automodification domain (3D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (1481) Google Scholar). PARP1 is initially responsible for DNA repair; the negative charge of PAR polymers covalently attached to PARP1 and histone loosen chromatin structure and recruit the scaffold protein XRCC1 and other DNA-remodeling enzymes (1Ame J.C. Spenlehauer C. de Murcia G. The PARP superfamily.Bioessays. 2004; 26: 882-893Crossref PubMed Scopus (1113) Google Scholar, 2Gupte R. Liu Z. Kraus W.L. PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes.Genes Dev. 2017; 31: 101-126Crossref PubMed Scopus (261) Google Scholar, 3D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (1481) Google Scholar). During caspase-dependent apoptosis, PARP1 is cleaved by caspases 3 and 7 at its caspase-cleavage site into 24-kDa and 89-kDa fragments (6Salvesen G.S. Dixit V.M. Caspases: intracellular signaling by proteolysis.Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1896) Google Scholar, 7Germain M. Affar E.B. D'Amours D. Dixit V.M. Salvesen G.S. Poirier G.G. Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7.J. Biol. Chem. 1999; 274: 28379-28384Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 8Kaufmann S.H. Desnoyers S. Ottaviano Y. Davidson N.E. Poirier G.G. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis.Cancer Res. 1993; 53: 3976-3985PubMed Google Scholar). The 24-kDa PARP1 fragment contains the DNA-binding motif and the NLS, whereas the 89-kDa PARP1 fragment contains the automodification and catalytic domains. After PARP1 cleavage by caspase, the 24-kDa PARP1 fragment irreversibly binds to DNA breaks and acts as a transdominant inhibitor of active PARP1, whereas the 89-kDa PARP1 fragment is translocated to the cytoplasm (9Soldani C. Lazze M.C. Bottone M.G. Tognon G. Biggiogera M. Pellicciari C.E. Scovassi A.I. Poly(ADP-ribose) polymerase cleavage during apoptosis: when and where?.Exp. Cell Res. 2001; 269: 193-201Crossref PubMed Scopus (111) Google Scholar, 10Smulson M.E. Pang D. Jung M. Dimtchev A. Chasovskikh S. Spoonde A. Simbulan-Rosenthal C. Rosenthal D. Yakovlev A. Dritschilo A. Irreversible binding of poly(ADP)ribose polymerase cleavage product to DNA ends revealed by atomic force microscopy: possible role in apoptosis.Cancer Res. 1998; 58: 3495-3498PubMed Google Scholar). Thereby, PARP1 fragmentation by caspase leads to its inactivation, which inhibits DNA repair and facilitates caspase-mediated DNA fragmentation in apoptosis. Parthanatos, a programmed cell death, is initiated by PARP1 over-reaction to DNA damage, which is seen in neurons in Parkinson's disease, after glutamate excitotoxicity and in brain ischemia (11Andrabi S.A. Kang H.C. Haince J.F. Lee Y.I. Zhang J. Chi Z. West A.B. Koehler R.C. Poirier G.G. Dawson T.M. Dawson V.L. Iduna protects the brain from glutamate excitotoxicity and stroke by interfering with poly(ADP-ribose) polymer-induced cell death.Nat. Med. 2011; 17: 692-699Crossref PubMed Scopus (144) Google Scholar, 12Lee Y. Karuppagounder S.S. Shin J.H. Lee Y.I. Ko H.S. Swing D. Jiang H. Kang S.U. Lee B.D. Kang H.C. Kim D. Tessarollo L. Dawson V.L. Dawson T.M. Parthanatos mediates AIMP2-activated age-dependent dopaminergic neuronal loss.Nat. Neurosci. 2013; 16: 1392-1400Crossref PubMed Scopus (123) Google Scholar, 13Wang Y. Kim N.S. Haince J.F. Kang H.C. David K.K. Andrabi S.A. Poirier G.G. Dawson V.L. Dawson T.M. Poly(ADP-ribose) (PAR) binding to apoptosis-inducing factor is critical for PAR polymerase-1-dependent cell death (parthanatos).Sci. Signal. 2011; 4: ra20Crossref PubMed Scopus (257) Google Scholar). Suppression of parthanatos through PARP1 inhibition may have therapeutic potential in these diseases (14Fatokun A.A. Dawson V.L. Dawson T.M. Parthanatos: mitochondrial-linked mechanisms and therapeutic opportunities.Br. J. Pharmacol. 2014; 171: 2000-2016Crossref PubMed Scopus (261) Google Scholar). On binding single- and double-stranded DNA breaks, activated PARP1 catalyzes the covalent addition of long and branched polymers of ADP-ribose to nuclear acceptor proteins, including PARP1 itself, XRCC1, and histones (1Ame J.C. Spenlehauer C. de Murcia G. The PARP superfamily.Bioessays. 2004; 26: 882-893Crossref PubMed Scopus (1113) Google Scholar, 2Gupte R. Liu Z. Kraus W.L. PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes.Genes Dev. 2017; 31: 101-126Crossref PubMed Scopus (261) Google Scholar). PAR translocation from the nucleus to the cytoplasm is a crucial step in the parthanatos pathway. After DNA damage, 90% of PAR polymers are synthesized by PARP1, and most PAR polymers are attached to PARP1 itself (1Ame J.C. Spenlehauer C. de Murcia G. The PARP superfamily.Bioessays. 2004; 26: 882-893Crossref PubMed Scopus (1113) Google Scholar). Poly(ADP-ribose) glycohydrolase, a primary enzyme for PAR degradation, is involved in PAR translocation from the nucleus to cytoplasm (15Mashimo M. Kato J. Moss J. ADP-ribosyl-acceptor hydrolase 3 regulates poly (ADP-ribose) degradation and cell death during oxidative stress.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 18964-18969Crossref PubMed Scopus (94) Google Scholar, 16Mashimo M. Kato J. Moss J. Structure and function of the ARH family of ADP-ribosyl-acceptor hydrolases.DNA Repair (Amst). 2014; 23: 88-94Crossref PubMed Scopus (41) Google Scholar, 17Mashimo M. Moss J. Functional role of ADP-ribosyl-acceptor hydrolase 3 in poly(ADP-ribose) polymerase-1 response to oxidative stress.Curr. Protein Pept. Sci. 2016; 17: 633-640Crossref PubMed Scopus (12) Google Scholar). Poly(ADP-ribose) glycohydrolase endoglycosidase activity generates protein-free and small PAR polymers that appear to pass thorough nuclear membranes (15Mashimo M. Kato J. Moss J. ADP-ribosyl-acceptor hydrolase 3 regulates poly (ADP-ribose) degradation and cell death during oxidative stress.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 18964-18969Crossref PubMed Scopus (94) Google Scholar, 17Mashimo M. Moss J. Functional role of ADP-ribosyl-acceptor hydrolase 3 in poly(ADP-ribose) polymerase-1 response to oxidative stress.Curr. Protein Pept. Sci. 2016; 17: 633-640Crossref PubMed Scopus (12) Google Scholar, 18Mashimo M. Bu X. Aoyama K. Kato J. Ishiwata-Endo H. Stevens L.A. Kasamatsu A. Wolfe L.A. Toro C. Adams D. Markello T. Gahl W.A. Moss J. PARP1 inhibition alleviates injury in ARH3-deficient mice and human cells.JCI Insight. 2019; 4: e124519Crossref PubMed Scopus (11) Google Scholar). After PAR polymers produced by PARP1 are translocated from the nucleus to the cytoplasm, they bind to apoptosis-inducing factor (AIF), which is anchored to the mitochondrial membrane (15Mashimo M. Kato J. Moss J. ADP-ribosyl-acceptor hydrolase 3 regulates poly (ADP-ribose) degradation and cell death during oxidative stress.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 18964-18969Crossref PubMed Scopus (94) Google Scholar, 19Andrabi S.A. Kim N.S. Yu S.W. Wang H. Koh D.W. Sasaki M. Klaus J.A. Otsuka T. Zhang Z. Koehler R.C. Hurn P.D. Poirier G.G. Dawson V.L. Dawson T.M. Poly(ADP-ribose) (PAR) polymer is a death signal.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 18308-18313Crossref PubMed Scopus (469) Google Scholar). PAR binding to AIF results in its release to the cytoplasm (13Wang Y. Kim N.S. Haince J.F. Kang H.C. David K.K. Andrabi S.A. Poirier G.G. Dawson V.L. Dawson T.M. Poly(ADP-ribose) (PAR) binding to apoptosis-inducing factor is critical for PAR polymerase-1-dependent cell death (parthanatos).Sci. Signal. 2011; 4: ra20Crossref PubMed Scopus (257) Google Scholar). As AIF has an NLS near its N terminus, released AIF is translocated to the nucleus and associates with DNAase, resulting in large-scale DNA fragmentation (20Wang Y. An R. Umanah G.K. Park H. Nambiar K. Eacker S.M. Kim B. Bao L. Harraz M.M. Chang C. Chen R. Wang J.E. Kam T.I. Jeong J.S. Xie Z. et al.A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1.Science. 