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Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish
A Picard1, J P Capony, D L Brautigan
1Centre National de la Recherche Scientifique, Montpellier, France.
Abstract:
Specific inhibition of types 1 and 2A protein phosphatases by microinjection of okadaic acid (OA) into starfish oocytes induced germinal vesicle breakdown and activation of M phase-promoting factor (MPF) and histone H1 kinase. The effects were evident in immature oocytes arrested at first meiotic prophase as well as in fully mature oocytes arrested at the pronucleus stage. In addition, MPF and histone H1 kinase were stabilized for several hours and protected from inactivation by inhibition of type 1 protein phosphatases with either OA or specific anti-phosphatase antibodies. Microinjection of okadaic acid was associated with unusual changes of the microtubule network, including the disappearance of spindles and extension of the cytoplasmic array of microtubules. MPF activation after OA injection was associated with dephosphorylation of phosphothreonine and phosphoserine residues in cdc2, showing that neither type 1 nor 2A protein phosphatases catalyzes these dephosphorylations. The effects of OA on MPF activation and inactivation appeared to involve the cyclin subunit. OA did not induce MPF activation in the absence of protein synthesis and it prevented degradation of cyclin. Therefore protein phosphatases types 1 and 2A appear to be involved in activation and inactivation of MPF involving mechanisms that operate after cyclin synthesis and before its degradation.
Insights
Inhibiting protein phosphatases 1 and 2A with okadaic acid activates M-phase promoting factor (MPF) and histone H1 kinase in starfish oocytes. This suggests these phosphatases regulate MPF activity through cyclin synthesis and degradation.
Area of Science:
- Cell Biology
- Molecular Biology
- Developmental Biology
Background:
- Protein phosphatases are crucial regulators of cell cycle progression.
- M-phase promoting factor (MPF) and histone H1 kinase are key regulators of meiotic maturation.
- Okadaic acid (OA) is a specific inhibitor of protein phosphatases type 1 and 2A.
Purpose of the Study:
- To investigate the role of protein phosphatases type 1 and 2A in regulating MPF and histone H1 kinase activity.
- To elucidate the mechanisms by which these phosphatases influence meiotic maturation in starfish oocytes.
Main Methods:
- Microinjection of okadaic acid (OA) into starfish oocytes.
- Assay of MPF and histone H1 kinase activity.
- Immunological inhibition of protein phosphatases.
- Analysis of microtubule network dynamics.
- Assessment of protein synthesis and cyclin degradation.
Main Results:
- OA injection induced germinal vesicle breakdown and activated MPF and histone H1 kinase in immature and mature oocytes.
- MPF and histone H1 kinase were stabilized by OA, indicating protection from inactivation.
- OA altered the microtubule network, causing spindle disappearance and cytoplasmic microtubule array extension.
- MPF activation involved dephosphorylation of cdc2, but not by phosphatases 1 or 2A.
- OA prevented cyclin degradation and MPF activation required protein synthesis.
Conclusions:
- Protein phosphatases type 1 and 2A are involved in both the activation and inactivation of MPF.
- These phosphatases regulate MPF activity via mechanisms acting after cyclin synthesis and before cyclin degradation.
- The findings highlight the critical role of phosphatases in controlling meiotic progression and cell cycle events.