Modification of the ADP-ribosyltransferase and NAD glycohydrolase activities of a mammalian transferase (ADP-ribosyltransferase 5) by auto-ADP-ribosylation.
Mono-ADP-ribosylation, a post-translational modification in which the ADP-ribose moiety of NAD is transferred to an acceptor protein, is catalyzed by a family of amino acid-specific ADP-ribosyltransferases. ADP-ribosyltransferase 5 (ART5), a murine transferase originally isolated from Yac-1 lymphoma cells, differed in properties from previously identified eukaryotic transferases in that it exhibited significant NAD glycohydrolase (NADase) activity. To investigate the mechanism of regulation of transferase and NADase activities, ART5 was synthesized as a FLAG fusion protein in Escherichia coli. Agmatine was used as the ADP-ribose acceptor to quantify transferase activity. ART5 was found to be primarily an NADase at 10 microM NAD, whereas at higher NAD concentrations (1 mM), after some delay, transferase activity increased, whereas NADase activity fell. This change in catalytic activity was correlated with auto-ADP-ribosylation and occurred in a time- and NAD concentration-dependent manner. Based on the change in mobility of auto-ADP-ribosylated ART5 by SDS-polyacrylamide gel electrophoresis, the modification appeared to be stoichiometric and resulted in the addition of at least two ADP-ribose moieties. Auto-ADP-ribosylated ART5 isolated after incubation with NAD was primarily a transferase. These findings suggest that auto-ADP-ribosylation of ART5 was stoichiometric, resulted in at least two modifications and converted ART5 from an NADase to a transferase, and could be one mechanism for regulating enzyme activity.
Weng
B
B
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1434, USA. wengb@gwgate.nhlbi.nih.gov
Thompson
W C
WC
Kim
H J
HJ
Levine
R L
RL
Moss
J
J
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D009842:Oligopeptides; D010430:Pentosyltransferases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011817:Rabbits
D009842:Oligopeptides; D010455:Peptides; D009243:NAD; D000246:Adenosine Diphosphate Ribose; C085706:FLAG peptide; D036002:ADP Ribose Transferases; D010430:Pentosyltransferases; D011065:Poly(ADP-ribose) Polymerases; C400745:ART5 protein, mouse; D009244:NAD+ Nucleosidase