Regulation of poly(ADP-ribose) polymerase 1 activity by the phosphorylation state of the nuclear NAD biosynthetic enzyme NMN adenylyl transferase 1

Abstract

Nuclear NAD ؉ metabolism constitutes a major component of signaling pathways. It includes NAD ؉ -dependent protein deacetylation by members of the Sir2 family and protein modification by poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 has emerged as an important mediator of processes involving DNA rearrangements. High-affinity binding to breaks in DNA activates PARP-1, which attaches poly(ADP-ribose) (PAR) to target proteins. NMN adenylyl transferases (NMNATs) catalyze the final step of NAD ؉ biosynthesis. We report here that the nuclear isoform NMNAT-1 stimulates PARP-1 activity and binds to PAR. Its overexpression in HeLa cells promotes the relocation of apoptosis-inducing factor from the mitochondria to the nucleus, a process known to depend on poly(ADP-ribosyl)ation. Moreover, NMNAT-1 is subject to phosphorylation by protein kinase C, resulting in reduced binding to PAR. Mimicking phosphorylation, substitution of the target serine residue by aspartate precludes PAR binding and stimulation of PARP-1. We conclude that, depending on its state of phosphorylation, NMNAT-1 binds to activated, automodifying PARP-1 and thereby amplifies poly(ADP-ribosyl)ation.apoptosis ͉ NAD biosynthesis ͉ poly(ADP-ribosyl)ation ͉ protein phosphorylation N MN adenylyl transferase (NMNAT) is an essential enzyme because it catalyzes the final step of NAD ϩ biosynthesis (1). There are three isoforms in humans that exhibit tissue-and organelle-specific expression (2). Because the biological role of NAD ϩ and NADH (collectively termed NAD) has long been thought to be confined to their function as electron carriers, the nuclear localization of the major isoforms, human NMNAT-1 (3) and yeast Nma2 (4), was somewhat puzzling. However, recent research has revealed many important regulatory functions of the pyridine nucleotides, some of which take place within the nucleus (5).NAD ϩ serves as substrate for covalent protein modifications such as mono(ADP-ribosyl)ation (6), poly(ADP-ribosyl)ation (7,8), and protein deacetylation by sirtuins, proteins of the silent information regulator 2 (Sir2) family (9). Pyridine nucleotides are also direct precursors of two calcium-mobilizing messengers, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate (NAADP ϩ ) (10 -13). The predominant NAD ϩ -consuming activity, poly(ADP-ribosyl)ation by poly(ADPribose) polymerase 1 (PARP-1), occurs within the nucleus, as does NAD ϩ -dependent protein deacetylation by sirtuins. Consequently, substrate supply by NMNAT-1 may directly influence these processes. Overexpression of NMNAT-1 extends the lifespan of eukaryotic cells, which has been attributed to augmented NAD ϩ -dependent histone deacetylation catalyzed by Sir2p or its homologs (4,(14)(15)(16)(17)(18). Increased NMNAT activity is also required for the axon-sparing activity of the chimeric slow Wallerian degeneration (Wld s ) protein, which is composed of 19 amino acids of the ubiquitin assembly protein Ufd2a and full-length NMNAT-1 (19).Although suggested previously (3, 20, 21), a functional intera...

Regulation of Poly(ADP-ribose) Polymerase-1-dependent Gene Expression through Promoter-directed Recruitment of a Nuclear NAD+ Synthase

“…can bind to automodified PARP-1 and stimulate its enzymatic activity (20). Our results agree with and further extend this conclusion with the observation that both PAR chain elongation and PAR synthesis activities of PARP-1 are allosterically regulated by NMNAT-1.…”

“…Based on the functional interactions that we observed between NMNAT-1 and PARP-1 in our in vitro assays (Fig. 1), as well as a previously published report (20), we decided to investigate the possibility that NMNAT-1 might interact with PARP-1 at target gene promoters to regulate its activity in cells. To this end, we used ChIP assays to examine binding of endogenous PARP-1 and ectopically expressed FLAG-tagged NMNAT-1 to target gene promoters.…”

“…We have recently shown that NMNAT-1 is recruited to target gene promoters through interactions with promoter-bound SIRT1, where it produces NAD ϩ that is used by SIRT1 to support its histone deacetylase activity (19). Furthermore, biochemical studies have shown that NMNAT-1 binds to automodified PARP-1 and stimulates its enzymatic activity in a phosphorylation-dependent manner (20), suggesting a signal-regulated role of NMNAT-1 in controlling PARP-1 function as well.…”

“…ϩ Production-Previous studies have shown that NMNAT-1 interacts with automodified PARP-1 and allosterically stimulates its enzymatic activity (20). To determine whether functional interplay between NMNAT-1 and PARP-1 occurs in our assay system, we carried out PARP-1 automodification reactions using [ 32 P]NAD ϩ for isotopic labeling.…”

“…NMNAT-1 may also affect the gene regulatory activity of PARP-1 in a manner that is independent of the enzymatic activity of both PARP-1 and NMNAT-1. The functional interactions of PARP-1 and NMNAT-1 with chromatin may be modulated as end points of cellular signaling pathways ("Signals") (20). P, PARP-1.…”