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Aurora B and Cdk1 Mediate Wapl Activation and Release of Acetylated Cohesin from Chromosomes by Phosphorylating Sororin

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Aurora B and Cdk1 Mediate Wapl Activation and Release of Acetylated Cohesin from Chromosomes by Phosphorylating Sororin Aurora B and Cdk1 mediate Wapl activation and release of acetylated cohesin from chromosomes by phosphorylating Sororin Tomoko Nishiyamaa,b,1, Martina M. Sykorab,2, Pim J. Huis in ’t Velda, Karl Mechtlera,c, and Jan-Michael Petersa,1 aResearch Institute of Molecular Pathology, A-1030 Vienna, Austria; bDivision of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan; and cInstitute of Molecular Biotechnology of the Austrian Academy of Science, A-1030 Vienna, Austria Edited by Douglas Koshland, University of California, Berkeley, CA, and approved July 9, 2013 (received for review March 19, 2013) Sister chromatid cohesion depends on Sororin, a protein that 19). A critical substrate of Plk1 in the prophase pathway is SA2, stabilizes acetylated cohesin complexes on DNA by antagonizing one of several SA/STAG paralogs, as nonphosphorylatable the cohesin release factor Wings-apart like protein (Wapl). Co- SA212A reduces cohesin dissociation from chromosome arms in hesion is essential for chromosome biorientation but has to be prometaphase (20). At centromeres, cohesin is protected from dissolved to enable sister chromatid separation. To achieve this, the prophase pathway by the MEI-S332/complex (21–24), which the majority of cohesin is removed from chromosome arms in dephosphorylates SA2 in vitro (21). SA212A suppresses the Sgo1- prophase and prometaphase in a manner that depends on Wapl depletion phenotype (25), suggesting that one key function of and phosphorylation of cohesin’s subunit stromal antigen 2 Sgo1–PP2A is to protect SA2 from phosphorylation. (SA2), whereas centromeric cohesin is cleaved in metaphase by Previous work had shown that Sororin is phosphorylated in the protease separase. Here we show that the mitotic kinases mitosis (10), that this phosphorylation prevents binding of Sororin Aurora B and Cyclin-dependent kinase 1 (Cdk1) destabilize inter- to Pds5A in vitro (2), that the phosphorylation of Sororin on actions between Sororin and the cohesin subunit precocious dis- putative Cdk1 sites is required for its dissociation from chro- sociation of sisters protein 5 (Pds5) by phosphorylating Sororin, matin and partial loss of cohesion (26), and that alanine muta- leading to release of acetylated cohesin from chromosome arms tion of these sites bypasses the requirement for Sgo1 in cohesion CELL BIOLOGY and loss of cohesion. At centromeres, the cohesin protector shu- (27). It has also been proposed that Sororin phosphorylation by goshin (Sgo1)-protein phosphatase 2A (PP2A) antagonizes Aurora Cdk1 enables Plk1 binding to Sororin, leading to SA2 phos- B and Cdk1 partly by dephosphorylating Sororin and thus main- phorylation by Plk1 (28). However, it remains unknown on which tains cohesion until metaphase. We propose that the stepwise loss sites Sororin is phosphorylated in vivo, by which mechanism of cohesion between chromosome arms and centromeres is caused Sororin phosphorylation enables the dissociation of cohesin from by local regulation of Wapl activity, which is controlled by the mitotic chromosomes, and if Sororin and SA2 phosphorylation phosphorylation state of Sororin. contribute to cohesin dissociation through the same mechanism. Here, we report the in vivo phosphosites on Sororin in human cell cycle | chromosome segregation | prophase pathway mitotic cells, and show that both Aurora B and Cdk1 are re- quired for the generation of these sites, for dissociation of eplicated DNA molecules (sister chromatids) remain physi- Sororin from cohesin and chromatin by releasing Sororin from Rcally connected with each other from S phase until meta- Pds5, and for release of acetylated “cohesive” cohesin from phase to enable biorientation of chromosomes on the mitotic mitotic chromosomes. We further show that Sgo1–PP2A pro- spindle. This association, called sister chromatid cohesion, is me- tects cohesin at centromeres in part by maintaining Sororin in diated by cohesin, whose core subunits Structural maintenance of a dephosphorylated state. In addition, we provide evidence that chromosomes 1 (Smc1), Smc3, Sister chromatid cohesion 1 (Scc1), SA2 phosphorylation by Plk1 and Sororin phosphorylation by and Scc3/stromal antigen (SA/STAG) form a molecular ring. This Aurora B and Cdk1 contribute to cohesin dissociation through structure is crucial for cohesion, presumably because cohesin to- distinct mechanisms. pologically embraces the two sister chromatids (reviewed in ref. 1). Results The core subunits, in turn, recruit the cohesin-binding proteins Identification of Mitotic Phosphorylation Sites on Sororin. Sororin Pds5, Wapl, and Sororin (1–3). Cohesion is established during DNA replication in S phase can be phosphorylated by Cdk1 in vitro but the sites on which Sororin is phosphorylated in vivo have so far only been inferred and depends on Smc3 acetylation (4–6) by the acetyltransferases from Sororin phosphopeptides found in large-scale proteomic Esco1 and Esco2 (2). This modification leads to recruitment of studies and from bioinformatic predictions of Cdk1 sites (26, Sororin, which stabilizes cohesin on DNA by inactivating the 28). We, therefore, performed a systematic and unbiased mass cohesin release factor Wapl (2, 7–11). Wapl binds to Pds5 pro- spectrometric analysis of Sororin’s phosphosites. We used HeLa teins (8) (Pds5A/B in vertebrates) (12) and, in the absence of cells stably expressing mouse Sororin with a localization and Sororin, the resulting Wapl–Pds5 heterodimers dissociate cohe- sin from chromatin (3). Sororin directly associates with Pds5 proteins and thereby dissociates Wapl from Pds5 in vitro (2), Author contributions: T.N. designed research; T.N. and M.M.S. performed research; T.N. implying that Sororin antagonizes Wapl by changing its in- and P.J.H.i.t.V. contributed new reagents/analytic tools; T.N., K.M., and J.-M.P. analyzed teraction with Pds5. data; and T.N. and J.-M.P. wrote the paper. In higher eukaryotes, cohesin is released from mitotic chro- The authors declare no conflict of interest. mosomes in two steps (13). Most cohesin complexes are removed This article is a PNAS Direct Submission. from chromosome arms in prophase and prometaphase via the 1 “ ” To whom correspondence may be addressed. E-mail: [email protected] or so-called prophase pathway (13, 14). The remaining cohesin, [email protected]. predominantly located at centromeres, is cleaved by the protease 2Present address: Boehringer Ingelheim RCV GmbH & Co KG, NBE Discovery, A-1120 Vienna, separase in metaphase, leading to loss of cohesion and anaphase Austria. onset (15). The prophase pathway depends on Wapl (7, 8) and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the mitotic kinases Polo-like kinase (Plk1) and Aurora B (16– 1073/pnas.1305020110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1305020110 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 affinity purification (LAP) tag at its carboxy teminus. In these A sup. beads B sup. beads “Sororin-LAP cells,” Sororin is expressed at near-endogenous wt 11A wt 11A input IMIM IMIM levels from its own promoter and is regulated in a cell-cycle– bufferCdk1bufferCdk1 wt 11A Wapl IMIM bufferCdk1 Wapl dependent manner like endogenous Sororin (2). These cells were Sororin Sororin Sororin p-Sororin synchronized in prometaphase with nocodazole, or in S/G2 Sororin Pds5A phase by thymidine release, and Sororin isolated by affinity pu- (autoradiograph) Pds5A rification was analyzed by mass spectrometry (MS). We identi- C extracts sup. beads fied 17 phosphosites in mouse Sororin, of which 15 were mitosis I M M-he I M M-he M-he I M specific. Of these, 11 and 9 sites are conserved in human and Sororin Wapl Xenopus Sororin, respectively (Fig. S1A). Only 5 of the 15 sites pH3S10 Sororin identified in our experiments were among the 9 putative Cdk1 H1K act. Pds5A sites that had been mutated in previous studies (26, 27). Next we tested if these phosphosites could be generated by Fig. 2. Aurora B and Cdk1 attenuate Wapl removal activity of Sororin. (A) Recombinant SororinWT or Sororin11A proteins were preincubated in either Cdk1, Aurora B, or Plk1. Human Sororin expressed in Esch- erichia coli interphase (I) or mitotic (M) egg extracts (Left). Anti-Pds5A antibody beads was incubated with ATP plus each of these kinases bound to Wapl–Pds5A were incubated with the I- or M-SororinWT/11A. Bead- and subsequently analyzed by MS. We found that all of the 11 bound proteins (beads) were separated from the supernatant (sup.) (Right). residues conserved between mouse and human Sororin, except (B) In vitro Cdk1-dependent phosphorylation is sufficient to inactivate the Ser181, could be phosphorylated by either Cdk1 or Aurora B Wapl removal activity of Sororin. SororinWT protein was incubated with ei- (Fig. 1). Plk1 also phosphorylated several sites on Sororin, but ther buffer or Cdk1 and purified (Left), and then mixed with anti-Pds5A – each of these could also be generated by Cdk1 or Aurora B (Fig. antibody beads bound to Wapl Pds5A. Bead-bound proteins (Right) were separated from the supernatant (Center). (C) Aurora B-dependent phos- 1), indicating redundancy in substrate targeting by these kinases, fi WT fi phorylation weakens the binding af nity of Sororin to Pds5. Sororin and that Cdk1 and Aurora B might be suf cient for mitotic protein was incubated with either I- or M extracts in the presence or absence Sororin phosphorylation. of Hesperadin (he). The extracts were analyzed by immunoblotting and histone H1 kinase activity assay (H1K act.). Subsequently, Sororin was puri- The Wapl Removal Activity of Sororin Is Attenuated by Aurora B- and fied from I- or M extracts, then mixed with anti-Pds5A antibody beads bound Cdk1-Dependent Phosphorylation. We showed previously that to Wapl–Pds5A. Bead-bound proteins were separated from the supernatant binding of Sororin to Pds5A dissociates Wapl from Pds5A in (Right). vitro, and that this Wapl removal activity is reduced by phos- phorylation of Sororin (2). To test if the mitotic phosphosites Next we asked which kinase is required for the inactivation of identified here are responsible for the attenuation of Sororin’s Sororin.
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