Role for non-proteolytic control of M-phase-promoting factor activity at M-phase exit.
M-phase Promoting Factor (MPF; the cyclin B-cdk 1 complex) is activated at M-phase onset by removal of inhibitory phosphorylation of cdk1 at thr-14 and tyr-15. At M-phase exit, MPF is destroyed by ubiquitin-dependent cyclin proteolysis. Thus, control of MPF activity via inhibitory phosphorylation is believed to be particularly crucial in regulating transition into, rather than out of, M-phase. Using the in vitro cell cycle system derived form Xenopus eggs, here we show, however, that inhibitory phosphorylation of cdk1 contributes to control MPF activity during M-phase exit. By sampling extracts at very short intervals during both meiotic and mitotic exit, we found that cyclin B1-associated cdk1 underwent transient inhibitory phosphorylation at tyr-15 and that cyclin B1-cdk1 activity fell more rapidly than the cyclin B1 content. Inhibitory phosphorylation of MPF correlated with phosphorylation changes of cdc25C, the MPF phosphatase, and physical interaction of cdk1 with wee1, the MPF kinase, during M-phase exit. MPF down-regulation required Ca(++)/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase (PKA) activities at meiosis and mitosis exit, respectively. Treatment of M-phase extracts with a mutant cyclin B1-cdk1AF complex, refractory to inhibition by phosphorylation, impaired binding of the Anaphase Promoting Complex/Cyclosome (APC/C) to its co-activator Cdc20 and altered M-phase exit. Thus, timely M-phase exit requires a tight coupling of proteolysis-dependent and proteolysis-independent mechanisms of MPF inactivation.
D'Angiolella
Vincenzo
V
Faculty of Biotechnological Sciences and Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, University of Napoli Federico II, Italy.
Palazzo
Luca
L
Santarpia
Concetta
C
Costanzo
Vincenzo
V
Grieco
Domenico
D
D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000502:physiology; D056744:Cyclin B1 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D008540:Meiosis; D008677:Metaphase / Q000502:physiology*; D008938:Mitosis; D009865:Oocytes / Q000166:cytology*; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D029867:Xenopus Proteins / Q000502:physiology; D014982:Xenopus laevis; D020687:cdc25 Phosphatases / Q000502:physiology
C534699:CCNB1 protein, human; D018797:Cell Cycle Proteins; D056744:Cyclin B1; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D010769:Phosphothreonine; D019000:Phosphotyrosine; C530005:WEE1 protein, Xenopus; D011505:Protein-Tyrosine Kinases; D016203:CDC2 Protein Kinase; D020687:cdc25 Phosphatases