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 Xenopus). 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.
Highlights
Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression
In this study we show that Gwl associates with protein phosphatase 1 (PP1), PP1␥, which mediates the dephosphorylation of Gwl Ser-883
To reveal Gwl regulation by protein phosphatases, we pulled down recombinant Gwl from interphase Xenopus egg extracts and probed for the presence of various PP1 catalytic subunits
Summary
Reversible phosphorylation of Gwl at multiple Ser/Thr residues plays a central role in the regulation of Gwl kinase activation [22,23,24]. We were unable to detect direct protein interaction using purified Gwl and PP1␥, whereas these proteins bound upon addition into interphase Xenopus egg extracts (Fig. 1F). We reasoned that a possible mechanism to account for the mitotic phosphorylation of Gwl Ser-883 is disruption of its PP1-association during M phase To this end, we analyzed the association between Gwl and PP1␥ in both interphase and CSF (M phase) extracts. The differential kinetics of Gwl Ser-883 dephosphorylation in comparison with other mitotic phosphoproteins likely reflects the orderly reactivation of distinct phosphatases during mitotic exit Such a pattern of dephosphorylation was not observed when the CSF extract was diluted in an interphase extract in which all antimitotic phosphatase activities are readily active (Fig. 4B). PPP1R3B does not influence mitotic exit indirectly by affecting endogenous PP1-containing complexes
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