A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis.

Yumi Ueki,A Arockia Jeyaprakash,Paula Sotelo‐Parrilla,Melanie B Weisser,Arminja Kettenbach,Tanmay Gupta,Emil P T Hertz,Isha Nasa,Guillermo Montoya,Susanne M A Lens,Michael A Hadders,Jakob Nilsson,Thomas Kruse,Lauren E Cressey

doi:10.15252/embr.202052295

Yumi Ueki, A Arockia Jeyaprakash + Show 12 more

Open Access

https://doi.org/10.15252/embr.202052295

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Abstract

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A‐B56 phosphatase through a coiled‐coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A‐B56‐hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A‐B56 substrates containing a canonical B56 binding motif. We find that PP2A‐B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis.

Highlights

The shugoshin proteins (hSgo1 (Sgol1) and hSgo2 (Sgol2) in humans) are conserved protectors of centromeric cohesion by preventing premature release of the cohesin complex (Marston, 2015)
Subsequent genetic screens identified the shugoshin proteins in yeast (Katis et al, 2004; Kitajima et al, 2004; Marston et al, 2004). Common to these proteins is the presence of an N-terminal coiled coil (CC) region that binds to B56 regulatory subunits, hereby localizing PP2A-B56 to centromeres (Kitajima et al, 2006; Riedel et al, 2006; Tang et al, 2006; Xu et al, 2009)
These results suggest that hSgo1 might engage the conserved LxxIxE binding pocket of B56a for binding in cells. To test this with purified components, we reconstituted a PP2AB56c-BubR1516-715 complex and isolated the complex by size exclusion chromatography. We incubated this complex with fivefold excess recombinant hSgo11–155 and following incubation characterized the complexes by size exclusion chromatography (Fig 1F)

Summary

Introduction

The shugoshin proteins (hSgo1 (Sgol1) and hSgo2 (Sgol2) in humans) are conserved protectors of centromeric cohesion by preventing premature release of the cohesin complex (Marston, 2015). G YFP-B56a pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56a and subsequent immunoblotting of indicated proteins. We first determined whether hSgo1 can bind to PP2A-B56 in complex with LxxIxE containing proteins.

Results

Conclusion