Cyclin B dissociation from CDK1 precedes its degradation upon MPF inactivation in mitotic extracts of Xenopus laevis embryos.
Cyclin B is a regulatory subunit of CDK1 within MPF complex. Degradation of cyclin B via ubiquitin-proteasome pathway seemed to be absolutely required for the M-phase exit. However, inhibition of the proteasome proteolytic activity upon the exit from the meiotic metaphase II-arrest in Xenopus cell-free extract revealed that the proteasome-dependent dissociation of cyclin B from CDK1 is sufficient to inactivate MPF without cyclin B degradation. In this study we analyze whether the same mechanism operates during the exit from mitotic M-phase. We show in Xenopus cell-free extract undergoing the first or the second embryonic mitosis that CDK1 oscillations are not affected by proteasome inhibition with MG132 or ALLN despite effective inhibition of cyclins B degradation. The majority of cyclins B1 and B2 surviving CDK1 inactivation is CDK-free and cyclin B2 becomes resistant to phosphatase lambda dephosphorylation. The pool of cyclins B remaining after CDK1 inactivation in the presence of MG132 is mitotically inert, while exogenous or newly synthesized cyclin B activates CDK1. This suggests that cyclins B remain sequestered within the proteasome upon MPF inactivation in the presence of MG132. Comparison of the dynamics of the decline of total and CDK-bound pools of cyclins B1, B2 and B4 upon mitotic exit in absence of protein synthesis reveals that CDK-bound cyclins B diminish clearly faster. Our results thus show that cyclin B dissociation from CDK1 precedes cyclins B degradation upon CDK1 inactivation in mitotic embryo extracts and that proteasome proteolytic activity is dispensable for both activation and inactivation of CDK1 in such extracts.
Chesnel
Franck
F
CNRS-UMR 6061, Biology and Genetics of Development, Mitosis and Meiosis Group, IFR140 GFAS, University of Rennes 1, Faculty of Medicine, Rennes, France.
Bazile
Franck
F
Pascal
Aude
A
Kubiak
Jacek Z
JZ
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002457:Cell Extracts; D002474:Cell-Free System; D019926:Cyclin B / Q000378:metabolism*; D056744:Cyclin B1; D016213:Cyclins / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D016200:Maturation-Promoting Factor / Q000378:metabolism*; D008938:Mitosis / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012617:Sea Urchins / Q000737:chemistry; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis / Q000378:metabolism*
C534699:CCNB1 protein, human; D002457:Cell Extracts; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins; D007976:Leupeptins; D029867:Xenopus Proteins; C438193:cyclin B4 protein, Xenopus; D011494:Protein Kinases; C102333:histone H1 kinase; D016203:CDC2 Protein Kinase; D016200:Maturation-Promoting Factor; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde