Distinct kinetics of serine and threonine dephosphorylation are essential for mitosis

Abstract

Protein phosphatase 2A (PP2A) in complex with B55 regulatory subunits reverses cyclin-dependent kinase 1 (Cdk1) phosphorylations at mitotic exit. Interestingly, threonine and serine residues phosphorylated by Cdk1 display distinct phosphorylation dynamics, but the biological significance remains unexplored. Here we demonstrate that the phosphothreonine preference of PP2A-B55 provides an essential regulatory element of mitotic exit. To allow rapid activation of the anaphase-promoting complex/cyclosome (APC/C) co-activator Cdc20, inhibitory phosphorylation sites are conserved as threonines while serine substitutions delay dephosphorylation and Cdc20 activation. Conversely, to ensure timely activation of the interphase APC/C co-activator Cdh1, inhibitory phosphorylation sites are conserved as serines, and threonine substitutions result in premature Cdh1 activation. Furthermore, rapid translocation of the chromosomal passenger complex to the central spindle is prevented by mutation of a single phosphorylated threonine to serine in inner centromere protein (INCENP), leading to failure of cytokinesis. Altogether, the findings of our work reveal that the inherent residue preference of a protein phosphatase can provide temporal regulation in biological processes.

PP2A‐B56 binds to Apc1 and promotes Cdc20 association with the APC/C ubiquitin ligase in mitosis

“…Pull-down assays showed only wild-type (WT) Apc1-loop 500 significantly bound 35 S-labelled in vitro-translated B56c in anaphase extracts (lane 14), compared with MBP-alone or MBP-fused Apc1-loop 500-Δ11 (lanes 13 and 15). This is consistent with the previous report that phosphorylation of the SP site in the middle of the B56 binding site increases binding strength [29]. This interaction was not observed in interphase extracts, which have low Cdk activity (lane 10), suggesting that the binding of B56 to Apc1-loop 500 depends on phosphorylation of the loop domain.…”

“…Phosphorylation of Cdc20 T79 was significantly reduced upon addition of PP2A-B56c, whereas phosphorylation of Apc1-loop 300 remained stable (Fig 4E, lanes 5-8). PP2A has been shown to be important for dephosphorylation of APC/C and Cdc20 [15,28,29]. This supports our model (Fig 4F) that PP2A-B56 bound to the APC/C ª 2019 The Authors EMBO reports 21: e48503 | 2020 enables efficient dephosphorylation of Cdc20, but not the APC/C, resulting in Cdc20 association with the APC/C in mitosis and initiating APC/C-dependent ubiquitylation.…”

“…At a higher concentration of PP2A-B56c, Apc1-loop 300 was dephosphorylated to some extent, but Cdc20 pT79 was again more efficiently dephosphorylated (Fig 4E, lanes 9-12). It has also been reported that in mitotic exit after cyclin B degradation, PP2A-B55 is crucial and preferentially dephosphorylates phospho-threonine over phospho-serine residues [29][30][31][32]. Thus, our study reveals for the first time the presence of a binding site for PP2A-B56 within the APC/C subunit and we propose a PP2A-B56-mediated mechanism controlling Cdc20-APC/C formation in mitosis.…”

“…It has been shown that PP2A can antagonise Cdk-dependent inhibitory phosphorylation of Cdc20 at the N-terminal domain (N-Cdc20) and promotes the engagement of Cdc20 with the APC/C for activation [15,29]. It has been shown that PP2A can antagonise Cdk-dependent inhibitory phosphorylation of Cdc20 at the N-terminal domain (N-Cdc20) and promotes the engagement of Cdc20 with the APC/C for activation [15,29].…”

“…It has been shown that PP2A can antagonise Cdk-dependent inhibitory phosphorylation of Cdc20 at the N-terminal domain (N-Cdc20) and promotes the engagement of Cdc20 with the APC/C for activation [15,29]. It has also been reported that Cdk1-dependent inhibitory phosphorylation sites within N-Cdc20 are exclusively threonine (T64, T68 and T79 in Xenopus Cdc20) [15,29], whereas Cdk1-dependent stimulatory phosphorylation sites in Apc1-loop 300 are exclusively serine (S314, S318, S335, S344, S358, S380 and S389 in Xenopus Apc1) [12]. It has also been reported that Cdk1-dependent inhibitory phosphorylation sites within N-Cdc20 are exclusively threonine (T64, T68 and T79 in Xenopus Cdc20) [15,29], whereas Cdk1-dependent stimulatory phosphorylation sites in Apc1-loop 300 are exclusively serine (S314, S318, S335, S344, S358, S380 and S389 in Xenopus Apc1) [12].…”