Dissociation of Cohesin from Chromosome Arms and Loss of Arm Cohesion during Early Mitosis Depends on Phosphorylation of SA2

Abstract

Cohesin is a protein complex that is required to hold sister chromatids together. Cleavage of the Scc1 subunit of cohesin by the protease separase releases the complex from chromosomes and thereby enables the separation of sister chromatids in anaphase. In vertebrate cells, the bulk of cohesin dissociates from chromosome arms already during prophase and prometaphase without cleavage of Scc1. Polo-like kinase 1 (Plk1) and Aurora-B are required for this dissociation process, and Plk1 can phosphorylate the cohesin subunits Scc1 and SA2 in vitro, consistent with the possibility that cohesin phosphorylation by Plk1 triggers the dissociation of cohesin from chromosome arms. However, this hypothesis has not been tested yet, and in budding yeast it has been found that phosphorylation of Scc1 by the Polo-like kinase Cdc5 enhances the cleavability of cohesin, but does not lead to separase-independent dissociation of cohesin from chromosomes. To address the functional significance of cohesin phosphorylation in human cells, we have searched for phosphorylation sites on all four subunits of cohesin by mass spectrometry. We have identified numerous mitosis-specific sites on Scc1 and SA2, mutated them, and expressed nonphosphorylatable forms of both proteins stably at physiological levels in human cells. The analysis of these cells lines, in conjunction with biochemical experiments in vitro, indicate that Scc1 phosphorylation is dispensable for cohesin dissociation from chromosomes in early mitosis but enhances the cleavability of Scc1 by separase. In contrast, our data reveal that phosphorylation of SA2 is essential for cohesin dissociation during prophase and prometaphase, but is not required for cohesin cleavage by separase. The similarity of the phenotype obtained after expression of nonphosphorylatable SA2 in human cells to that seen after the depletion of Plk1 suggests that SA2 is the critical target of Plk1 in the cohesin dissociation pathway.

Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome

“…Immunofluorescence Microscopy-After harvesting and washing with PBS, cells were cytospun (Thermo Fisher Scientific, Shandon Brand) and fixed onto microscopy slides (12). The nuclear envelopes of the cells were stained with a Lamin A antibody and DNA was counterstained with 4Ј-6-Diamidino-2-phenylindole (DAPI, Molecular Probes, Invitrogen, UK).…”

“…Using 250 nM of BI 4834 we tested five different treatment durations of 15, 30, 45, 60, and 120 min. To measure PLK1 inhibition and subsequent dephosphorylation of PLK1 substrates we analyzed cell lysates by immunoblotting, using phospho-specific antibodies that recognize a PLK1-dependent phosphorylation site (S1224ph) on the cohesin subunit STAG2 (12,31). This phosphoepitope became undetectable after 15 min of BI 4834 treatment (Fig.…”

“…In prometaphase, PLK1 is required for the generation of stable kinetochoremicrotubule attachments (7)(8)(9)(10). PLK1 also promotes dissociation of cohesin from chromosome arms in prophase and prometaphase by phosphorylating cohesin's STAG2 subunit (11)(12)(13)(14), as well as multiple aspects of cytokinesis by phosphorylating activators and effectors of RhoA (1,15).…”

Shugoshin regulates cohesion by driving relocalization of PP2A in Xenopus extracts

“…This population is essential in preventing precocious separation of the sister chromatids and allowing proper chromosome alignment (Vass et al 2003;Vagnarelli et al 2004;. The dissociation of cohesin in prophase requires Polo and Aurora B kinases as well as a number of additional factors that include Wapl Sumara et al 2002;Hauf et al 2005;Gandhi et al 2006;Kueng et al 2006). At the onset of anaphase, cleavage of Scc1/Rad21 by separase drives dissociation of the remaining cohesin and sister chromatid separation ensues (Hauf et al 2001).…”

C-terminus of Sororin interacts with SA2 and regulates sister chromatid cohesion

“…Plk1 is required for the non-proteolytic arm cohesin removal via SA2 phosphorylation in prophase. 19,[23][24][25][26] Our published results suggest that after the Sororin ST 159 P motif is phosphorylated by Cdk1/cyclin B (becomes SpT 159 P) at prophase/prometaphase, Sororin serves as a docking protein for Plk1 to bind to the SpT 159 P motif so that the enzymatic catalytic domain of Plk1 is brought proximately to its substrate SA2 to phosphorylate. 3,15 Sororin 7 Wapl can remove cohesin from chromatin when it binds to Pds5 via FGF motif.…”