2016; 354: aad6872Crossref PubMed Scopus (133) Google Scholar). PAR also interacts with hexokinase 1, which is the first enzyme in the glycolytic pathway, and inhibits its activity, leading to energy depletion (21Andrabi S.A. Umanah G.K. Chang C. Stevens D.A. Karuppagounder S.S. Gagne J.P. Poirier G.G. Dawson V.L. Dawson T.M. Poly(ADP-ribose) polymerase-dependent energy depletion occurs through inhibition of glycolysis.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10209-10214Crossref PubMed Scopus (174) Google Scholar). These pathways, including PAR synthesis and its translocation to induce parthanatos, are caspase independent (22Yu S.W. Wang H. Poitras M.F. Coombs C. Bowers W.J. Federoff H.J. Poirier G.G. Dawson T.M. Dawson V.L. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor.Science. 2002; 297: 259-263Crossref PubMed Scopus (1489) Google Scholar). To the contrary, AIF release and its translocation to the cytoplasm during apoptosis also has been demonstrated in response to several stimuli in diverse cell types (23Bano D. Prehn J.H.M. Apoptosis-inducing factor (AIF) in physiology and disease: the tale of a repented natural born killer.EBioMedicine. 2018; 30: 29-37Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Proapoptotic Bcl-2 members, such as Bax and Bak, induce a selective process of outer membrane permeabilization through the formation of channels, which allows AIF release from mitochondria (24Otera H. Ohsakaya S. Nagaura Z. Ishihara N. Mihara K. Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space.EMBO J. 2005; 24: 1375-1386Crossref PubMed Scopus (278) Google Scholar). Calpain, a Ca2+-activated protease, releases AIF from mitochondria by cleaving membrane-bound AIF, which then induces AIF-mediated DNA fragmentation (25Polster B.M. Basanez G. Etxebarria A. Hardwick J.M. Nicholls D.G. Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria.J. Biol. Chem. 2005; 280: 6447-6454Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). In this study, we report a novel route for AIF release during apoptosis. Caspase-3 activation after exposure to staurosporine and actinomycin D, conventional apoptosis inducers, resulted in PARP1-mediated PAR production and then PARP1 cleavage into 89-kDa and 24-kDa PARP1 fragments. The 89-kDa PARP1 fragments, with attached PAR polymers, were translocated from the nucleus to the cytoplasm, whereas 24-kDa fragments were associated with DNA breaks. The 89-kDa PARP1 fragments in the cytoplasm interacted with AIF via their PAR polymers, leading to AIF release and translocation to the nucleus, resulting in nuclear shrinkage. Thus, the 89-kDa PARP1 fragment generated by caspase-3 acts as a PAR carrier from the nucleus to the cytoplasm to induce AIF-mediated DNA fragmentation in caspase-mediated apoptosis. We investigated whether staurosporine, an apoptosis inducer, stimulated PARP1-dependent cell death. Exposure of HeLa cells to staurosporine for 6 h resulted in cytotoxicity (Fig. 1A). Pharmacological inhibition of PARP by PJ34 and ABT888 increased significantly the number of viable cells, whereas inhibition of caspase by zVAD-fmk suppressed cell death completely (Fig. 1A). PJ34 did not augment the improved survival rate seen with zVAD-fmk (Fig. 1B). In contrast to the effects of PARP1 inhibition by PJ34, pharmacological inhibition of tankyrase (PARP5) by XAV939 did not alter the sensitivity to staurosporine-induced cytotoxicity to the extent seen with PJ34 (Fig. S1A). Stable expression of PARP1 shRNA reduced PARP1 protein to 10% of that in HeLa cells transfected with control shRNA (Fig. 1C). HeLa cells expressing PARP1 shRNA showed reduced staurosporine-induced cytotoxicity, compared with control shRNA, similar to that seen with PJ34 (Fig. 1D). In addition, pretreatment with PJ34 did not improve the viability of Hela cells expressing PARP1 shRNA (Fig. S1B), suggesting that PJ34 has specificity to PARP1-dependent cell death induced by staurosporine. Exposure to N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent, resulted in PARP1-dependent and caspase-independent cell death, because PJ34, but not zVAD-fmk, reduced N-methyl-N'-nitro-N-nitrosoguanidine-induced cytotoxicity (Fig. S1C). PARP1 activation results in PAR synthesis and then AIF release from mitochondria (22Yu S.W. Wang H. Poitras M.F. Coombs C. Bowers W.J. Federoff H.J. Poirier G.G. Dawson T.M. Dawson V.L. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor.Science. 2002; 297: 259-263Crossref PubMed Scopus (1489) Google Scholar). Western blot analysis revealed that PAR was generated as early as 1 h after exposure to staurosporine, and approached the peak at 4 h, with increased PAR lasting at least past 6 h (Fig. 1E). After exposure to staurosporine for 6 h, AIF accumulated in HeLa cell nuclei, leading to nuclear shrinkage (Fig. 1, F–H). The pharmacological inhibition of either caspase or PARP1 prevented PAR synthesis as well as AIF-mediated nuclear shrinkage (Fig. 1, E–H). Moreover, HeLa cells expressing PARP1 shRNA did not exhibit PAR synthesis, AIF translocation to nuclei, and nuclear shrinkage (Fig. S2, A–C). All findings are consistent with the notion that PARP1 activation and then AIF release from mitochondria appears to be partially required downstream of caspase-mediated apoptosis after staurosporine exposure. PARP1 is proteolytically cleaved near the third zinc-finger domain by caspases-3 and 7 into 24-kDa N-terminal and 89-kDa C-terminal PARP1 fragments (6Salvesen G.S. Dixit V.M. Caspases: intracellular signaling by proteolysis.Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1896) Google Scholar). Western blot analysis using the PARP1 antibody, which recognizes full-length PARP1 and its 89-kDa PARP1 fragments, detected 89-kDa PARP1 fragments after a 1-h exposure to staurosporine, which resulted in caspase-3 activation (Fig. 2A). zVAD-fmk, but not PJ34, inhibited caspase-3 activation as well as PARP1 fragmentation after 4 and 6 h of staurosporine exposure (Fig. 2A). PARP1 knockdown by shRNA did not affect caspase-3 activation (Fig. S2D). Using immunocytochemistry to evaluate intracellular localization of PARP1 and PAR, we found that, after exposure to staurosporine, PARP1 was translocated from the nucleus to the cytoplasm after 3 h; in contrast, PAR was located primarily in the nuclei of HeLa cells at 1 h and translocated to the cytoplasm after 3 h (Fig. 2B). zVAD-fmk and PJ34 reduced cytoplasmic PAR content after 6-h exposure to staurosporine, whereas zVAD-fmk, but not PJ34, inhibited the cytoplasmic localization of PARP1 (Fig. 2C). Moreover, HeLa cells expressing PARP1 shRNA exhibited reduced translocation of PAR to the cytoplasm after 6-h exposure to staurosporine compared with control shRNA (Fig. S2E). We next investigated the molecular state of PARP1. Subcellular fractionation confirmed that, after 6-h exposure to staurosporine, only the 89-kDa PARP1 fragment, but not full-length PARP1, was localized in the cytoplasm (Fig. 2D). zVAD-fmk, but not PJ34, prevented both PARP1 fragmentation and the cytoplasmic localization of 89-kDa PARP1 fragments (Fig. 2E). Densitometric analysis indicates that approximately 20% of PARP1 was present as the cleaved form in the cytoplasm (Fig. 2E). These results indicate that the 89-kDa PARP1 fragment is translocated to the cytoplasm after PARP1-mediated PAR production in nuclei. To investigate the subcellular localization of 89-kDa and 24-kDa PARP1 fragments after exposure to staurosporine, GFP and mCherry were linked to the C-terminal and N-terminal sites, respectively, of PARP1, generating PARP1-GFP and mCherry-PARP1, respectively (Fig. 3, A–B). After 6-h exposure to staurosporine, the PARP1-GFP signal was localized in the cytoplasm and nucleus, whereas mCherry-PARP1 signal was confined to the nucleus (Fig. 3C). All findings are consistent with the notion that 89-kDa PARP1 fragments, but not 24-kDa PARP1 fragments, are translocated to the cytoplasm after PARP1 cleavage by caspase. The recruitment of PARP1 at DNA lesions is required for PAR synthesis (26Mortusewicz O. Ame J.C. Schreiber V. Leonhardt H. Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells.Nucleic Acids Res. 2007; 35: 7665-7675Crossref PubMed Scopus (210) Google Scholar). We next investigated whether PARP1 is cleaved after it is recruited to DNA lesions. To address this issue, we performed a 405-nm laser microirradiation to induce DNA lesions in HeLa cells expressing PARP1 constructs. After microirradiation, PARP1-GFP and mCherry-PARP1 rapidly accumulated at DNA-damage sites and gradually disappeared (Fig. 3, D–G). Short-term exposure (2 h) to staurosporine suppressed PARP1-GFP recruitment and accelerated dissociation at DNA lesions, whereas zVAD-fmk blocked the effect (Fig. 3, D–E). In contrast, mCherry-PARP1 accumulated at DNA lesions in the presence of staurosporine for a longer time than occurred in the absence of staurosporine, whereas staurosporine exposure did not affect its accumulation level (Fig. 3, F–G). The prolonged accumulation of mCherry-PARP1 was blocked by the addition of zVAD-fmk to the extent seen with control (Fig. 3, F–G). These results suggest that caspases can cleave PARP1 that is recruited at DNA lesions and, after fragmentation, 89-kDa PARP1 fragments dissociate, whereas 24-kDa PARP1 fragments remain at the DNA lesions. Based on the facts that caspase can cleave PARP1 after it is recruited to DNA lesions and that the 89-kDa PARP1 fragment possesses the automodification domain, we hypothesized that 89-kDa PARP1 fragments were translocated to the cytoplasm with attached PAR polymers. To address this issue, we performed a pull-down assay using a glutathione-S-transferase (GST)–tagged macrodomain immobilized on Glutathione Sepharose. The macrodomain is a PAR-binding motif in Histone H2A1.1. GST macrodomain specifically binds ADP-ribosylated proteins, which can then be isolated in a pull-down assay. Recombinant PARP1 proteins automodified by PAR polymers were incubated with recombinant capsase-3 proteins for 1 h. Pull-down assay using the GST macrodomain detected recombinant full-length PARP1 and 89-kDa PARP1 fragments (Fig. 4A). PAR polymers with attached 89-kDa PARP1 fragments were shifted to a lower molecular weight range than the full-length PARP1 (Fig. 4A). These results suggest that 89-kDa PARP1 fragments generated by caspase-3 still retained PAR polymers. In HeLa cells, after 4-h exposure to staurosporine, 89-kDa PARP1 fragments present in the cytoplasm were poly(ADP-ribosyl)ated (Fig. 4B, left). PJ34 did not suppress the generation of 89-kDa PARP1 fragments and its translocation to the cytoplasm but inhibited formation of poly(ADP-ribosyl)ated 89-kDa PARP1 fragments (Fig. 4B, right). To confirm whether 89-kDa PARP1 fragments generated after staurosporine exposure carry PAR polymers from the nucleus to the cytoplasm, we prepared a PARP1 D214G-GFP construct, which is a mutant lacking the caspase-cleavage site (Fig. 4, C–D). In PARP1 shRNA-expressing HeLa cells, staurosporine exposure did not result in the translocation of PARP1 D214G-GFP and PAR to the cytoplasm (Fig. 4E). Moreover, after microirradiation, PARP1 D214G-GFP rapidly accumulated at DNA-damage sites and gradually disappeared regardless of the presence of staurosporine (Fig. 4, F–G). These results are consistent with the fact that generation of the 89-kDa PARP1 fragment by caspase cleavage is required for PAR translocation to the cytoplasm. In the cytoplasm, PAR binding to AIF is a critical step involved in the release of AIF from mitochondrial membranes (19Andrabi S.A. Kim N.S. Yu S.W. Wang H. Koh D.W. Sasaki M. Klaus J.A. Otsuka T. Zhang Z. Koehler R.C. Hurn P.D. Poirier G.G. Dawson V.L. Dawson T.M. Poly(ADP-ribose) (PAR) polymer is a death signal.