Localization of PP2A-B56 to centromeres in Drosophila

KEYWORDS:

• B56
• Cohesin
• Cohesion
• Dalmatian
• Drosophila
• LxxIxE
• PP2A
• Shugoshin
• Sororin
• Wdb

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Protection of chromosome cohesion through Shugoshin-dependent recruitment of the PP2A-B56 phosphatase to the pericentromeric region has become a cornerstone of the chromosome segregation model in eukaryotes. Shugoshin is essential for meiotic chromosome segregation in all tested eukaryotes but only found to be essential for mitotic chromosome segregation in vertebrates. Nishiyama and colleagues have now found that the protein Dalmatian, an ortholog of the vertebrate cohesion regulator Sororin, performs the function of Shugoshin in Drosophila melanogaster by recruiting PP2A-B56 to the pericentromeric region supporting an unified model of mitotic chromosome segregation in the animal kingdom. Here we speculate how Dalmatian might bind to PP2A-B56.

Accurate segregation of chromosomes is ensured by centromeric cohesion between sister chromatids until anaphase onset.¹ Sister chromatid cohesion is established during S phase when replicated chromatids become entrapped by the cohesin ring complex. In the vertebrate mitosis, cohesin is removed in 2 steps. In the first step, most cohesin is removed along chromosome arms during prophase and prometaphase. This is driven by Wapl, which is believed to catalyze opening of the cohesin ring and thus release of entrapped chromatids. Before mitosis Wapl activity is antagonized by Sororin, which prevents Wapl binding to the cohesin complex. During mitosis phosphorylation of Sororin and the cohesin complex by mitotic kinases excludes Sororin from the cohesin complex allowing Wapl activity. Importantly, Sororin activity remains in the pericentromeric region due to recruitment of the PP2A-B56 phosphatase by the centromeric protein Shugoshin. In the second step of cohesin removal, Separase cleaves the kleisin subunit of the cohesin complex once the Separase inhibitor Securin is degraded at the onset of anaphase.

The role of Shugoshin and Sororin in the mitosis of invertebrates has remained less clear. Nishiyama and colleagues² find that Dalmatian protects centromeric cohesion in mitosis through recruitment of the PP2A-B56 phosphatase to the pericentromeric region analogous to the function of Shugoshin in vertebrates. The authors identify a region of Dalmatian between residues 275–299 as necessary for binding to the Drosophila ortholog of B56 by truncation analysis and speculate that the binding mechanism of Dalmatian to PP2A-B56 might be related to that of Shugoshin.

The PP2A holoenzyme is a heterotrimer composed of a catalytic subunit, a scaffolding subunit, and a regulatory subunit. The regulatory subunits belong to 4 distinct gene families, B (B55), B′(B56), B,“ and B”', each encoding 2 to 5 family members. Substrate specificity is conferred by the regulatory subunits through binding to substrates and regulators.³ Shugoshin binding to the PP2A-B56 holoenzyme involves dimerization of the Shugoshin N-terminal coiled coil region which makes contacts with the catalytic subunit of PP2A as well as the B56 regulatory subunit.⁴ Recently⁵ we described a general mechanism of binding to PP2A-B56 involving B56 recognition of a binding motif with the consensus sequence LxxIxE (where x denotes any amino acid). The LxxIxE motif is degenerate allowing most hydrophobic residues in position 1 and 4, while position 6 is strictly a glutamic acid. Composition of the motif dictates the affinity for B56, which usually lies in the low micromolar range. Importantly phosphorylation of the motif in position 2 and positions 7–9 increases affinity for B56 enabling kinase regulation of PP2A-B56 recruitment.

In light of the findings by Nishiyama and colleagues² we performed a bioinformatics analysis of Dalmatian orthologs to identify putative LxxIxE motifs (Fig. 1). Strikingly we identified a motif conserved within the Drosophila genus residing in Drosophila melanogaster residues 299–304 in a region of the protein predicted to be intrinsically unstructured. The binding capacity of this motif for PP2A-B56 is consistent with the truncation analysis of Nishiyama and colleagues, since truncation of residues 1–299 deletes position 1 of the LxxIxE motif, which is necessary for binding.⁵ We therefore speculate that the binding mechanism of Dalmatian to PP2A-B56 may be distinct from that of Shugoshin. Interestingly, position 2 of the Dalmatian LxxIxE motif is conserved as a phosphorylatable serine or threonine, which could represent a mechanism for spatial and temporal control of PP2A-B56 binding to Dalmatian. From an evolutionary point of view, acquisition of a short binding motif in a disordered region not otherwise conserved is a less demanding path than acquisition of structural features⁶ like those of Shugoshin. The LxxIxE motif of Dalmatian is not conserved in orthologs from the closely related species⁷ Bactrocera cucurbitae (melon fly) and Musca domestica (house fly) pointing to a unique Drosophila adaptation. Therefore, if the LxxIxE motif of Drosophila Dalmatian is responsible for PP2A-B56 binding this leaves open the question of how protection of chromosome cohesion is achieved in other invertebrates.

Figure 1. Putative PP2A-B56 binding LxxIxE motif of Dalmatian proteins from the Drosophila genus. The Drosophila melanogaster Dalmatian protein sequence was blasted against annotated protein sequences from the Diptera order of flies using the Ensembl Metazoa database (http://metazoa.ensembl.org). Alignment shows the identity of a putative PP2A-B56 binding LxxIxE motif conserved within the Drosophila genus.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed