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A new role for Mos in Xenopus oocyte maturation: targeting Myt1 independently of MAPK

Marion Peter^*, Jean-Claude Labbé, Marcel Dorée and Elisabeth Mandart

CNRS-CRBM, 1919 route de Mende, 34293 Montpellier cedex 05, France
^* Present address: ICRF/Richard Dimbleby Department of Cancer Research, St Thomas Hospital, Lambeth Palace Road, London SE1 7EH, UK

View larger version (23K): [in a new window]   Fig. 1. A faint amount of Mos protein is synthesized in Mos antisense-injected oocytes. (A) Early process of MAPK monophosphorylation is allowed in Mos antisense-injected oocytes. Timecourse of Mos accumulation and MAPK phosphorylation during maturation of oocytes injected (+ AS Mos) or not (– AS Mos) with Mos antisense oligonucleotides. At the indicated time (in minutes or hours) after progesterone addition, a group of five oocytes were homogenized and analyzed by western blot with the indicated antibodies. The same membrane was used for each antibody after stripping. M indicates that oocytes underwent GVBD. The equivalent of one oocyte was loaded per lane. (B) Only the diphosphorylated form of MAPK is active. Timecourse of MAPK phosphorylation in non injected oocytes was analyzed by western blot using specific antibodies that recognize the monophosphorylated (Ab pY), the diphosphorylated (Ab pTpY) or the total MAPK (Ab ERK). The same samples (equivalent of two oocytes) were analyzed for MAPK activity, using the in gel MBP kinase assay. (C) Quantification of Mos protein levels in Mos antisense-injected oocytes. Successive dilutions of homogenates from non-injected mature oocytes with stage VI oocyte homogenates (to keep the same protein concentration in each sample) were performed to obtain the equivalent of one matured oocyte to 1/20th of a matured oocyte as indicated. These samples were compared with Mos antisense-injected oocytes (equivalent of one oocyte) that underwent (AS Mos M) or not (AS Mos NM) GVBD for the expression of Mos protein. All the oocytes came from the same experiment and the same frog. A control for loading is achieved using ß-tubulin detection.

View larger version (24K): [in a new window]   Fig. 2. Mos triggers GVBD independently of MAPK activation. Oocytes were incubated in U0126 1 hour before oligonucleotide injection, and progesterone was added 1 hour after microinjection. (A) Oocytes were microinjected with Mos antisense oligonucleotides in presence (AS Mos + U0126) or absence (AS Mos) of U0126, or were non-injected in the presence (U0126) or absence (control) of U0126. Homogenates from resting oocytes (stage VI) or oocytes stimulated for 20 hours by progesterone were analyzed by western blot with different antibodies as indicated. (B) Mos antisense-injected oocytes undergo GVBD with or without U0126. Oocytes treated as in A were scored as a function of time for GVBD (observed as appearance of a distinct white spot).

View larger version (25K): [in a new window]   Fig. 3. Mos expression is required for oocyte exit from G2 arrest. (A) Overexpression of Eg2 facilitates synthesis of Mos protein in Mos antisense-injected oocytes. Oocytes were first injected with Eg2 mRNA, and, 16 hours later, with (AS Mos + Eg2 mRNA) or without (Eg2 mRNA) Mos antisense oligonucleotides, or were injected with Mos antisense oligonucleotides alone (AS Mos), or were non-injected (Control). The timecourse of Mos accumulation was analyzed by western blot as indicated in Fig. 1. At 180 minutes and 8 hours, all the oocytes underwent GVBD except for those under AS Mos conditions at 180 minutes. (B) Injected antisense Eg2 oligonucleotides prevent the Mos antisense-injected oocytes from undergoing GVBD. Oocytes were microinjected with Mos antisense (AS Mos) or antisense Eg2 (AS Eg2), or both (AS Mos + AS Eg2) oligonucleotides, or were non injected (control) 1 hour before progesterone addition. They were scored for GVBD 20 hours after stimulation. The average of three experiments with different frogs is shown. (C) Injected antisense Eg2 oligonucleotides prevent the synthesis of Mos protein in Mos antisense-injected oocytes. Homogenates from stage VI oocytes (non stimulated) or oocytes treated differently, as in B, that were stimulated by progesterone for 20 hours were analyzed for the presence of Mos, Eg2 and the different phosphorylated forms of MAPK by Western blot using correspondent antibodies as indicated. The same homogenates were tested for histone H1 kinase assays (H1). Equivalent of one oocyte was loaded per lane. (D) Oocytes co-injected with Mos and Eg2 antisense oligonucleotides are rescued by injection of Mos protein. Oocytes incubated in U0126 were microinjected or not (U0126) with Mos and Eg2 antisense oligonucleotides 1 hour before progesterone addition and were supplied (AS Mos + AS Eg2 + U0126 + Mos), or not (AS Mos + AS Eg2 + U0126), with MBP-Mos protein (50 µg/ml). The oocytes were scored for GVBD 20 hours after stimulation. In this experiment, we verified that oocytes injected with only the same amount of MBP-Mos protein and incubated in U0126 underwent GVBD only when progesterone was added. Control: oocytes stimulated by progesterone.

View larger version (27K): [in a new window]   Fig. 4. Cyclin B1 does not require the MAPK cascade pathway to induce GVBD. Oocytes injected with recombinant GST-cyclin B1 (1 mg/ml) in absence (B1) or presence (B1+CHX) of cycloheximide were homogenized 2 hours after they underwent GVBD and analyzed by immunoblot for diphosphorylated MAPK and Mos. Non-injected oocytes stimulated with progesterone (control M) or not (stage VI) are shown as control. Groups of three oocytes were homogenized and the equivalent of one oocyte was loaded per lane.

View larger version (20K): [in a new window]   Fig. 5. Mos facilitates activation of MPF in the first meiosis, independently of the MAPK cascade activation. (A) Mos increases the amount of injected GST-cyclin B1 oocytes undergoing GVBD. Oocytes incubated in U0126 were injected with either GST-cyclin B1 (25 µg/ml) alone (B1+U0126), or both GST-cyclin B1 and MBP-Mos (B1+Mos+U0126) in presence or absence of cycloheximide (CHX). Oocytes were scored for GVBD 5 hours after injection. The average of several experiments with different frogs is shown. Oocytes microinjected with MBP-Mos alone (Mos), at the same concentration (20 µg/ml), in absence of U0126 did not undergo GVBD. (B) Oocytes injected with GST-cyclin B1 and MBP-Mos underwent GVBD without activating MAPK. Oocytes microinjected as in A were collected at different times, and analyzed by western blot for diphosphorylated MAPK. Oocytes injected with MBP-Mos alone (Mos) were collected at the end of the experiment. Non-injected immature (stage VI) and progesterone treated mature (control M) oocytes are shown as control. Groups of three oocytes were homogenized and the equivalent of one oocyte was loaded per lane. The band present in all lanes that migrates more slowly than active MAPK corresponds to unspecific staining. (C) Mos facilitates Tyrosine dephosphorylation of Cdc2 in GST-cyclin B1-Cdc2 complexes. Groups of five oocytes treated as in A were collected 1 hour after injection and submitted to GST pull-down assay. The elution of the beads was analyzed by immunoblot with anti-Cdc2 antibodies. The arrow indicates the accumulation of the dephosphorylated form of Cdc2 kinase. Stage VI, one immature oocyte; control M, one progesterone-induced mature oocyte.

