Hara M et al. (2012), Greatwall kinase and cyclin B-Cdk1 are both cri...

XB-ART-46651

Nat Commun 2012 Jan 01;3:1059. doi: 10.1038/ncomms2062.

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Greatwall kinase and cyclin B-Cdk1 are both critical constituents of M-phase-promoting factor.

Hara M , Abe Y , Tanaka T , Yamamoto T , Okumura E , Kishimoto T .

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Maturation/M-phase-promoting factor is the universal inducer of M-phase in eukaryotic cells. It is currently accepted that M-phase-promoting factor is identical to the kinase cyclin B-Cdk1. Here we show that cyclin B-Cdk1 and M-phase-promoting factor are not in fact synonymous. Instead, M-phase-promoting factor contains at least two essential components: cyclin B-Cdk1 and another kinase, Greatwall kinase. In the absence of Greatwall kinase, the M-phase-promoting factor is undetectable in oocyte cytoplasm even though cyclin B-Cdk1 is fully active, whereas M-phase-promoting factor activity is restored when Greatwall kinase is added back. Although the excess amount of cyclin B-Cdk1 alone, but not Greatwall kinase alone, can induce nuclear envelope breakdown, spindle assembly is abortive. Addition of Greatwall kinase greatly reduces the amount of cyclin B-Cdk1 required for nuclear envelope breakdown, resulting in formation of the spindle with aligned chromosomes. M-phase-promoting factor is thus a system consisting of one kinase (cyclin B-Cdk1) that directs mitotic entry and a second kinase (Greatwall kinase) that suppresses the protein phosphatase 2A-B55 which opposes cyclin B-Cdk1.

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Species referenced: Xenopus laevis
Genes referenced: ccnb1.2 cdk1 mapk1 mastl mbp mcm2 mink1 myt1 nucb1 plk1 ptpa rasgrf1

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	Figure 1. Cyclin B–Cdk1 (cycB–Cdk1) is not identical to MPF in either frog or starfish oocytes.(a,b) Purified cycB–Cdk1 requires higher H1K activity for the meiotic G2/M-phase transition than does cytoplasmic MPF. H1K activity required for NEBD induction after injection into recipients was compared in Xenopus oocytes between CSF extracts (MPF) and purified cycB–Cdk1 (a), and in starfish oocytes between maturing oocyte cytoplasm (MPF) and purified cycB–Cdk1 (b). H1K activity in the abscissa was calculated from an injection volume and an activity that catalyses the transfer of phosphate from ATP to histone H1 per minute per volume (Methods). (c,d) Enucleated, as well as nucleated, starfish oocytes respond to 1-MeAde by undergoing changes in phosphorylation of Cdc25, Myt1, Cdk1, and MAPK (c), and an increase in cycB–Cdk1 activity, as measured by H1K (d). Mcm2 is a marker for nucleated oocytes. The lower band of Cdk1 corresponds to its Tyr15-dephosphorylated, active form. The upper band of MAPK corresponds to its active form. NEBD (arrow head) occurred at 18–20 min in nucleated oocytes. (e) Cytoplasm from enucleated starfish oocytes exhibits no MPF activity. Cytoplasm (250 pl) of nucleated or enucleated oocytes during metaphase of meiosis I (nucleated) or the equivalent period after 1-MeAde addition (enucleated) was injected into recipient immature oocytes, and NEBD was inspected (numbers with NEBD/total recipients).
	Figure 2. Gwl is present exclusively in the nucleus and is activated downstream of cyclin B–Cdk1 at meiotic resumption in starfish oocytes.(a) Gwl kinase activity cycles in parallel with Cdk1 (H1K) activity during meiotic and cleavage cycles. Immature oocytes were treated with 1-MeAde, followed by insemination 40 min later. After immunoprecipitation, Gwl kinase activity was assayed by phosphorylation of myelin basic protein (MBP). To compare amounts of Gwl proteins, oocyte extracts were treated with λ-protein phosphatase (PPase). Quantified activities of H1K and Gwl (normalized to its protein levels) are shown at the bottom. (b) Gwl activation after 1-MeAde treatment does not occur in the presence of the Cdk1 inhibitor, roscovitine. Dimethylsulphoxide (DMSO) indicates the control without roscovitine. (c) Amounts of Gwl protein were compared with immunoblots between the indicated numbers of nucleated or enucleated immature oocytes. Plk1 is a cytoplasmic marker.
	Figure 3. Gwl is necessary for MPF in starfish oocytes.(a,b) ZZ-IBB causes nuclear accumulation of IgG. Schematic diagram of the ZZ-IBB construct (a). Immature oocytes were injected with fluorescently labelled donkey IgG (Alexa488-IgG) in the absence or presence of ZZ-IBB, and then examined by confocal microscopy (b). (c) Anti-Gwl-C antibody (anti-Gwl) along with ZZ-IBB inhibits activity of Gwl, but not cyclin B–Cdk1. Immature oocytes, which had been injected with either anti-Gwl or control IgG in the absence or presence of ZZ-IBB, were treated with 1-MeAde, followed by examination of Gwl and Cdk1 (H1K) activities at 40 min. (d,e) Gwl is not essential for meiotic resumption. Gwl-inhibited oocytes, which had been injected with anti-Gwl along with ZZ-IBB, were treated with 1-MeAde. Thereafter, the time courses of NEBD (d) and Cdk1 activation (e), and changes in the phosphorylations of Gwl, Cdc25, Myt1, Cdk1 and MAPK (e) were monitored. Asterisks indicate non-specific bands. (f) MPF is undetectable from Gwl-inhibited oocytes. For MPF assay, cytoplasm from Gwl-inhibited and 1-MeAde-treated oocytes (anti-Gwl+ZZ-IBB), from control IgG-injected and 1-MeAde-treated oocytes (Cont IgG+ZZ-IBB), or from oocytes treated with 1-MeAde alone (None) was transferred into immature oocytes in which NEBD was inspected (numbers with NEBD/total recipients).
	Figure 4. Gwl restores MPF in enucleated starfish oocytes.(a,b) Gwl is sufficient for restoration of MPF in enucleated oocytes (Enuc). Enuc were injected with recombinant Gwl protein (rGwl-WT) or its kinase-dead mutant (rGwl-G52S), in the same amounts as the endogenous Gwl in an oocyte (0.06 fmol). These oocytes and control nucleated (Nuc) or Enuc oocytes were treated with 1-MeAde, and then examined for the amount of Gwl protein (upper) and the level of H1K (lower) at the indicated times (a), or for MPF activity by cytoplasmic transfer into immature oocytes (numbers with NEBD/total recipients) (b). (c) Gwl together with the cytoplasm of 1-MeAde-treated enucleated oocytes, but not Gwl alone, exhibits MPF activity. Immature oocytes were injected with 1.2 fmol of active rGwl-WT, whose activity is sevenfold higher than that of the total endogenous Gwl in a meta-I oocyte; but no NEBD occurred (active rGwl-WT). In contrast, immature oocytes were injected with 250 pl of cytoplasm from an enucleated, 1-MeAde-treated oocyte along with 14 amol of active rGwl-WT, whose activity is equivalent to the endogenous Gwl in 250 pl of cytoplasm from a nucleated, meta-I oocyte; and then NEBD occurred (active rGwl-WT+Enuc cytoplasm). As controls, the same amounts of kinase-dead rGwl-G52S, with or without cytoplasm from Enuc and 1-MeAde-treated oocytes, were injected. In parentheses, numbers with NEBD/total recipients.
