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Development
2004 Apr 01;1317:1543-52. doi: 10.1242/dev.01050.
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Polo-like kinase confers MPF autoamplification competence to growing Xenopus oocytes.
Karaiskou A
,
Leprêtre AC
,
Pahlavan G
,
Du Pasquier D
,
Ozon R
,
Jessus C
.
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During oogenesis, the Xenopus oocyte is blocked in prophase of meiosis I. It becomes competent to resume meiosis in response to progesterone at the end of its growing period (stage VI of oogenesis). Stage IV oocytes contain a store of inactive pre-MPF (Tyr15-phosphorylated Cdc2 bound to cyclin B2); the Cdc25 phosphatase that catalyzes Tyr15 dephosphorylation of Cdc2 is also present. However, the positive feedback loop that allows MPF autoamplification is not functional at this stage of oocyte growth. We report that when cyclin B is overexpressed in stage IV oocytes, MPF autoamplification does not occur and the newly formed cyclin B-Cdc2 complexes are inactivated by Tyr15 phosphorylation, indicating that Myt1 kinase remains active and that Cdc25 is prevented to be activated. Plx1 kinase (or polo-like kinase), which is required for Cdc25 activation and MPF autoamplification in full grown oocytes is not expressed at the protein level in small stage IV oocytes. In order to determine if Plx1 could be the missing regulator that prevents MPF autoamplification, polo kinase was overexpressed in stage IV oocytes. Under these conditions, the MPF-positive feedback loop was restored. Moreover, we show that acquisition of autoamplification competence does not require the Mos/MAPK pathway.
Expression of the regulators of Cdc2 activation in stage IV oocytes. Prophase oocytes at stage IV (750-800 μm), stage V1 (900-1000μ m), stage V2 (1000-1100 μm) and stage VI (≥1200 μm) were incubated or not in the presence of progesterone and collected 18 hours afterwards. Occurrence of GVBD is indicated by (+). Oocyte extracts were either assayed for H1 kinase activity of Cdc2 (A) or western blotted (B) with antibodies against Cdc2, cyclin B1, cyclin B2, Cdc25, Plx1 and the Tyr15-phosphorylated form of Cdc2 (P-Tyr-Cdc2).
Expression of the regulators of Cdc2 activation during oogenesis. (A) Prophase oocytes at stage IV (750-800 μm), stage V (900-11,000 μm) and stage VI (≥1200 μm) were incubated or not in the presence of progesterone and collected 18 hours afterwards. Oocyte extracts were western blotted with antibodies against cyclin B2, the active phosphorylated form of MAPK (P-MAPK), Mos, MAPK, the Thr161-phosphorylated form of Cdc2 (P-Thr161-Cdc2), Plkk1 and Myt1. (B) RT-PCR analysis of Plx1 transcripts. Total RNA of oocytes at various stages was subjected to RT-PCR analysis using Plx1 or Myt1 oligonucleotides as primers. Myt1 was used as the loading control. Without reverse transcription (Plx1 RT-), no amplification products were detectable using Plx1 primers.
Mos is able to activate MAPK and Rsk in stage IV oocytes but does not promote MPF activation. Stage IV and stage VI oocytes were injected with mouse Mos mRNA. After overnight incubation, they were incubated or not in the presence of progesterone. Stage IV oocytes were collected 4 hours after GVBD occurred in stage VI oocytes. Oocyte extracts were western blotted with antibodies against Rsk, Cdc25, Xenopus Mos (XeMos), the active phosphorylated form of MAPK (P-MAPK), the Tyr15-phosphorylated form of Cdc2 (P-Tyr-Cdc2), cyclin B1 and cyclin B2.
Stage IV and stage VI oocytes were stimulated by progesterone or injected with either GST-cyclin B or Xenopus Mos protein or both GST-cyclin B and Mos. Oocytes were collected and western blotted with antibodies against cyclin B2, the Tyr15-phosphorylated form of Cdc2 (P-Tyr-Cdc2), the active phosphorylated form of MAPK (P-MAPK) and Rsk.
Okadaic acid is unable to induce MPF auto-amplification in stage IV oocyte extracts. Stage IV and stage VI oocyte extracts were incubated in the presence (+) or in the absence (–) of 1 μM okadaic acid (OA) and an ATP-regenerating system for 3 hours. (A) Histone H1 kinase assay of Cdc2. (B) Western blots revealed with antibodies against the active phosphorylated form of MAPK (P-MAPK), Cdc25 and the Tyr15-phosphorylated form of Cdc2 (P-Tyr-Cdc2). (C) Oocyte extracts were supplemented with [35S]methionine labeled Myt1 at time of OA addition. Extracts were then submitted to SDS-PAGE and to autoradiography.
Presence of Plk1 restores MPF auto-amplification induced by cyclins or okadaic acid in stage IV oocyte extracts. (A) Stage IV and stage VI oocyte extracts were incubated in the presence or in the absence of either His-cyclin B1 (B1) or GST-cyclin A (A) and ATP for 3 hours. They were western blotted with antibodies against Cdc25 and the Tyr15-phosphorylated form of Cdc2 (P-Tyr-Cdc2). (B) Stage IV and stage VI oocyte extracts were incubated for 3 hours in the presence of increasing amounts of human His-cyclin B1 and were then assayed for H1 kinase activity of Cdc2. (C) Stage IV oocytes were injected with human Plk1 mRNA. After overnight incubation, oocyte extracts were prepared and supplemented with 1 μM okadaic acid (OA) or His-cyclin B1 (B1) and ATP. Three hours later, extracts were western blotted with antibodies directed against Myc (indicating the presence of the Myc-tagged Plk protein), Cdc25 and the Tyr15-phosphorylated form of Cdc2 (P-Tyr-Cdc2). They were also assayed for H1 kinase activity of Cdc2 (lower panel shows autoradiograph of [32P]histone H1).
In vivo rescue of MPF auto-amplification by Plk1 in stage IV oocytes. (A) Stage IV oocytes were injected or not with Plk1 mRNA. After overnight incubation, they were either incubated in the presence or not of progesterone, or injected with His-cyclin B1. Stage VI oocytes were used as control. Extracts were western blotted with antibodies against Cdc25, the Tyr15-phosphorylated form of Cdc2 (P-Tyr-Cdc2), Myc (indicating the presence of the Myc-tagged Plk protein), Myt1 and the active phosphorylated form of MAPK (P-MAPK). They were also assayed for H1 kinase activity of Cdc2 (lower panel shows autoradiograph of [32P]histone H1). (B) Stage IV oocytes were injected or not with Plk1 mRNA. After overnight incubation, they were either incubated in the presence or not of progesterone (Pg), or injected with okadaic acid (OA). Extracts were western blotted with antibodies against Cdc25 and the Tyr15-phosphorylated form of Cdc2 (P-Tyr-Cdc2). (C) Stage IV oocytes were injected with mouse Mos mRNA and human Plk1 mRNA. After overnight incubation, they were incubated or not in the presence of progesterone. Oocytes were collected 4 hours after GVBD occurred in stage VI oocytes. Oocyte extracts were western blotted with antibodies against the active phosphorylated form of MAPK (P-MAPK), Cdc25, the Tyr15-phosphorylated form of Cdc2 (P-Tyr-Cdc2) and cyclin B2.
