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Protein kinase A acts at multiple points to inhibit Xenopus oocyte maturation
W Matten1, I Daar, G F Vande Woude
1ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Maryland 21702.
Abstract:
In Xenopus oocytes, initiation of maturation is dependent on reduction of cyclic AMP-dependent protein kinase (PKA) activity and the synthesis of the mos proto-oncogene product. Mos is required during meiosis I for the activation of both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Here we show that injection of the catalytic subunit of PKA (PKAc) prevented progesterone-induced synthesis of endogenous Mos as well as downstream MPF and MAPK activation. However, PKAc did not prevent injected soluble Mos product from activating MAPK. While MAPK is activated during Mos-PKAc coinjection, attendant MPF activation is blocked. Additionally, PKAc caused a potent block in the electrophoretic mobility shift of cdc25 that is associated with phosphatase activation. This inhibition of cdc25 activity was not reversed by progesterone, Mos, or MPF. We conclude that PKAc acts as a negative regulator at several points in meiotic maturation by preventing both Mos translation and MPF activation.
Insights
High PKA activity prevents frog oocyte maturation by blocking Mos synthesis and MPF activation. This protein kinase acts as a negative regulator, inhibiting key steps in meiosis.
Area of Science:
- Cellular and Developmental Biology
- Molecular Biology
- Reproductive Biology
Background:
- Oocyte maturation initiation requires decreased cyclic AMP-dependent protein kinase (PKA) activity and Mos proto-oncogene product synthesis.
- Mos is essential for activating maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) during meiosis I.
Purpose of the Study:
- To investigate the role of the catalytic subunit of PKA (PKAc) in regulating progesterone-induced meiotic maturation in Xenopus oocytes.
- To determine how PKAc affects Mos synthesis, MPF and MAPK activation, and cdc25 activity.
Main Methods:
- Injection of the catalytic subunit of PKA (PKAc) into Xenopus oocytes.
- Assessment of endogenous Mos synthesis, MPF and MAPK activation following progesterone stimulation.
- Analysis of cdc25 activity through electrophoretic mobility shift assays.
Main Results:
- PKAc injection inhibited progesterone-induced Mos synthesis and subsequent MPF and MAPK activation.
- PKAc did not block MAPK activation by exogenous Mos but prevented MPF activation.
- PKAc potently inhibited cdc25 activity, an effect not reversed by progesterone, Mos, or MPF.
Conclusions:
- PKAc acts as a negative regulator of meiotic maturation in Xenopus oocytes.
- PKAc inhibits maturation by preventing Mos translation and blocking MPF activation.
- PKAc interferes with cdc25 phosphatase activity, further disrupting the maturation process.