Requirement for proteolysis in spindle assembly   checkpoint silencing.
Anaphase initiation requires ubiquitin-dependent proteolysis of crucial substrates through activation of the ubiquitin ligase Anaphase promoting Complex/Cyclosome (ApC/C) in association with its coactivator Cdc20. To prevent chromosome segregation errors, effector proteins of a safeguard mechanism called spindle assembly checkpoint (SAC), Mad2 and BubR1, bind Cdc20 and restrain ApC/C(Cdc20) activation until spindle assembly. Coordinated chromosome segregation also requires timely SAC inactivation. Spindle assembly appears necessary to silence SAC, however, how resolution of the SAC effector branch is achieved is still largely unknown. We show here that the complex between Mad2 and Cdc20 peaked at prometaphase in mammalian cells, while its dissociation proceeded along with spindle assembly and required proteolysis. proteolysis did not appear required for assembly of metaphase spindles but rather needed for Mad2-Cdc20 complex resolution by promoting reversal of phosphorylations that maintain the complex. Indeed, in the absence of proteolysis, Mad2-Cdc20 complex dissociation was reversed by treatment with cyclin-dependent kinase or Aurora kinase inhibitors. Mad2-Cdc20 disassembly was, however, resistant to the potent pp1 and pp2A phosphatases inhibitor okadaic acid. We propose that SAC silencing in mammalian cells requires proteolysis-dependent activation of okadaic acid-resistant phosphatase(s) to reverse phosphorylations that lock the Mad2-Cdc20 complex.
Visconti
Roberta
R
CEINGE Biotecnologie Avanzate, Naples, Italy.
Palazzo
Luca
L
Grieco
Domenico
D
D002135:Calcium-Binding Proteins / Q000378:metabolism; D064199:Cdc20 Proteins; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D064246:Mad2 Proteins; D008954:Models, Biological; D015739:Nocodazole / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012097:Repressor Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects
D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D047428:Protein Kinase Inhibitors; D012097:Repressor Proteins; C577488:CDC20 protein, human; D019319:Okadaic Acid; D015739:Nocodazole