Phosphorylation of Cdc20 by Bub1 provides a catalytic mechanism for APC/C inhibition by the spindle checkpoint.
To ensure the fidelity of chromosome segregation, the spindle checkpoint blocks the ubiquitin ligase activity of APC/C(Cdc20) in response to a single chromatid not properly attached to the mitotic spindle. Here we show that HeLa cells depleted for Bub1 by RNA interference are defective in checkpoint signaling. Bub1 directly phosphorylates Cdc20 in vitro and inhibits the ubiquitin ligase activity of APC/C(Cdc20) catalytically. A Cdc20 mutant with all six Bub1 phosphorylation sites removed is refractory to Bub1-mediated phosphorylation and inhibition in vitro. Upon checkpoint activation, Bub1 itself is hyperphosphorylated and its kinase activity toward Cdc20 is stimulated. Ectopic expression of the nonphosphorylatable Cdc20 mutant allows HeLa cells to escape from mitosis in the presence of spindle damage. Therefore, Bub1-mediated phosphorylation of Cdc20 is required for proper checkpoint signaling. We speculate that inhibition of APC/C(Cdc20) by Bub1 in a catalytic fashion may partly account for the exquisite sensitivity of the spindle checkpoint.
Tang
Zhanyun
Z
Department of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Shu
Hongjun
H
Oncel
Dilhan
D
Chen
She
S
Yu
Hongtao
H
D064173:Anaphase-Promoting Complex-Cyclosome; D002384:Catalysis; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D018816:Genes, cdc / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases; D034622:RNA Interference; D012694:Serine / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism
D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D025801:Ubiquitin; C577488:CDC20 protein, human; D013912:Threonine; D012694:Serine; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases