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© The Rockefeller University Press,
0021-9525/2000//293 $5.00
The Journal of Cell Biology, Volume 150, Number 2,
, 2000 293-308
Original Article |
A Fast Signal–Induced Activation of Poly(Adp-Ribose) Polymerase
: A Novel Downstream Target of Phospholipase C
b Department of Agricultural Botany, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel
c Laboratory of Molecular and Structural Biology, Ecole Superieure de Biotechnologie de Strasbourg, F-67400 Illkirch-Graffenstaden, France
d Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva 84105, Israel
e Department of Molecular and Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
Cardiac Research Institute, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel.972-3-535-1139972-3-535-4865
marmon{at}post.tau.ac.il
We present the first evidence for a fast activation of the nuclear protein poly(ADP-ribose) polymerase (PARP) by signals evoked in the cell membrane, constituting a novel mode of signaling to the cell nucleus. PARP, an abundant, highly conserved, chromatin-bound protein found only in eukaryotes, exclusively catalyzes polyADP-ribosylation of DNA-binding proteins, thereby modulating their activity. Activation of PARP, reportedly induced by formation of DNA breaks, is involved in DNA transcription, replication, and repair. Our findings demonstrate an alternative mechanism: a fast activation of PARP, evoked by inositol 1,4,5,-trisphosphate–Ca2+ mobilization, that does not involve DNA breaks. These findings identify PARP as a novel downstream target of phospholipase C, and unveil a novel fast signal–induced modification of DNA-binding proteins by polyADP-ribosylation.
Key Words: poly(ADP-ribose) polymerase • calcium signaling • inositol 1,4,5-trisphosphate • electrical stimulation • brain neurons
© 2000 The Rockefeller University Press
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Introduction |
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Known substrates of PARP include topoisomerase I (Ferro and Olivera 1984; Kasid et al. 1989), RNA-polymerase II (Hanawalt et al. 1994, Li Oei et al. 1998), DNA polymerases (Simbulan et al. 1993), transcription factors (Rawling and Alvarez-Gonzalez 1997; Li Oei et al. 1998), histones (Boulikas 1990; D'Amours et al. 1999), high mobility group proteins (Tsai et al. 1992; D'Amours et al. 1999), p53 (Li Oei et al. 1998), and DNA-dependent kinase (Ruscetti et al. 1998). PolyADP-ribosylation modulates their activity, influencing DNA replication (Cesarone et al. 1990), transcription (Meisterernst et al. 1997, D'Amours et al. 1999), and repair (Satoh and Lindahl 1992; Lazebnik et al. 1994; Nicholson et al. 1995; Schreiber et al. 1995; Martinou 1996; Trucco et al. 1998).
The findings presented here demonstrate a fast signal–induced activation of PARP in brain cortical neurons, mediated by inositol 1,4,5,-trisphosphate (IP3)–induced Ca2+ mobilization, which does not involve DNA damage. Thus, PARP acts as a downstream target of phospholipase C.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Crude Nuclei
Crude nuclei were isolated from lysed brain cortical neurons (Cohen-Armon et al. 1996). Cultured cortical neurons were homogenized on ice in isotonic 0.32 M sucrose containing PMSF (0.1 mM), using a glass/glass homogenizer, and were centrifuged at 900 g for 10 min at 4°C. Cells in the resulting pellet were lysed in hypotonic solution (50 mM Tris-Cl, pH 7.4) and centrifuged as described above. This procedure was repeated in 0.32 M sucrose (900 g for 10 min at 4°C) and in 50 mM Tris-Cl, pH 7.4 (12,000 g for 10 min, 4°C). The resulting pellet contained isolated crude nuclei (see electromicrograph in Fig. 8 a).
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Immunoprecipitation
PolyADP-ribosylated proteins were immunoprecipitated from nuclear protein extracts by monoclonal antibody directed against ADP-ribose polymers containing >10 ADP-riboses (10H; Lamarre et al. 1988; Shah et al. 1995) (see Materials). PARP was immunoprecipitated from the nuclear protein extracts by an affinity-purified goat polyclonal antibody raised against amino acids 1–20 at the NH2 terminus of human PARP (N-20; see Materials). For immunoprecipitation, nuclear proteins (
400 µg protein/sample) were extracted during incubation of crude nuclei (30 min, 4°C) with 50 µl buffered solution containing 500 mM NaCl, 1.5 mM MgCl2, 10 mM Tris-Cl (pH 7.4). Samples were then centrifuged (10,000 g, 5 min) and the supernatants were diluted in buffered solution containing 1.5 mM MgCl2 and 10 mM Tris-Cl. Nuclear proteins were exposed in this solution (overnight, 4°C) to the first antibody (dilution 1:20). Proteins bound to the antibody were precipitated during overnight incubation with protein G–conjugated agarose beads at 4°C, and then extracted from the beads after several washes with PBS by boiling for 2 min in sample buffer.
In Situ Immunofluorescent Labeling of PolyADP-ribosylated Proteins in Cultured Cortical Neurons
Tissue cultures were prepared on coverslips. Monoclonal 10H antibody (dilution 1:10) was introduced into rapidly fixed neurons (fixed for 10 min in ice-cold methanol/acetone 1:1, vol/vol). After overnight incubation with the first antibody at 4°C, neurons were washed with PBS containing 0.1% Tween 20 and exposed to the secondary antibody (dilution 1:500) for 3 h at room temperature. ADP-ribose polymers bound to the nuclear proteins were visualized by the FITC-conjugated affinity pure goat anti–mouse IgG3
secondary antibody, using a fluorescence confocal inverted microscope (ZEISS LSM 410).
[32P]PolyADP-ribosylation of Proteins in Isolated Crude Nuclei
Unless indicated otherwise, crude nuclei isolated from cortical neurons were incubated for 1–5 min with [32P]NAD (1,000 Ci/mmol; 1µ Ci/sample) and 2.3 mM MgATP at 37°C in a solution containing (mM): 0.045 EDTA, 60 Tris-Cl, 1 MgCl2, and 0.8 DTT (pH 7.4). Deionized water contained 25–30 nM Ca2+ (determined by atomic absorbtion). [32P]Poly-ADP-ribosylation was terminated by high salt extraction of the nuclear proteins (500 mM NaCl, 10 mM Tris-Cl [pH 7.4], 4°C, 30 min). [32P]polyADP-ribosylated PARP was immunoprecipitated from the nuclear proteins extracts, subjected to SDS-PAGE, electroblotted, and autoradiographed. [32P]PolyADP-ribosylation was quantified by densitometry.
Thymidine Incorporation into DNA during Stimulation
Cultured neurons were incubated with [3H]thymidine (1 µCi/ml) for 1 h before stimulation. 4 h after stimulation, neurons were lysed and harvested onto filters (GF/C, Whatman). The tritium β emission of incorporated [3H]thymidine was counted in scintillation mixture (Friedberg et al. 1995).
Incorporation of modified thymidine, 5-bromodeoxyuridine (BrdUrd) was measured by immunolabeling with anti-BrdUrd monoclonal antibody IU-4 (Caltag Laboratories). BrdUrd (50 µM) was added to cultured neurons 1 h before stimulation. 6 h after stimulation, the neurons were fixed and treated with RNase A. A limited DNA denaturation was performed to allow access of anti-BrdUrd antibody into the DNA (Selden and Dolbeare 1994). Immunolabeled neurons were then incubated with 5 µg/ml propidium iodide, which intercalates into native DNA (Selden and Dolbeare 1994). The amount of incorporated BrdUrd labeled by FITC-conjugated secondary antibody (green fluorescence), indicating DNA synthesis, and the amount of intercalated propidium iodide (red fluorescence), indicating the amount of double stranded DNA, were measured by flow cytometry (FACSort machine operated by CellQest software; Becton Dickinson).
Single Strand DNA Breaks Examined by Alkaline Gel Electrophoresis
This method provides a sensitive and rapid method for direct quantitation of breaks in DNA single strands (Sutherland et al. 1999). DNA was isolated from the nuclei of cortical neurons using the Hirt procedure (Hirt 1967). The migration of equivalent amounts of DNA was analyzed by electrophoresis on 1% alkaline agarose gel (Sutherland et al. 1999). DNA was stained with ethidium bromide (1 µg/ml) and photographed under UV illumination.
Selective Extraction of Fragmented DNA from Nuclei
Fragmented DNA was selectively extracted from prefixed nuclei in high molarity phosphate–citrate buffer (Darzynkiewicz and Juan 1999). High molecular weight DNA and DNA attached to the nuclear matrix resisted extraction, but fragmented DNA was extracted from the nuclei and identified on agarose gel by staining with ethidium bromide (1 µg/ml).
Displacement of Bound [3H]IP3 by IP3
Samples (20 µl) of crude nuclei (1.5 mg protein/ ml) were incubated (10 min, 4°C) with [3H]IP3 (200 pmol/20 µl sample) in the solution used for [32P]polyADP-ribosylation. Crude nuclei were then rapidly washed under pressure on Whatman GF/B glass-fiber filters, with ice-cold solution containing 25 mM Tris-Cl, 5 mM NaHCO3 and 1 mM EDTA, pH 8.0 (Challiss et al. 1990). The amount of [3H]IP3 bound to the crude nuclei was assayed by counting their β emission in scintillation fluid. Nonspecific binding of [3H]IP3 was determined in the presence of 10 µM IP3.
Topoisomerase I Activity
Topoisomerase I activity was measured in nuclear protein extracts as described previously (Liu and Miller 1984). Extracted nuclear proteins (0.1 µg/sample) were added to a reaction mixture containing, at a final volume of 25 µl (mM): 20 Tris-Cl (pH 8.1), 1 DTT, 20 KCl, 10 MgCl2, 0.5 EDTA, 20 µg/ml BSA, and (as substrate) 250 ng of pUC-19, a supercoiled DNA plasmid (Promega). After incubation at 37°C for 30 min, the reaction was terminated by the addition of 5 µl of buffer containing: 50 mM EDTA (pH 8.0), 1% SDS, 15% glycerol, and 0.05% bromophenol blue. The reaction products were analyzed by electrophoresis on 1% agarose gel. Under these experimental conditions topoisomerase II is not activated (Liu and Miller 1984).
Electron Microscopy
Nuclei isolated from cultured brain cortical neurons were fixed with glutaraldehyde/paraformaldehyde (3:1%) in Krebs-Henseleit buffer (pH 7.4) containing 30% BSA. They were then washed at 4°C with 0.1 M PBS (pH 7.4) and postfixed with 1% OsO4 and 1.5% potassium ferricyanide in PBS at 4°C for 2 h. The samples were examined under a Jeol Jem-100CX electron microscope.
Simultaneous Recording of Rhod-2 Fluorescence
Isolated crude nuclei were loaded with the Ca2+ indicator rhod-2/AM (4.5 µM, 30 min incubation, 25°C, at dark), washed, and attached to poly-L-lysine–coated coverslips. Ca2+-induced fluorescent signal of rhod-2 (excitation, 540 nm; emission, >570 nm) was collected through appropriate filters above 520 nm and monitored by confocal inverted microscope (ZEISS LSM 410), equipped with a 25 mW krypton–argon laser (488- and 568-nm lines) and 10 mW He–Ne laser (633-nm line). A 40x NA/1.2 C-apochromat water-immersion lens (Axiovert 135 M, ZEISS) was used for imaging.
DNAse I Activity in Nuclei Isolated from Cultured Neurons
DNAse I activity in nuclei isolated from cultured neurons was assayed according to the procedure described by Boulikas 1990. Nuclei were incubated with DNAse I (RNAse-free; D 7291, Sigma-Aldrich) in buffered solution containing 20 mM Mn2+, 10% glycerol, 10 mM Tris-Cl, and 1 mM DTT (pH 7.4). The reaction was terminated by the addition of 25 mM EDTA (pH 8.0). Fragmented DNA was examined by gel agarose electrophoresis.
Materials
[Adenylate-32P]nicotinamide-adenindinucleotide, di(tri-ethyl-ammonium) salt ([32P]NAD) (1,000 Ci/mmol) was purchased from DuPont or from Amersham Pharmacia Biotech. D-myo-[3H]Inositol 1,4,5,-trisphosphate, potassium salt ([3H]IP3) (20–60 Ci/mmol) was from Amersham Pharmacia Biotech. [Methyl-3H]thymidine 5'-triphosphate, tetrasodium salt (70–90 Ci/mmol) and [phenyl-3H]tetraphenyl phosphonium bromide ([3H]TPP+) (35 Ci/mmol) were from DuPont. IP3 (hexapotassium salt) was from BIOMOL. Ethylenediamine-tetraacetic acid (EDTA) and ethyleneglycol-bis(β-amino-ethyl) N,N,N',N'-tetraacetic acid (EGTA) were from Merck. D(–)-2-amino-5-phosphovaleric acid (APV) was from Cambridge Research Biochemicals. Ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA AM) and rhod-2 AM were from Molecular Probes. (+)-MK-801 hydrogen maleate was from Biotrend. The polyclonal anti–human PARP antibody Vic-5 and monoclonal antibody 10H, directed against ADP-ribose polymers, were kind gifts from Dr. Sugimura, Tokyo Cancer Center, Japan. Anti–human PARP antibody (N-20) and secondary antibodies were from Santa Cruz Biotechnology. Other materials were from Sigma-Aldrich.
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Results |
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In Situ Immunolabeling of PolyADP-ribosylated Nuclear Proteins by Antibody Directed against ADP-ribose Polymers.
PolyADP-ribosylated proteins were immunolabeled in situ by monoclonal antibody directed against ADP-ribose polymers (10H; Shah et al. 1995) in neurons, permeabilized by a rapid fixing procedure (see Materials and Methods). Immunolabeling of ADP-ribose polymers indicated an increased polyADP-ribosylation of proteins in the nuclei of depolarized neurons, relative to that in nuclei of unstimulated neurons (Fig. 1 a). Moreover, in situ polyADP-ribosylated PARP was immunoprecipitated by 10H antibody from nuclear extracts of depolarized or electrically stimulated neurons, indicating its enhanced polyADP-ribosylation during depolarization (Fig. 1 b). A significantly higher polyADP-ribosylation of PARP was observed in nuclei of neurons pretreated by H2O2, an agent producing DNA breaks (Dizdaroglu 1992; de Murcia et al. 1994; Fig. 1 b). PARP in nuclei of unstimulated neurons was not immunoprecipitated by 10H antibody (Fig. 1 b).
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PolyADP-ribosylation of PARP in intact cells can be assayed by its [32P]polyADP-ribosylation in their isolated nuclei (back-[32P]polyADP-ribosylation), since PARP activity is preserved in the isolated nuclei, and only DNA-bound PARP is polyADP-ribosylated (Satoh et al. 1994). Extensively polyADP-ribosylated PARP is released from DNA, and its ADP-ribose polymers are immediately subjected to a partial degradation (Satoh et al. 1994). PARP carrying partially degraded ADP-ribose polymers is not rebound to DNA (Udea 1990; Satoh et al. 1994; Lindahl et al. 1995). Since a complete degradation of ADP-ribose polymers, which would enable PARP de novo binding to DNA, is very slow relative to the time course of its [32P]polyADP-ribosylation (see Materials and Methods; Satoh et al. 1994), extensively polyADP-ribosylated PARP in situ may not undergo further [32P]polyADP-ribosylation. Thus, although PARP was extensively [32P]polyADP-ribosylated during DNA-nicks formation by DNAse I, it was scarcely [32P]polyADP-ribosylated in nuclei pretreated with DNAse I before [32P]polyADP-ribosylation (Fig. 2, compare lanes 3 and 6 with lanes 8 and 10).
Thus, for DNA-bound PARP undergoing [32P]polyADP-ribosylation in the isolated nuclei, the more extensive the PARP endogenous polyADP-ribosylation, the lower its measured [32P]poly-ADP-ribosylation. This is illustrated in Fig. 3, based on the schematic presentation of polyADP-ribosylation by Satoh et al. 1994. The low extent of PARP [32P]polyADP-ribosylation in nuclei isolated from depolarized neurons (Fig. 1 c) is in accordance with its high endogenous polyADP-ribosylation (Fig. 1, a and b, and 3).
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Cortical neurons in culture were stimulated by pulsed electrical stimuli (see Materials and Methods). Evoked action potentials and postsynaptic potentials were recorded in individual neurons during stimulation by using the patch-clamp whole cell configuration (see Materials and Methods; Hamill et al. 1981) (Fig. 4 a). Immediately after stimulation, neurons were lysed and their nuclei were isolated (see Materials and Methods). [32P]polyADP-ribosylation was conducted in the isolated nuclei (see Materials and Methods). Generally, a continuous electrical activity in the cortical neurons resulted in a low back-[32P]polyADP-ribosylation of PARP in their isolated nuclei (Fig. 4 b). This was consistent with the directly assayed enhanced polyADP-ribosylation of PARP in depolarized neurons (Fig. 1, a and b). The effect of depolarization on PARP activity was reversed by repolarization (Fig. 4 b); the more effective the stimulation, the longer the repolarization period required for reversal (Fig. 4 b). Stimulated neurons preserved their resting potential (Fig. 4 a), evidence that they were not damaged by the depolarizing stimulations. Also, depolarized neurons survived in their cultures for 10 d after stimulation, similar to the survival period of unstimulated neurons.
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We examined the effect of membrane depolarization on both activity and polyADP-ribosylation of topoisomerase I. Incubation of the supercoiled DNA-plasmid with proteins extracted from nuclei of depolarized neurons resulted in a significantly reduced topoisomerase I activity (Fig. 5 a, lanes 7–11), as compared with its activity in protein extracts of unstimulated or repolarized neurons (Fig. 5 a, lanes 3–6 and 12). Moreover, inhibition of topoisomerase I activity in depolarized neurons was prevented by suppression of PARP activity with 3-aminobenzamide (3-AB; Udea 1990) (Fig. 5 a, lanes 8 and 10). This result was in line with polyADP-ribosylation of topoisomerase I in the depolarized neurons (Fig. 5 b), thereby indicating that topoisomerase I is inhibited in depolarized neurons by polyADP-ribosylation.
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Depolarizing stimulation induced a transient polyADP-ribosylation of PARP, which disappeared as the resting potential was restored (Fig. 4 b). Therefore, we used methods suitable for detecting DNA repair in intact neurons during this transient effect. Induction of DNA breaks should be reflected in an increased DNA repair in depolarized neurons (Friedberg et al. 1995). We therefore examined DNA synthesis in the stimulated neurons by measuring the incorporation of thymidine (Friedberg et al. 1995) or the thymidine analogue BrdUrd (Selden and Dolbeare 1994) into DNA (see Materials and Methods).
[3H]Thymidine was incorporated only in nicked DNA of neurons pretreated with H2O2 (Fig. 6 a). There was no significant incorporation of [3H]thymidine or BrdUrd into DNA of depolarized or unstimulated neurons (Fig. 6, a and b, respectively).
Lack of DNA breaks in the depolarized neurons was further confirmed by two sensitive methods for DNA breaks detection: (i) alkaline gel electrophoresis of DNA, for detecting breaks in single stranded DNA (Sutherland et al. 1999) and (ii) selective extraction of fragmented DNA from isolated nuclei (Darzynkiewicz and Juan 1999) (see Materials and Methods).
The results depicted in Fig. 6 c show no evidence of breaks in single DNA strands of depolarized or unstimulated neurons. Moreover, there was no evidence of DNA fragmentation in either unstimulated or depolarized neurons (Fig. 6 d). Nicked DNA single strands or fragmented DNA were extracted only from neurons pretreated by H2O2 or from nuclei pretreated with DNAse I (Fig. 6c and Fig. d). These results strongly suggest that the enhanced polyADP-ribosylation of PARP in depolarized neurons is not derived from the formation of DNA breaks.
Lack of breaks in the DNA of depolarized neurons (Fig. 6) is consistent with the lack of NAD consumption in the depolarized neurons (Fig. 2 a) (Satoh and Lindahl 1992).
Evidence Associating Activation of PARP with IP3-mobilized Ca2+
Extranuclear Ca2+ Promotes Activation of PARP.
An increased intracellular Ca2+ concentration ([Ca2+]) is measured in neurons during membrane depolarization (Al-Mohanna et al. 1994). We therefore examined the possibility that Ca2+ is a mediator of depolarization-induced PARP activation. The effect of extranuclear [Ca2+] on [32P]polyADP-ribosylation of nuclear proteins was examined in isolated nuclei of cortical neurons in the presence of ATP (Methods). Nuclei were exposed to increasing [Ca2+], added before or after the addition of [32P] NAD, which initiates [32P]polyADP-ribosylation.
Increasing the extranuclear [Ca2+] during [32P]polyADP-ribosylation enhanced, by a dose-dependent manner, the [32P]polyADP-ribosylation of PARP (Fig. 7, a and b). The effect of Ca2+ on polyADP-ribosylation was very fast. It was therefore identified better at 25°C (rather than at 37°C; Fig. 7 a, lanes 1–6). Accordingly, when Ca2+ was added to the nuclei before [32P]polyADP-ribosylation, the [32P]polyADP-ribosylation of PARP decreased in a dose-dependent manner by increasing extranuclear [Ca2+] (Fig. 7 a, lanes 7–12), indicating a decreased back-[32P]polyADP-ribosylation of the activated PARP (see Fig. 1 and Fig. 3).
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Ca2+ Release into the Nucleoplasm in Isolated Nuclei of Cortical Neurons.
We next examined the possibility that Ca2+, mobilized from intracellular stores, is released into the nucleoplasm. Crude nuclei (Fig. 8 a; see Materials and Methods) were isolated from brain cortical neurons and loaded with the permeant fluorescent Ca2+ indicator rhod-2 AM (Minta et al. 1989) in the absence of extranuclear Ca2+ (see Materials and Methods). Capture of Ca2+ by rhod-2 was visualized by confocal microscopy (see Materials and Methods; Fig. 8, b–d). [Ca2+] was markedly increased in the nucleoplasm of nuclei isolated from depolarized neurons (Fig. 8 b), in line with the transient increase in nuclear [Ca2+] in depolarized neurons (Al-Mohanna et al. 1994; Hardingham et al. 1997).
An increase of extranuclear [Ca2+] did not induce Ca2+ release into the nucleoplasm unless ATP (2.5 mM) was added (Fig. 8 d); extranuclear Ca2+, in its physiological concentration range, was instantaneously accumulated in perinuclear compartments by adding ATP (Fig. 8c and Fig. d). Under these experimental conditions, Ca2+ was instantaneously released into the nucleoplasm by the addition of IP3 (1–2 µM; Fig. 8 c). In the presence of ATP, Ca2+ was also moderately released into the nucleoplasm when extranuclear [Ca2+] was elevated (Fig. 8 d). cADP-ribose (5–20 µM), reportedly inducing Ca2+-dependent Ca2+ release from perinuclear stores (Gerasimenko et al. 1995), had a very small effect on Ca2+ release into the nucleoplasm under these experimental conditions (data not shown). These findings are consistent with a growing body of evidence indicating that extranuclear Ca2+ permeates the nuclear membrane mainly via Ca-ATPase–induced Ca2+ accumulation in IP3-gated perinuclear stores (Gerasimenko et al. 1995; Hennager et al. 1995; Malviya and Rogue 1998) and with evidence indicating phosphatidylinositol signaling in the nucleus (Boronenkov et al. 1998).
The release of Ca2+ into the nucleoplasm was prevented by caffeine, added to the crude nuclei at concentrations suppressing IP3-induced Ca2+ mobilization (Ehrlich et al. 1994) (Fig. 8 c). Release of Ca2+ into the nucleoplasm was also prevented in nuclei isolated from neurons pretreated by thapsigargin that inhibits Ca-ATPase activity (Takemura et al. 1989), thereby preventing Ca2+ accumulation in the perinuclear stores (Malviya and Rogue 1998) (Fig. 8 c).
A Fast Activation of PARP by IP3 in Isolated Nuclei of Cortical Neurons.
We next examined the possibility that PARP is polyADP-ribosylated by IP3-induced Ca2+ mobilization. [32P]polyADP-ribosylation of PARP was examined in the presence of IP3 added to nuclei isolated from unstimulated neurons. EDTA was omitted from the incubation solution (see Materials and Methods), to avoid chelation of free Ca2+. In addition, [32P]polyADP-ribosylation was carried out at 25°C to enable detection of fast changes in the activity of PARP. For the same reason, IP3 was added after the addition of [32P]NAD. [32P]polyADP-ribosylated proteins were extracted 1 min after the addition of IP3. IP3 (at concentrations of 50 nM to 5 µM) enhanced the [32P]polyADP-ribosylation of PARP in a dose-dependent manner. Maximal 10-fold enhancement was measured with a half maximal effect induced by 100 ± 30 nM IP3 (Fig. 9 a). At the same concentration range, IP3 displaced specifically bound [3H]IP3 from its receptors in the crude nuclei (IC50 = 30 ± 5 nM; Fig. 9 b).
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Addition of FK-506 (1 µM) altered the dose-dependent effect of IP3 on [32P]polyADP-ribosylation of PARP; the concentration of IP3 required for enhancement of [32P]polyADP-ribosylation in the presence of FK-506 was 10 times lower than that required in untreated nuclei (Fig. 9 d). Hence, the fast polyADP-ribosylation of PARP by IP3 (Fig. 9, a and b), and its modulation by agents affecting IP3-gated Ca2+ release (Fig. 8 c and 9, c and d), strongly suggest that PARP in the isolated nuclei was activated via IP3-induced Ca2+ mobilization.
IP3-induced [32P]polyADP-ribosylation of PARP in isolated nuclei was neither affected by the addition of calmodulin (10–20 µM; Mackrill 1999; data not shown), nor by preventing Ca-calmodulin binding to CAM-kinase II in the presence of saturating amounts (1.5 µM) of the Ca-calmodulin binding peptide on CAM-kinase II (Payne et al. 1988; data not shown). cADP-ribose (5–20 µM) did not alter the basal [32P]poly-ADP-ribosylation of PARP in the crude nuclei (data not shown).
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Discussion |
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High-[K+]–induced membrane depolarization promoted polyADP-ribosylation of nuclear proteins in the absence of extracellular Ca2+ (Fig. 1, Fig. 4 b, and 5). Findings indicating that PARP is activated by intracellular Ca2+ mobilization in the depolarized neurons include: a fast dose-dependent activation of PARP by extranuclear [Ca2+] (Fig. 7, a and b), independent of extracellular Ca2+ influx (Fig. 7 c); and a fast dose-dependent PARP activation by physiological concentrations of IP3 (Fig. 9, a and b), modulated by agents affecting IP3-induced Ca2+ mobilization (Fig. 9c and Fig. d).
IP3-induced Ca2+ release into the nucleoplasm (Fig. 8 c) may underlie the depolarization-induced activation of PARP (Fig. 1 and Fig. 4 b). This is supported by data indicating Ca2+ release into the nucleoplasm of depolarized cortical neurons (Fig. 8 b; Al-Mohanna et al. 1994; Hardingham et al. 1997) and IP3-induced Ca2+ release into the nucleoplasm of cortical neurons (Fig. 8 c), also reported in other cell types (Malviya and Rogue 1998).
An enhanced IP3 production has been measured in depolarized neurons (Gusovsky et al. 1986). It may be attributed to an accelerated phosphoinositide turnover (Gusovsky et al. 1986; Gurwitz and Sokolovsky 1987), as well as to the stimulation of receptor tyrosine kinases (Castren et al. 1992; Huang et al. 1999), or activation of trimeric G-proteins (Banno et al. 1987; Berridge and Irvine 1989; Exton 1990; Sierro et al. 1992; Anis et al. 1999).
The fast polyADP-ribosylation of PARP by IP3 in the isolated nuclei (Fig. 9) is compatible with the time course of Ca2+ release from IP3-gated stores (Ferris and Snyder 1992). Moreover, IP3 stimulated polyADP-ribosylation in the isolated nuclei of cortical neurons (Fig. 9 b) at concentrations compatible with the affinity of IP3-receptors IP3-R1 and IP3-R2 (Mignery et al. 1992; Miyakawa et al. 1999), identified in the brain (Mignery et al. 1992; Ross et al. 1992). IP3-gated Ca2+ stores have been identified in the inner nuclear membrane (Nicotera et al. 1990; Gerasimenko et al. 1995; Malviya and Rogue 1998). Phosphatidylinositol signaling pathways have been identified in the nuclei of several cell types (Boronenkov et al. 1998).
The enhanced activity of PARP in depolarized neurons was independent of extracellular [Ca2+] (see Materials and Methods; Fig. 1, Fig. 4 b, and 5) and resisted Ca2+ influx blockers, including agents suppressing NMDA-induced Ca2+ influx (Fig. 7 c). We therefore consider it unlikely that PARP is activated in depolarized neurons by DNA damage, caused by nitric oxide formation (Zhang et al. 1994; Shah et al. 1996). It is also unlikely that the fast signal–induced activation of PARP was mediated by Ca2+-induced activation of endonucleases, producing DNA breaks (Arends et al. 1990). The activation of Ca,Mg-endonuclease would require extranuclear Ca2+ concentrations 100–1,000-fold higher (Peitsch et al. 1993; Peitsch et al. 1994) than those inducing PARP activation (Fig. 7, a and b). Endonuclease activity at [Ca2+] <1 µM has a much slower time course (>30 min; Jones et al. 1989). Accordingly, DNA breaks or NAD depletion (Satoh and Lindahl 1992) were not detected in the depolarized cortical neurons (Fig. 6 and Fig. 2 a, respectively).
