Division of labour between different PP2A-B56 complexes during mitosis

Abstract

DD1 9SY *correspondence: a.saurin@dundee.ac.uk PP2A-B56 is a serine/threonine phosphatase complex that regulates several major mitotic processes, including sister chromatid cohesion, kinetochore-microtubule attachment and the spindle assembly checkpoint. We show here that these key functions are shared between B56 isoforms which localise differentially to either the centromere or kinetochore. The centromeric B56 isoforms rely on a specific interaction with Sgo2, whereas the kinetochore isoforms bind preferentially to BubR1 and other proteins containing an LxxIxE motif. The molecular basis for this differential localisation can be mapped to a small C-terminal region in B56 that regulates these key interactions and defines whether PP2A-B56 complexes bind to either the centromere or kinetochore. Together, this study describes how different PP2A-B56 complexes utilise isoform-specific interactions to control distinct processes during mitosis.

PP1 and PP2A use opposite phospho-dependencies to control distinct processes at the kinetochore

“…pcDNA5-YFP-B561-Knl1 ΔNT (Knl1 ΔNT fused to B561) and pcDNA5-YFP-B56 1 CD -Knl1 ΔNT (Knl1 ΔNT fused to a version of B561 with a S296D mutation that disrupts PP2A binding (Vallardi et al, 2018)) were produced by restriction cloning using fragments generated by PCR from pcDNA5-YFP-B561 and pcDNA5-YFP-B561 CD , respectively (Vallardi et al, 2018), both were inserted using NotI and KasI restriction sites creating a 28 amino acid linker before Knl1 ΔNT . To create pcDNA5-YFP-Knl1 ΔNT-PP1mid a fragment containing the PP1-binding SILK and RVSF motifs (amino acids 24 to 70 of Knl1 WT ) was inserted at a BlpI site of pcDNA5-YFP-Knl1 ΔNT by Gibson assembly (between MELT-10 and MELT-11).…”

“…pcDNA5-YFP-BubR1 ΔCT -B561 was also subcloned with the same strategy but using a fragment containing B56 amplified from pcDNA5-YFP-B561 (Vallardi et al, 2018), inserting B561 and a 7 amino acid linker after amino acid 658 of BubR1.…”

“…In the same way, pcDNA4-Turq2-BubR1 Long was created by restriction cloning using BstBI and Bsu36I, and pcDNA4-Turq2 versions of BubR1 ΔCT-PP1:Knl1 and BubR1 ΔCT-Knl1 were created by restriction cloning using BstBI and NotI. pMESVΨ-mCherry-B561 was produced by Gibson assembly of pMESVΨ-mCherry backbone (amplified from pMESVΨ-mCherry-CenpB-Mad1 ) and B561 (amplified from pcDNA5-YFP-B561) (Vallardi et al, 2018). All plasmids were fully sequenced to verify the transgene was correct.…”

“…fig.2a). This requires PP2A catalytic activity, because fusion of a B56 mutant that cannot bind the catalytic domain (B56 CD -Knl1 NT (Vallardi et al, 2018)) Figure 1. PP1-Knl1 and PP2A-B56 exert control over different pathways and processes at the kinetochore.…”

Phosphatases in Mitosis: Roles and Regulation

“…Peptide phage display of intrinsically disordered regions of the human proteome identified 126 ligands of B56 and confirmed substantial specificity overlap between PP2A-B56α and PP2A-B56γ holoenzymes as expected from the highly conserved LxxIxE binding surface in all B56 isoforms [68]. Nevertheless, B56 isoforms were recently shown to bind differentially to LxxIxE motifs on mitotic proteins [69]. This apparent selectivity is proposed to allow B56α and B56γ to localize at discrete subcellular compartments and control separate mitotic processes.…”