SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis

Abstract

Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMOinteracting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the Cterminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres.

PICH promotes SUMOylated TopoisomeraseIIα dissociation from mitotic centromeres for proper chromosome segregation

“…Intriguingly, the translocase deficient PICH mutant further 293 suppressed the SUMOylated TopoIIα decatenation activity (Figure 7B,D). This suggests that the 294 translocase deficient mutant forms a stable complex with SUMOylated TopoIIα and the catenated kDNA 295 because the translocase mutant retains its DNA binding ability (Kaulich et al, 2012, Nielsen et al, 2015 296 Sridharan and Azuma, 2016). Notably, neither mutant showed any significant effect on non-SUMOylated 297 TopoIIα ( Figure 7B) similar to wild-type PICH.…”

“…Because the translocase activity of PICH removes proteins from DNA, PICH inhibits decatenation 283 activity of SUMOylated TopoIIα by removing SUMOylated TopoIIα from kDNA. To gain insight into that 284 potential mechanism, we utilized a PICH mutant that has defects in either the SUMO-binding activity 285 or in translocase activity (PICH-K128A) ( Figure 7A) (Sridharan et al, 2016). If 286 PICH/SUMO interaction is critical for inhibiting decatenation activity of SUMOylated TopoIIα, the PICH-287 d3SIM mutant would lose its inhibitory function.…”

“…We previously showed that PICH interacts with 349 SUMOylated PARP1 as well as a tetrameric SUMO chain, suggesting that PICH promiscuously binds 350 SUMOylated proteins . Both loss of translocase activity and SUMO-binding activity 351 of PICH leads to chromosome bridge formation (Sridharan and Azuma, 2016) which could derive from the 352 increased incidence of UFBs due to stalled SUMOylated TopoIIα. However, other SUMO2/3 modified 353 chromosomal proteins remodeled by PICH might contribute to chromosome bridge formation in loss of 354 PICH cells (Baumann et al, 2007, Kurasawa and Yu-Lee, 2010, Nielsen et al, 2015.…”

“…Recombinant TopoIIα and PICH proteins were prepared as previously described (Ryu et al, 2010b, 406 Sridharan and Azuma, 2016). In brief, the pPIC 3. dnUbc9, and SUMO paralogues were expressed in Rosetta2(DE3) and purified as described previously 423 (Ryu et al, 2010a).…”

Resolving Chromatin Bridges With SIMs, SUMOs and PICH

“…A recent study from Sridharan and Azuma provides new insights by identifying small ubiquitin-related modifier (SUMO)-interacting motifs (SIMs) in PICH that are essential for both its localization and function in preventing chromatin bridges. 4 SUMOs function as posttranslational protein modifications and are essential during mitosis. Sumoylation regulates the localization and activities of a variety of centromere and kinetochore-associated proteins, including CENP-E and Topoisomerase IIa (TopoIIa).…”