THE PROTEOLYSIS-DEPENDENT METAPHASE TO ANAPHASE TRANSITION - CALCIUM CALMODULIN-DEPENDENT PROTEIN-KINASE-II MEDIATES ONSET OF ANAPHASE IN EXTRACTS PREPARED FROM UNFERTILIZED XENOPUS EGGS/

It has been shown, using spindles assembled in vitro in extracts conta ining CSF (the cytostatic factor responsible for arresting unfertilize d vertebrate eggs at metaphase), that onset of anaphase requires Ca2+- dependent activation of the ubiquitin-dependent proteolytic pathway t hat destroys both mitotic cyclins and an unknown protein responsible f or metaphase arrest (Holloway et al., 1993, Cell, 73, 1382-1402). We s howed recently that Ca2+/calmodulin-dependent protein kinase II (CaM K II) activates the ubiquitin-dependent cyclin degradation pathway in CS F extracts (Lorca et al., 1993, Nature, 366, 270-273), but did not inv estigate its possible effect on sister chromatid segregation. In this work we identify CaM KII as the only target of Ca2+ in inducing anapha se in CSF extracts, and further show that transition to anaphase does not require the direct phosphorylation of metaphase spindle components by CaM KII. A possible interpretation of the above results could have been that the ubiquitin-dependent degradation pathway is required for onset of anaphase only when spindles are clamped at metaphase due to CSF activity, and not in the regular cell cycle that occurs in the abs ence of CSF activity. We ruled out this possibility by showing that co mpetitive inhibition of the ubiquitin-dependent degradation pathway st ill prevents the onset of anaphase in cycling extracts that lack CSF a nd do not require Ca2+ for sister chromatid separation.