Degradation of the proto-oncogene product p39mos is not necessary for cyclin proteolysis and exit from meiotic metaphase: requirement for a Ca(2+)-calmodulin dependent event.
Exit from M phase, which requires cyclin degradation, is prevented from occurring in unfertilized eggs of vertebrates arrested at second meiotic metaphase due to a cytostatic factor recently identified as p39mos, the product of the proto-oncogene c-mos. Calpain can destroy both p39mos and cyclin in vitro in extracts prepared from metaphase-arrested Xenopus eggs, but only when free Ca2+ concentration is raised to the millimolar range. When free Ca2+ concentration is raised for only 30 s to the micromolar range, as occurs in physiological conditions after fertilization, cyclin degradation is induced, byt p39mos is not degraded. Cyclin proteolysis at micromolar free Ca2+, is not inhibited by calpastatin, and therefore does not involve calpain. A cyclin mutant modified in the destruction box is found to be resistant at micromolar, but not millimolar free Ca2+, suggesting that the ubiquitin pathway mediates cyclin degradation at micromolar Ca2+ concentration whereas calpain is involved at the millimolar level. A synthetic peptide which binds Ca(2+)-calmodulin with high affinity suppresses cyclin degradation at micromolar but not millimolar free Ca2+, and this only when it is present in the extract during the first 30 s after raising free Ca2+ concentration. The inhibition of the cyclin degradation pathway by the Ca(2+)-calmodulin binding peptide can be overcome by adding calmodulin. These results strongly suggest that a Ca(2+)-calmodulin process is required as an early event following fertilization to release the cyclin degradation pathway from inhibition in metaphase-arrested eggs. In contrast, p39mos degradation is not required.
Lorca
T
T
CNRS and INSERM, BP 505, Montpellier, France.
Galas
S
S
Fesquet
D
D
Devault
A
A
Cavadore
J C
JC
Dorée
M
M
D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007150:Immunohistochemistry; D008540:Meiosis; D008677:Metaphase; D009865:Oocytes / Q000166:cytology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D017231:Proto-Oncogene Proteins c-mos; D014981:Xenopus
D002147:Calmodulin; D016213:Cyclins; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; D017231:Proto-Oncogene Proteins c-mos; D002118:Calcium