PIASy mediates SUMO-2/3 conjugation of poly(ADP-ribose) polymerase 1 (PARP1) on mitotic chromosomes.
PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins.
Ryu
Hyunju
H
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Al-Ani
Gada
G
Deckert
Katelyn
K
Kirkpatrick
Donald
D
Gygi
Steven P
SP
Dasso
Mary
M
Azuma
Yoshiaki
Y
D000818:Animals; D002843:Chromatin / Q000235:genetics; D002875:Chromosomes / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D008938:Mitosis; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D050828:Protein Inhibitors of Activated STAT / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D012097:Repressor Proteins / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism*
D002843:Chromatin; C495202:PIAS4 protein, Xenopus; D050828:Protein Inhibitors of Activated STAT; D011994:Recombinant Proteins; D012097:Repressor Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D029867:Xenopus Proteins; D011065:Poly(ADP-ribose) Polymerases