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First published online 27 August 2003
doi: 10.1242/dev.00731

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DOC1R: a MAP kinase substrate that control microtubule organization of metaphase II mouse oocytes

M. Emilie Terret1, Christophe Lefebvre1, Alexandre Djiane1, Pascale Rassinier1, Jacques Moreau2, Bernard Maro1 and Marie-Hélène Verlhac1,*

1 UMR 7622, CNRS, Université Paris VI, 9 quai Saint Bernard, Bat. C, 75252 Paris, cedex 05, France
2 Institut Jacques Monod, CNRS, Université Paris VII, 2 place Jussieu, 75251 Paris, cedex 05, France



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  		Fig. 1. Interaction between DOC1R and MAPK; alignments of DOC1R sequences. (A) Two-hybrid interaction between DOC1R and MAPK. DOC1R interacts with ERK2WT and ERK2KD but not with the negative control Su(Fu). Yeast strains transformed with LexAERK2WT, LexA-ERK2KD (Waskiewicz et al., 1997Go), LexA-53 (positive control), LexASu(Fu) or LexA alone were mated with yeast strains transformed respectively with the B42-DOC1R, B42-AD-T (positive control), or empty B42. The diploids obtained were tested for transactivation of both the ß-galactosidase and the LEU2 reporter genes on glucose (Glu)- or galactose (Gal/Raf)-containing mediums. The B42 constructs are under the control of the galactose promoter. The B42-DOC1R fusion protein clearly interacts both with LexA fusions of ERK2WT and ERK2KD as strongly as the positive control, whereas it does not interact with the negative control Su(Fu). (B) DOC1R co-immunoprecipitates with endogenous p42mapk (ERK2) from immature Xenopus oocyte extracts. Lanes 1 and 2: total immature Xenopus oocyte extracts expressing either MYC-WNT11 (Xenopus WNT11, a negative control, lane 1) or MYC-DOC1R mRNA (lane 2). Lanes 3 and 4: anti-p42mapk (ERK2) immunoprecipitates prepared from the MYC-WNT11 (lane 3) and MYC-DOC1R (lane 4) expressing oocyte lysates. All samples were analysed by immunoblotting using the anti-MYC antibody. This experiment was repeated twice. (C) Amino acid sequence alignments of the DOC1R protein from different vertebrate species. DOC1R is rich in proline in its N-terminal end and contains one potential MAPK phosphorylation site (blue), one CDK2 binding site (red) and one cyclin/CDK-binding site (green). (D) Percentage of identities (I) and similarities (S) between the amino acid sequences of DOC1R from different vertebrate species (h, human; m, mouse; xt, Xenopus tropicalis; xl, Xenopus laevis).

 


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  		Fig. 2. DOC1R mRNA and protein are present in mouse oocyte. (A) Expression of DOC1R mRNA in immature oocytes (lanes 1 and 2) and ovaries (lane 3), treated or not with reverse transcriptase (RT, + or -). (B) The anti-DOC1R antibody recognizes a band at 28 kDa in 200 oocytes arrested in metaphase II (lane 2) and recognizes 1 µg of purified DOC1R protein (lane 4). The antibody pre-incubated with the DOC1R peptide (peptide, + or -) does not recognize either endogenous DOC1R (lane 1) or purified DOC1R (lane 3). These experiments were repeated twice.

 


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  		Fig. 3. DOC1R is regulated by phosphorylation. For A,B, immature oocytes were injected with RNA encoding the MYC-DOC1R protein and further cultured for different length of time. Each sample corresponds to a pool of 25 oocytes from the same injection and to oocytes that expressed the mRNA for at least 6 hours. (A) MYCDOC1R regulation during meiotic maturation. MYC-DOC1R mRNA was injected into wild-type oocytes that were collected at different stages of meiotic maturation: immature (lane 1, GV), at 1 hour (lane 2), 3 hours (lane 3), 6 hours (lane 4) and 14 hours (lane 5, Metaphase II, MII) after GVBD. (B) In vitro dephosphorylation of MYCDOC1R from mouse oocyte extracts. Thirty oocytes were injected with MYC-DOC1R mRNA, cultured 14 hours after GVBD, collected and incubated without (lane 1) or with (lane 2) 400 U of {lambda}-phosphatase (PPase + or -). All samples were analyzed by immunoblotting with the anti-MYC antibody. Experiments have been repeated four times.

 


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  		Fig. 4. cyclin B/CDC2 and MAP kinase phosphorylate DOC1R. (A) Purified cyclin B/CDC2 and MAPK phosphorylate DOC1R in vitro. Purified cyclin B/CDC2 (lanes 1, 2 and 3) and recombinant active rat ERK2 (lanes 4, 5 and 6) were incubated with (+) or without (-) 6His-DOC1R or Histone H1 (lanes 2 and 5) in the presence of [{gamma}-32P]-ATP. The [32P] incorporation was detected by autoradiography. This experiment has been repeated twice. (B) The MOS/.../MAPK pathway phosphorylates DOC1R. MYC-DOC1R-injected oocytes from wild-type (lane 1) or Mos-/- (lane 2) mice were cultured for 12 hours after GVBD and collected. This experiment has been repeated three times. (C) MYC-DOC1R expression after microinjection of RNA encoding MYC-DOC1R into immature wild-type or Mos-/- oocytes. Forty wild-type oocytes cultured for 12 hours after GVBD (top panel) or 40 Mos-/- oocytes cultured for 12 hours (bottom panel) after GVBD were collected and analysed by 2D gel electrophoresis. The migration of MYC-DOC1R is shifted towards the acidic pole (H+) in wild-type oocytes compared with its migration in Mos-/- oocytes. To position the different MYC-DOC1R isoforms from one sample to the other, we re-probed all the blots using Ezrin as an internal control (Louvet-Vallee et al., 2001Go). This experiment has been repeated three times. Samples in B and C were analyzed by immunoblotting using an anti-MYC antibody.

 


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  		Fig. 5. DOC1R localizes to microtubules during meiotic maturation. (A-F) Localization of the endogenous DOC1R protein. Groups of 20 oocytes at different stages of meiotic maturation were fixed with formaldehyde and further stained with the anti-DOC1R antibody (AF) or with the antibody blocked with 30 µM of immunogenic peptide (G). (H,I) Oocytes microinjected with DOC1R-GFP encoding RNA and collected in GV (H) or MII (I). All oocytes were analyzed by confocal microscopy using identical settings. (A) Immature oocyte in GV, (B) oocyte collected 1 hour after GVBD, (C) 5 hours after GVBD, (D) 8 hours after GVBD, (E,G-I) 14 hours after GVBD in metaphase II (MII), (F) cultured for 13 hours after GVBD then treated with nocodazole for 1 hour (MII+NZ). The DOC1R staining or DOC1R-GFP localization appear in green and chromosomes in red. Two-hundred and fifty oocytes were scored for this experiment. Scale bar: 10 µm.

 


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  		Fig. 6. DOC1R depletion induces formation of numerous microtubule asters at spindle poles as well as in the cytoplasm of MII oocytes. Immature oocytes were microinjected with antisense RNA (asDOC1R) or double-stranded RNA (dsDOC1R or dsFrz) directed against DOC1R or Xenopus Frizzled mRNA, or they were injected with RNA encoding Xenopus DOC1R with or without dsDOC1R, further cultured and collected at different stages of meiotic maturation. Oocytes were fixed with formaldehyde and analyzed by confocal microscopy. (A) Control non-injected oocyte (NI) collected in metaphase II. (B) Control oocyte injected with dsFrz collected in metaphase II. (C) Control oocyte injected with RNA encoding Xenopus DOC1R and collected in metaphase II. (D) Oocyte injected with asDOC1R collected in metaphase II. (E) Oocyte injected with dsDOC1R collected in metaphase II. (F) Oocyte injected with dsDOC1R and with RNA encoding Xenopus DOC1R, then collected in metaphase II. (G,H) Non-injected oocyte collected in metaphase I (MI, G) or metaphase II (MII, H). (I,J) Oocyte injected with the dsDOC1R collected in metaphase I (I) or II (J). For A-F, microtubules appear in green, for G,H, the endogenous DOC1R staining appears in green. For all images, chromosomes appear in red. Scale bars: 10 µm in A-F; 6 µm in G-J. (K) Statistics of the experiment described above. Percent MII: percentage of oocytes presenting a normal bipolar metaphase II spindle. Percent phenotypes: percentage of oocytes presenting spindle defects and numerous asters in the cytoplasm. The numbers in brackets correspond to the total number of oocytes analyzed. These experiments have been repeated between three to five times.

 

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© The Company of Biologists Ltd 2003