A fast signal–induced PARP activation via IP3-induced Ca2+ mobilization constitutes a novel mode of signaling to the cell nucleus: PARP, being a downstream target of phospholipase C, modulates by polyADP-ribosylation the activity of nuclear proteins in response to signals promoting phosphoinositides turnover and phosphatidyl-inositol 4,5-bisphosphate (PIP2) hydrolysis (Berridge and Irvine 1989; Fruman et al. 1998; Toker 1998). A fast modification of transcription factors by polyADP-ribosylation (Li Oei et al. 1998) during electrical activity in brain cortical neurons may associate depolarization-induced polyADP-ribosylation with "memory storage" (Kandel 1997).
The role of PARP in DNA repair and transcription (Satoh and Lindahl 1992; Oliver et al. 1998; Trucco et al. 1998) may underlie the effect of depolarization in protecting growth factor–deprived neurons from apoptotic cell death (D'Mello et al. 1993; Galli et al. 1995). This mechanism suggests a crucial influence of neuronal activity in preserving the viability of brain cortical neurons, thereby implementing the rule of "use it or lose it."
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Acknowledgments |
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This work was supported by grants that Dr. Cohen-Armon received from the Israel Academy of Science, Adams Super-Center for Brain Research (Tel-Aviv University), and the Israel Ministry of Science.
Submitted: 22 March 2000
Revised: 7 June 2000
Accepted: 8 June 2000
Abbreviations used in this paper: BrdUrd, 5-bromodeoxyuridine; IP3, inositol 1,4,5-triphosphate; PARP, poly(ADP-ribose) polymerase.
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) and depolarized (•) neurons due to increasing extranuclear NAD concentration (n = 6). (b) [32P]polyADP-ribosylation of PARP (2 min, 37°C) was conducted in nuclei isolated from unstimulated (lanes 1–3, 7, 8) and depolarized (lanes 4–6, 9, 10) neurons. (Lanes 2 and 5) Neurons treated by H2O2 (1 mM, 10 min, 25°C). (Lanes 3 and 6) Nuclei treated with DNAse I (20 µg/ml, 2 min, 37°C) during [32P]polyADP-ribosylation. (Lanes 8 and 10) Nuclei treated with DNase I (80 µg/ml, 2 min, 37°C) before [32P]polyADP-ribosylation (n = 4).
). Values are means of seven experiments, expressed for each experiment as a percentage of the maximal enhancement in [32P]polyADP-ribosylation of PARP. (c, top) Autoradiograms of [32P]polyADP-ribosylated PARP in crude nuclei (2 min at 25°C) in the absence (lanes 1, 6, 9–11, and 15) or presence (lanes 2–5, 7, 8, 12–14, and 16) of IP3, BAPTA (lanes 6 and 8), or caffeine (lanes 10, 11, 13, and 14), and in crude nuclei of neurons pretreated with thapsigargin (10 min, 37°C, lanes 15 and 16). Preincubation with BAPTA or caffeine lasted 5 min at 25°C. [32P]polyADP-ribosylated PARP was immunoprecipitated from nuclear proteins extracts by N-20 antibody, subjected to SDS-PAGE, electroblotted (Western blot), autoradiographed, and immunolabeled (bottom) with anti-PARP, Vic-5 antibody (n = 3). (d, top) Autoradiograms of [32P]polyADP-ribosylated PARP (2 min at 25°C) in crude nuclei of cortical neurons in the absence (lanes 1 and 8) and presence (lanes 2–7 and 9–13) of IP3 or FK-506 (lanes 8–13). Since FK-506 was dissolved in ethanol, all the samples contained 0.03% ethanol. [32P]polyADP-ribosylated PARP was immunoprecipitated from nuclear protein extracts by N-20 antibody, subjected to SDS-PAGE, electroblotted (Western blots), autoradiographed, and immunolabeled (bottom) with anti-PARP Vic-5 antibody (n = 3).