PP2A^Cdc55dephosphorylates Pds1 to inhibit spindle elongation

Abstract

DNA replication stress stalls replication forks leading to chromosome breakage and Intra-S checkpoint activation. In S. cerevisiae, this checkpoint arrests the cell cycle by stabilizing securin (Pds1) and inhibiting the cyclin dependent kinase (CDK) through multiple pathways.Pds1 inhibits separase (Esp1) which cleaves the cohesin subunit Scc1 and also functions in spindle elongation. However, the role of Pds1-Esp1 in spindle elongation during replication stress response is unknown. Here, we show that Pds1 phosphorylation plays a positive role in spindle elongation through the Pds1-Esp1 interaction in unperturbed and replication stress conditions. PP2A Cdc55 directly dephosphorylates Pds1 both in vivo and in vitro. Pds1 hyperphosphorylation in a cdc55∆ mutant enhanced the Pds1-Esp1 interaction, which accelerated spindle elongation. This PP2A Cdc55 -dependent Pds1 dephosphorylation plays a role during replication stress and acts independently of the known Mec1, Swe1 or Spindle Assembly Checkpoint (SAC) checkpoint pathways. We propose a model where PP2A Cdc55 dephosphorylates Pds1 to disrupt the Pds1-Esp1 interaction that inhibits spindle elongation during replication stress.

Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

“…Previous studies indicate that PP2A along with Cdc14 counteracts CDK-imposed phosphorylation (Godfrey et al, 2017). Interestingly, a recent work showed increased Pds1 phosphorylation in cdc55Δ cells and that PP2A Cdc55 directly dephosphorylates Pds1 to reverses Cdk1-mediated phosphorylation (Khondker et al, 2020). However, it is unlikely that PP2A Cdc55 promotes cell cycle recovery from replication stress by directly reversing CDK-dependent Pds1 phosphorylation, because pds1-38 mutant lacking the CDK phosphorylation sites was unable to suppress the HU sensitivity of PP2A mutants (Figure S4A).…”

“…In addition to Chk1, Cdk1 also phosphorylates Pds1, which facilitates the interaction of Pds1 with separase (Esp1) for its nuclear accumulation (Agarwal and Cohen-Fix, 2002). A recent study indicates that PP2A directly reverses Cdk1-mediated Pds1 phosphorylation (Khondker et al, 2020). Therefore, Cdk1-dependnet Pds1 phosphorylation might play a role in replication stress recovery.…”

“…PP2A Cdc55 dephosphorylates Net1, a nucleolar protein that anchors Cdc14 to the nucleolus, and loss of function of PP2A Cdc55 results in premature release of Cdc14 from the nucleolus (Queralt et al, 2006;Wang and Ng, 2006;Yellman and Burke, 2006). Recent evidence also indicates that PP2A Cdc55 reverses Cdk1-mediated phosphorylation of Pds1, which is expected to abolish Pds1-Esp1 interaction for anaphase progression (Khondker et al, 2020).…”