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Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro
Abstract:
Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.
Insights
Mos protein activates key signaling molecules like MAP kinase and MPF in Xenopus oocytes, initiating meiosis. This study reveals Mos acts upstream of MAP kinase kinase (MAPKK), directly phosphorylating it to drive meiotic maturation.
Area of Science:
- Cellular biology
- Molecular biology
- Developmental biology
Background:
- Xenopus oocytes undergo meiotic maturation involving protein kinases like Mos, MPF, MAP kinase, and MAPKK.
- Mos protein synthesis is crucial for progesterone-induced meiotic maturation.
- Bacterial fusion proteins of Mos can initiate meiosis and MPF activation without protein synthesis.
Purpose of the Study:
- To investigate the role of Mos in activating MAP kinase and MPF in Xenopus oocytes.
- To determine if Mos directly or indirectly activates MAP kinase kinase (MAPKK).
- To elucidate the upstream signaling pathway involving Mos in meiotic maturation.
Main Methods:
- Injection of wild-type and kinase-inactive MBP-Mos fusion proteins into Xenopus oocytes.
- Monitoring phosphorylation and activation of MAP kinase and MPF.
- Assaying oocyte extracts for MAP kinase-phosphorylating activity.
- In vitro phosphorylation assays using purified MAPKK.
Main Results:
- Wild-type MBP-Mos rapidly phosphorylates and activates MAP kinase within 20 minutes.
- MBP-Mos activates MPF approximately 2 hours post-injection.
- Oocyte extracts from MBP-Mos injected oocytes show MAPKK-activating activity.
- MBP-Mos directly phosphorylates and activates purified MAPKK in vitro.
Conclusions:
- Mos acts as an upstream activator of MAP kinase in oocytes.
- Mos likely functions by directly phosphorylating MAPKK.
- This Mos-MAPKK-MAPK pathway is critical for initiating meiotic maturation.