Calmodulin-dependent protein kinase II mediates inactivation of MPF and CSF upon fertilization of Xenopus eggs

Abstract

IN vertebrates, unfertilized eggs are arrested at second meiotic metaphase by a cytostatic factor (CSF)¹, an essential component of which is the product of the c-mos proto-oncogene². CSF prevents ubiquitin-dependent degradation of mitotic cyclins and thus inactivation of the M phase-promoting factor (MPF)^3,4.Fertilization or parthenogenetic activation triggers a transient increase in the cytoplasmic free Ca²⁺ (reviewed in refs 5 and 6), inactivates both CSF and MPF, and releases eggs from meiotic metaphase arrest^7–10. A calmodulin-dependent process is required for cyclin degradation to occur in cell-free extracts prepared from metaphase II-arrested eggs (CSF extracts) when the free Ca²⁺concentration is transiently raised in the physiological micromolar range¹⁰. Here we show that when a constitutively active mutant of calmodulin-dependent protein kinase II (CaM K_II) is added to a CSF extract, cyclin degradation and Cdc2 kinase inactivation occur even in the absence of Ca²⁺, and the extract loses its ability to cause metaphase arrest when transferred into embryos. Furthermore, specific inhibitors of CaM K_IIprevent cyclin degradation after calcium addition. Finally, the direct microinjection of constitutively active CaM KH into unfertilized eggs inactivates Cdc2 kinase and CSF, even in the absence of a Ca²⁺ transient. The target for Ca²⁺calmodulin is thus CaM K_II.