	Figure 5. Cyclin B–Cdk1 (cycB–Cdk1) plus Gwl is nearly identical to MPF in starfish and frog oocytes.(a) In starfish, various volumes of cytoplasm from maturing oocytes were injected into immature oocytes, and the volume of cytoplasm that induces NEBD in 50% of recipients was estimated to be 105 pl. This volume of cytoplasm was calculated to contain Gwl activity equivalent to 6.1 amol of active rGwl-WT (see Methods in detail) and an H1 kinase (H1K) activity of 2.56 fmol P min−1 (Methods; for below (c)). (b) Gwl reduces the dose dependence of cycB–Cdk1 for NEBD induction in starfish oocytes. Various amounts of purified cycB–Cdk1, having kinase activities of 2.5–50 fmol P min−1 (each dissolved in a volume of 100 pl; Methods), were injected into immature oocytes along with (magenta squares) or without (green diamonds) active rGwl-WT (6.1 amol in 35 pl solution; see (a) above). Kinase defective rGwl-G52S (blue triangles) was used as a control. (c) Supplementation of purified cycB–Cdk1 with the endogenous level of Gwl reduced the level of cycB–Cdk1 required for NEBD induction in starfish oocytes by ~75% (from 25.1 to 6.12 fmol P min−1). From graphs (a,b), the amounts of H1K activity required for 50% NEBD were estimated. (d) Supplementation with Gwl transforms cycB–Cdk1 to MPF in Xenopus oocytes. For MPF assay, immature oocytes were injected with purified cycB–Cdk1 at a level of H1K activity, which is insufficient for NEBD induction (60 fmol P min−1 in 0.23 nl), without (green) or with various amounts of active rGwl-WT (magenta, 0.01–1.0 ng in 2.1 nl) or control kinase defective rGwl-G52S (blue, 1.0 ng in 2.1 nl). Neither rGwl-WT nor rGwl-G52S alone (gray, 1.0 or 30 ng in 2.1 nl) induced NEBD. As a positive control, CSF extract (black; H1K activity, 60 fmol P min−1 in 18.4 nl) invariably induced NEBD. (e) Gwl is localized mostly in the cytoplasm of immature Xenopus oocytes. Amounts of Gwl were compared by immunoblotting among whole immature oocytes (whole), enucleated immature oocytes (cytoplasm) and isolated GVs (nucleus). Mcm2 and α-tubulin are nuclear and cytoplasmic markers, respectively.
	Figure 6. Excess cyclin B–Cdk1 is deleterious for spindle assembly in starfish oocytes.(a–e) Meiotic spindle is assembled in starfish oocytes, which have undergone NEBD following injection with the reduced level of cyclin B–Cdk1 plus Gwl, but not in those with the excess level of cyclin B–Cdk1 alone. Live-cell images of microtubules (green) and chromosomes (magenta) for a control 1-MeAde-treated oocyte (a), and oocytes injected with the excess cyclin B–Cdk1 alone (b), the excess cyclin B–Cdk1 plus Gwl (c), the reduced cyclin B–Cdk1 plus Gwl (d) and cytoplasmic MPF (e). Maximum intensity Z projections using ImageJ are shown at a time point equivalent to metaphase of meiosis I. More than ten oocytes from different females were examined for each of b–e. For injection of purified cyclin B–Cdk1, minimum amounts of kinase activity required for 100% NEBD were estimated to be 50 fmol P min−1 for cyclin B–Cdk1 alone (excess) and 15 fmol P min−1 for cyclin B–Cdk1 (reduced) plus Gwl, respectively (each dissolved in a volume of 100 pl for cyclin B–Cdk1). The same amount of active rGwl-WT (10 amol in 50 pl) was injected in c,d. All figures were taken at the same magnification, and bar indicates 10 μm. (f) Comparison of Cdk1 activity contained in oocytes after NEBD. Levels of H1K activity contained in starfish oocytes were measured at NEBD and NEBD plus 20 min (equivalent to metaphase of meiosis I) after injection with the excess cyclin B–Cdk1 alone (green), the reduced cyclin B–Cdk1 plus Gwl (magenta) or cytoplasmic MPF (gray), or after control treatment with 1-MeAde (black). Im indicates immature oocytes. Each H1K activity was measured from an oocyte. Bars indicate s.e. of six separate experiments.
	Figure 7. MPF as a core component in the autoregulatory loop for cyclin B–Cdk1 activation.Cyclin B–Cdk1 is by itself very inefficient in triggering the autoregulatory loop in recipient oocytes, but MPF, consisting of both cyclin B–Cdk1 and Gwl, can efficiently initiate the activation loop, leading to full activation of cyclin B–Cdk1 in recipients.

References [+] :

Abe, A single starfish Aurora kinase performs the combined functions of Aurora-A and Aurora-B in human cells. 2010, Pubmed