View larger version (28K): [in a new window]   Fig. 6. Mos does not facilitate MPF activation in the second meiosis. Oocytes were stimulated by progesterone. At 50% GVBD, the oocytes that did not undergo GVBD were transferred in cycloheximide, and left in this medium for all the experiment. In these conditions, oocytes that underwent GVBD were arrested in interphase 1 hour later. They were then injected with GST-cyclin B1 alone (B1), or both GST-cyclin B1 and MBP-Mos (B1+Mos). Groups of three oocytes were collected at the indicated times and submitted to a GST pull-down assay, followed by a histone H1 kinase assay. The whole final reaction mixture was resolved by SDS-PAGE, and phosphorylated histone H1 was revealed by autoradiography. As a control, the equivalent of one immature oocyte (stage VI) and one mature oocyte (control M) are shown. Lanes 3-8, GST-cyclin B1 at 100 µg/ml was injected per oocyte; lanes 9-14, GST-cyclin B1 at 50 µg/ml was injected per oocyte; lanes 6-8 and 12-14, MBP-Mos at 20 µg/ml was injected per oocyte.

View larger version (27K): [in a new window]   Fig. 7. Mos targets Myt1. (A) Mos associates with Myt1 in vivo: stage VI oocytes were injected with mRNA encoding GST protein, GST-Mos wild type (WT) or GST-Mos kinase dead (KD) protein. In some cases, as indicated, oocytes were incubated in U0126 during the whole experiment. Twelve hours after microinjection, oocytes were treated (or not) with progesterone. Four hours later, groups of 40 oocytes were collected and submitted to a GST pull-down assay. The elution of the beads was analyzed by immunoblot with the indicated antibodies. The equivalent of one immature oocyte (stage VI) and one mature oocyte (control M) are shown as control. (B) In vitro phosphorylation of Myt1. mRNAs of GST-Mos WT or GST-Mos KD (as a negative control) were injected into stage VI oocytes. Fourteen hours later, extracts of 30 oocytes of each type were prepared. Myt1 translated in reticulocyte extract (without radiolabeled amino acids) was added (20 µl) to the GST-pull down performed from injected oocytes. Myt1 phosphorylation was tested in presence of 32PATP, MgCl2 and microcystin. After several washes of the beads, the samples were loaded on a 8% acrylamide gel and analyzed by autoradiogram and western blotting using anti-Myt1 antibodies. Lanes 1-4: extracts (equivalent of one oocyte) of stage VI oocytes (lane 1), metaphase II oocytes (lane 2), GST-Mos KD-injected oocytes (lane 3), and GST-Mos WT-injected oocytes (lane 4). Lanes 5,6: GST pull-down on oocytes injected with GST-Mos KD (lane 5) or Mos WT (lane 6) after the phosphorylation reaction. Lanes 7-9: Myt1 translation in vitro without radiolabeled amino acids (lane 7; 4 µl), with 35S Met (lane 8; 2 µl) or without Myt1 plasmid (lane 9; 2 µl negative control). Lane 8 is a control for Myt1 translation and for the size of non phosphorylated Myt1.

View larger version (44K): [in a new window]   Fig. 8. Myt1 is phosphorylated in vivo, although MAPK activation is inhibited. (A) Timecourse of Myt1 phosphorylation during maturation of oocytes incubated in U0126 1 hour before progesterone addition. (B) Timecourse of Myt1 phosphorylation during maturation of oocytes non-treated (Control) or incubated in U0126 and injected (AS Mos + U0126) or not (U0126) with Mos antisense oligonucleotides before progesterone addition. Groups of five oocytes were collected per point. The equivalent of one oocyte was loaded per lane and was analyzed by immunoblot with the indicated antibodies. Control M: non-treated mature oocyte.

View larger version (9K): [in a new window]   Fig. 9. Model for the dual control of Mos on MPF activation in Xenopus oocytes. Mos associates with Myt1 independently of MAPK cascade activation (broken arrow). This interaction could facilitate Myt1 inactivation by restricting the access of Myt1 phosphatase (PPase) and/or by Myt1 phosphorylation by Mos on some sites. Thus, the Mos/Myt1 interaction could lead to Myt1 inactivation by allowing its complete phosphorylation by other(s) kinase(s) and therefore enhancing MPF activation.

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