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.

GTP-binding protein(s) recognized by antibodies against the ␣-subunits of G i -and G o -proteins were detected in crude nuclei isolated from rat brain stem and cortex. Immunohistochemical staining indicated that in the cortex these proteins are perinuclear, or are embedded in the nuclear membrane. Evidence is presented for an endogenous ADP-ribosylation of these proteins, which competes with their PTX-catalyzed ADP-ribosylation. The endogenous reaction has the characteristics of nonenzymatic ADP-ribosylation of cysteine residues, known to involve NAD-glycohydrolase activity. In vitro experiments showed that the ␣-subunit of G o -proteins in the cell membrane also acts as a substrate of this endogenous ADP-ribosylation. The in situ effect of membrane depolarization on the nuclear GTP-binding proteins may be attributable to their depolarization-induced endogenous ADP-ribosylation, suggesting a novel signaling mechanism in neuronal cells in the central nervous system. NAD is the substrate for enzymes that catalyze ADP-ribosylation, i.e. cleavage of the bond between nicotinamide and ribose and the transfer of ADP-ribose to nucleophilic acceptors (1). ADP-ribosylation represents a mechanism for post-translational modification of proteins. Among the known acceptors for ADP-ribose are GTP-binding proteins in cell membranes (2). ADP-ribosyltransferases that catalyze ADP-ribosylation of arginine residues in GTP-binding proteins (similar to cholera toxin) (2) have been detected in eukaryotic cells (3). An endogenous ADP-ribosylation of cysteine residues of membrane Gproteins (similar to pertussis toxin (PTX) 1 -catalyzed ADP-ribosylation) (2) has been suggested to occur in erythrocytes (4,5).In this report we present evidence for an endogenous ADPribosylation of cysteine residues in GTP-binding protein(s) in the nuclei of cells in rat brain stem and cortex. These proteins also act as substrates of PTX-catalyzed ADP-ribosylation and react with antibodies against the ␣-subunits of G i -and G oproteins (G␣ i -and G␣ o -proteins). Unlike these membrane Gproteins, however, the nuclear GTP-binding proteins are apparently modified by a nonenzymatic ADP-ribosylation of cysteine residues, which competes with their PTX-catalyzed ADPribosylation. Also, unlike membrane G-proteins, the nuclear GTP-binding proteins are not extractable by detergents, nor are they activated by membrane depolarization (6 -9). The evidence presented here is consistent with a depolarization-induced ADP-ribosylation of these nuclear GTP-binding proteins, suggesting a novel signaling mechanism in neuronal cells in the central nervous system. MATERIALS AND METHODSReagents-Nicotinamide-adenine dinucleotide (grade I), adenosine triphosphate (grade I), GDP␤S, GTP␥S, tetrodotoxin, dithiothreitol (DTT), azidoaniline, sodium nitroprusside, 3-aminobenzamide (3-AB), and thymine were all purchased from Sigma. PTX and the A-protomer of PTX (ADP-ribosyltransferase) were purchased from List Biological Laboratories. [ 14 C]NAD (57 mCi/mmol) was purchased from Amersham Corp...