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 18308-18313Crossref PubMed Scopus (469) Google Scholar). We next investigated whether 89-kDa PARP1 fragments interact with AIF via PAR polymers after translocation to the cytoplasm. After 4-h exposure to staurosporine, the 89-kDa PARP1 fragments, but not the full-length PARP1, interacted with AIF (Fig. 5A). Moreover, in cytoplasm, the 89-kDa PARP1 fragments interacted with AIF, which was suppressed by PJ34 (Fig. 5B, left). Reciprocal immunoprecipitation with anti-AIF antibody confirmed that 89-kDa–cleaved PARP1 fragments interacted with AIF in cytoplasmic and mitochondrial fractions after a 4-h exposure to staurosporine (Fig. 5B, right). These results indicate that the 89-kDa PARP1 fragments interacted with AIF in the cytoplasm via PAR polymers. PARP1 E988K mutant has a mutation in the catalytic domain and loses the ability to catalyze poly(ADP-ribosyl)ation (Fig. S3A). In PARP1 shRNA-expressing HeLa cells, expression of PARP1 E988K-GFP did not produce PAR after 4-h exposure to staurosporine, as was seen with PARP1-GFP (Fig. S3B, upper). As PARP1 E988K-GFP retains a caspase-cleavage site, staurosporine exposure resulted in the cleavage of PARP1 E988K-GFP as was seen with PARP1-GFP (Fig. S3B, lower). In HeLa cells, PARP1 E988K-GFP was cleaved after a 2-h exposure to staurosporine (Fig. S3C). Under these conditions, pull-down assay using the GST macrodomain detected the poly(ADP-ribosyl)ation of full-length and cleaved forms of PARP1 E988K-GFP (Fig. S3C). Pretreatment with zVAD-fmk and PJ34 suppressed staurosporine-induced poly(ADP-ribosyl)ation of PARP1 E988K-GFP. These results indicate that caspase, which is activated by staurosporine, activates endogenous PARP1, resulting in its dimerization with PARP1 E988K-GFP and transautomodification with PAR. In PARP1 shRNA-expressing HeLa cells, staurosporine exposure resulted in the translocation of PARP1 E899K-GFP to the cytoplasm but was not accompanied by PAR production (Fig. S3D). Long-term exposure (8 h) to staurosporine resulted in the cleavage of both PARP1-GFP and PARP1 E988K-GFP, which were translocated to the cytoplasm (Fig. S3, E–F). Approximately 20% of their cleaved forms were localized in the cytoplasm (Fig. S3F). Unlike PARP1-GFP, the cleaved form of PARP1 E988K-GFP localized in the cytoplasm was not automodified by PAR and did not interact with AIF (Fig. S3, G–H). These results indicate that transautomodification of PARP1 by PAR and then its fragmentation by caspase are required for AIF binding. We next investigated whether the 89-kDa PARP1 fragment acts as a carrier for PAR translocation from the nucleus to the cytoplasm in caspase-dependent apoptosis induced by another apoptosis inducer, actinomycin D, which inhibits DNA-dependent RNA polymerase. PJ34 and PARP1 shRNA partially suppressed actinomycin D-induced cytotoxicity. In contrast, zVAD-fmk completely blocked the cytotoxicity (Fig. 6, A–B). Actinomycin D-induced apoptosis was partially mediated through AIF-mediated cell death; in agreement, actinomycin D induced PAR synthesis in the nucleus and its translocation to the cytoplasm, AIF translocation from mitochondria to the nucleus, and nuclear shrinkage, whereas zVAD-fmk and PJ34 inhibited these effects (Fig. 6, C–H). Exposure to actinomycin D resulted in PARP1 fragmentation along with caspase-3 activation (Fig. 7A). After PARP1 fragmentation by actinomycin D exposure, 89-kDa PARP1 fragments, but not 24-kDa PARP1 fragments, were translocated to the cytoplasm (Fig. 7B). S

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