Cell cycle-dependent regulation of Greatwall kinase by protein phosphatase 1 and regulatory subunit 3B

Abstract

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in ). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation.

PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20

“…However, due to the temporal properties of its regulatory mechanism PP2A-B55 only becomes active in anaphase B when cyclin B1 levels fall below a threshold level (Cundell et al, 2013;Cundell et al, 2016). PP1 has been reported to be involved in the activation of PP2A-B55 through the dephosphorylation of the Gwl/MASTL kinase (Heim et al, 2015;Ma et al, 2016;Ren et al, 2017;Rogers et al, 2016), and could thus be considered an upstream regulator of PP2A-B55. PP2A-B55 action on CDC20 is therefore most likely restricted to a later point in anaphase, perhaps at the switch to APC/C regulation by CDH1 and destruction of late anaphase substrates.…”

“…This APC/C CDC20 active state is then consolidated by the action of PP2A-B55, the activation of which is initiated by PP1 (Heim et al, 2015;Ma et al, 2016;Ren et al, 2017;Rogers et al, 2016), ensuring that CDC20 is completely dephosphorylated in anaphase ( Figure 9).…”

Greatwall kinase at a glance

“…PP2A-B55 and PP1 are inactivated during mitosis; the former by Arpp19 and/or ENSA, and the latter through its direct phosphorylation by cyclin-B-Cdk1 (Wu et al, 2009). The reactivation of PP1 by auto-dephosphorylation on its cyclin-B-Cdk1 site (T320), which stems from cyclin B degradation, is likely the major factor that prompts Gwl inactivation (Heim et al, 2015;Ma et al, 2016;Ren et al, 2017). Upon anaphase entry, active PP1 promotes dephosphorylation of S875 and partial inactivation of Gwl, which in turn results in a partial activation of PP2A-B55.…”

Phosphatase 1 Nuclear Targeting Subunit (PNUTS) Regulates Aurora Kinases and Mitotic Progression

“…SDS-PAGE and immunoblotting were performed as described previously (36). For immunoprecipitation, anti-rabbit magnetic beads (New England Biolabs) were conjugated to the primary antibody, and then incubated in cell lysates for 2 hours.…”

“…To this end, the antimitotic function of PP1 has been well established as an essential mechanism that promotes mitotic exit (42,49). As an example, recent studies discovered a role of PP1 in the dephosphorylation of MASTL (also known as Greatwall kinase) during mitotic exit (36,(52)(53)(54). On the other hand, portions of PP1 bind specific mitotic structures and substrates, and play an active role in promoting several key steps of mitotic progression.…”