Abstract
Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres.
Highlights
Mitotic SUMOylation is critical for faithful chromosome segregation in eukaryotes
In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation
Either SUMOylated or non-SUMOylated PARP1 was captured on agarose beads, and the beads were incubated with Xenopus egg extract (XEE) to isolate binding proteins
Summary
Mitotic SUMOylation is critical for faithful chromosome segregation in eukaryotes. Results: PICH (Plk1-interacting checkpoint helicase) is a SUMO-interacting protein and a mitotic SUMO substrate. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. Using the XEE assays, we have previously identified two major PIASy-dependent mitotic chromosomal SUMO2/3 substrates: DNA topoisomerase II␣ (TopoII␣) and poly(ADP-ribose) polymerase 1 (PARP1) [11, 12]. Accumulating evidence indicates that SUMOylation is important for the regulation of TopoII␣ activity [14, 15] Another robust mitotic SUMOylation substrate, PARP1 [12], is a member of the PARP family that catalyzes the formation of poly(ADP-ribose) on target proteins, leading to multifaceted biological consequences [16]. We propose a novel regulation of PICH function at mitotic centromeres by SUMOylation
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