Yamamoto TM et al. (2011), Regulation of Greatwall kinase during Xenopus o...

XB-ART-43230

Mol Biol Cell 2011 Jul 01;2213:2157-64. doi: 10.1091/mbc.E11-01-0008.

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Regulation of Greatwall kinase during Xenopus oocyte maturation.

Yamamoto TM , Blake-Hodek K , Williams BC , Lewellyn AL , Goldberg ML , Maller JL .

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Greatwall kinase has been identified as a key element in M phase initiation and maintenance in Drosophila, Xenopus oocytes/eggs, and mammalian cells. In M phase, Greatwall phosphorylates endosulfine and related proteins that bind to and inhibit protein phosphatase 2A/B55, the principal phosphatase for Cdk-phosphorylated substrates. We show that Greatwall binds active PP2A/B55 in G2 phase oocytes but dissociates from it when progesterone-treated oocytes reach M phase. This dissociation does not require Greatwall kinase activity or phosphorylation at T748 in the presumptive T loop of the kinase. A mutant K71M Greatwall, also known as Scant in Drosophila, induces M phase in the absence of progesterone when expressed in oocytes, despite its reduced stability and elevated degradation by the proteasome. M phase induction by Scant Greatwall requires protein synthesis but is not associated with altered binding or release of PP2A/B55 as compared to wild-type Greatwall. However, in vitro studies with Greatwall proteins purified from interphase cells indicate that Scant, but not wild-type Greatwall, has low but detectable activity against endosulfine. These results demonstrate progesterone-dependent regulation of the PP2A/B55-Greatwall interaction during oocyte maturation and suggest that the cognate Scant Greatwall mutation has sufficient constitutive kinase activity to promote M phase in Xenopus oocytes.

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Species referenced: Xenopus
Genes referenced: ccnb1 ccnb2 cdc25c cdk1 mastl mbp mink1 ppp2ca ptpa

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	FIGURE 1:. Regulated binding of PP2A/B55 to Greatwall. (A) Greatwall releases PP2A/B55 in M phase. Oocytes were injected with mRNA encoding FLAG-Gwl and incubated overnight as described in Materials and Methods. Some oocytes were then treated with progesterone and monitored for entry into meiosis I (MI, GVBD). Extracts from the control (uninjected), G2, and GVBD oocytes were immunoprecipitated with anti-FLAG antibody beads, and the immunoprecipitates were Western blotted for FLAG and the B55 subunit of PP2A. C, control; WT, wild type. (B) Association of Greatwall with PP2A in G2 phase. Oocytes were injected with mRNA encoding FLAG-WT or T748V Gwl and incubated overnight. Some oocytes were then treated with progesterone and monitored for white-spot formation (GVBD, MI). Lysates from the oocytes were precipitated with microcystin–Sepharose beads (MC), and the precipitates were Western blotted for FLAG and for the catalytic subunit of PP2A (bottom). Lysates for Western blotting were also analyzed before immunoprecipitation (top). (C) Kinase-dead Greatwall releases PP2A in M phase. An experiment like that in B was performed, except that G41S Gwl was analyzed in MI (GVBD) oocytes. (D) The C-terminal region of Greatwall binds PP2A in G2. mRNA encoding FLAG-tagged N-terminal (NT) or C-terminal (CT) Gwl was injected into oocytes, followed by overnight incubation. Oocyte lysates were immunoprecipitated with anti-FLAG antibody beads, and the precipitate was Western blotted with FLAG and B55 antibodies, as indicated. (E) The C-terminal region of Greatwall releases PP2A in M phase. Oocytes were injected with CT Gwl mRNA as in D, except that some were treated with progesterone to stimulate entry into meiosis I (MI), as indicated. At GVBD, FLAG-Gwl was immunoprecipitated on anti-FLAG beads from progesterone-treated (MI) or nontreated (G2) oocytes, and the immunoprecipitates were Western blotted for FLAG and B55.
	FIGURE 2:. Greatwall-bound PP2A is catalytically active. (A) Dephosphorylation and deactivation of Gwl. Oocytes were injected with mRNA encoding WT or G41S Gwl. After overnight incubation to allow protein expression, the oocytes were treated with progesterone. At GVBD, oocyte lysates were prepared and anti-FLAG bead immunoprecipitates incubated with either PP2Ac or lambda phosphatase as described in Materials and Methods. The precipitates were then washed and assayed for autophosphorylation or MBP kinase activity (bottom, autoradiographs), or blotted with anti-FLAG antibodies after SDS–PAGE (top), as indicated. (B) Phosphatase activity associated with Gwl. Oocytes were injected with mRNA encoding WT FLAG-tagged Gwl and incubated overnight. Some oocytes were then treated with progesterone to enter M phase. At GVBD, anti-FLAG beads were used to precipitate Gwl, and after washing, the beads were incubated with or without OA and 32P-labeled histone H1 prepared as described in Materials and Methods. The reaction was terminated by addition of 30% trichloracetic acid, and the counts per minute released were quantified by Cerenkov counting in a liquid scintillation counter. Similar results were obtained in several independent experiments. (C) Anti-FLAG immunoblot of the immunoprecipitates in B. C, control oocytes not injected with mRNA but subjected to anti-FLAG bead precipitation and assay. Activity associated with Gwl in B was corrected for background seen with control FLAG bead immunoprecipitates from uninjected oocytes.
	FIGURE 3:. K71M Greatwall induces oocyte maturation in the absence of progesterone. (A) Oocyte morphology. Maturation (GVBD) was assessed by white-spot formation in oocytes incubated overnight after injection of mRNA encoding K71M Gwl or K71M/G41S Gwl, as indicated. (B) Electrophoretic mobility. Lysates from the oocytes in A were Western blotted for FLAG. (C) Germinal vesicle breakdown. Oocytes were treated with progesterone (blue) or microinjected with equal amounts of mRNA encoding either WT Gwl (green) or K71M Gwl (red). Maturation was monitored at the indicated times by the appearance of a white spot signifying GVBD. (D) K71M Gwl kinase activity. Top, the oocytes in (C) expressing WT or K71M Gwl at 240 min (G2) after injection or expressing K71M Gwl at 420 min after injection with a white spot (GVBD, MI) were analyzed by Western blot for Gwl expression. Bottom, the same oocytes were lysed, and Gwl was immunoprecipitated on anti-FLAG beads and analyzed for kinase activity against MBP as described in Materials and Methods. An autoradiograph is shown. (E) K71M Gwl releases PP2A in M phase. Oocytes were injected with mRNA encoding either FLAG-WT or FLAG-K71M Gwl and treated with the proteosome inhibitor MG132. Some FLAG-WT Gwl mRNA-injected oocytes were treated with progesterone to undergo GVBD (MI). G2 and MI lysates were prepared and analyzed by MC bead precipitation and Western blotting as described in Figure 1B.
	FIGURE 4:. K71M Gwl protein induces oocyte maturation. (A) Oocyte morphology. Oocytes were treated with progesterone or injected with buffer or WT or K71M Gwl proteins purified from non-OA treated (interphase) Sf9 cells. After incubation overnight, GVBD was assessed by white-spot formation. An oocyte that did not undergo GVBD with K71M Gwl was designated “G2” (e.g., upper oocyte, right panel). (B) Analysis of K71M Gwl expressing oocytes. The oocytes in A were lysed and Western blotted for Gwl, cyclin B1, and pY15 Cdc2, as indicated. At this exposure level, the shifted form of endogenous Gwl in progesterone-treated (GVBD) oocytes is less apparent. (C) Maturation induced by K71M Gwl requires protein synthesis. Active Gwl was purified from OA-treated Sf9 cells as described previously and microinjected into oocytes, followed by incubation in the absence and presence of cycloheximide (CHX, 10 μg/ml). After 6 h, GVBD was assessed by white-spot formation.
	FIGURE 5:. Constitutive activity of Scant. Recombinant Drosophila Gwl proteins, either WT or K97M/Scant (equivalent to K71M Xenopus Gwl), were purified from non-OA–treated Sf9 cells and assayed for activity against a fragment of endosulfine fused to maltose-binding protein as described in Materials and Methods. The first and third rows show Coomassie staining of Gwl and endosulfine proteins used in the assay. The amount of Gwl proteins assayed varied from 1 to 50 ng. The autoradiographs in the second and fourth rows show, respectively, autophosphorylation of Gwl and phosphorylation of the endosulfine fragment fused to maltose-binding protein.
	FIGURE 6:. K71M Gwl is an unstable protein. (A) Greatwall accumulation. mRNA encoding either WT FLAG-Gwl or FLAG-K71M Gwl was injected into oocytes, and after 4 h of incubation in the absence or presence of the protesome inhibitor MG132 the level of expressed Gwl was assessed by SDS–PAGE and immunoblotting for FLAG-Gwl. (B) Maturation with K71M Gwl and MG132. Oocytes were injected with mRNA encoding FLAG-WT or K71M Gwl and incubated with medium containing 50 μM MG132. At the indicated times, oocyte lysates were prepared and Western blotted for FLAG-Gwl, active Cdc25C (pSer287), and cyclin B2. GVBD (unpublished data) began at 6 h after injection. (C) Acceleration of K71M-induced GVBD by MG132. Oocytes were injected with mRNA encoding K71M Gwl and incubated in the presence or absence of MG132 or treated with progesterone. The time course of GVBD was assessed by white-spot formation at the indicated times.

References [+] :

Archambault, Mutations in Drosophila Greatwall/Scant reveal its roles in mitosis and meiosis and interdependence with Polo kinase. 2007, Pubmed, Xenbase