Uncoupling of the spindle-checkpoint and chromosome-congression functions of BubR1.
The BubR1 checkpoint protein performs multiple functions in mitosis. We have carried out a functional analysis of conserved motifs of human BubR1 (also known as BUB1B) and demonstrate that spindle assembly checkpoint (SAC) and chromosome attachment functions can be uncoupled from each other. Mutation of five proline-directed serine phosphorylation sites, identified in vivo by mass spectrometry, essentially abolishes attachment of chromosomes to the spindle but has no effect on SAC functionality. By contrast, mutation of the two conserved KEN boxes required for SAC function does not impact chromosome congression. Interestingly, the contribution of the two KEN-box motifs is not equal. Cdc20 associates with the N-terminal but not C-terminal KEN box, and mutation of the N-terminal KEN motif results in more severe acceleration of mitotic timing. Moreover, the two KEN motifs are not sufficient for maximal binding of Cdc20 and APC/C, which also requires sequences in the BubR1 C-terminus. Finally, mutation of the GLEBS motif causes loss of Bub3 interaction and mislocalization of BubR1 from the kinetochore; concomitantly, BubR1 phosphorylation as well as SAC activity and chromosome congression are impaired, indicating that the GLEBS motif is strictly required for both major functions of human BubR1.
Elowe
Sabine
S
Department of Cell Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany. elowe@biochem.mpg.de
Dulla
Kalyan
K
Uldschmid
Andreas
A
Li
Xiuling
X
Dou
Zhen
Z
Nigg
Erich A
EA
D020816:Amino Acid Motifs / Q000235:genetics*; D016203:CDC2 Protein Kinase / Q000378:metabolism; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018816:Genes, cdc / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000187:drug effects; D007976:Leupeptins / Q000494:pharmacology; D008938:Mitosis / Q000187:drug effects / Q000235:genetics; D009154:Mutation / Q000235:genetics; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D008941:Spindle Apparatus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D050257:Tubulin Modulators / Q000494:pharmacology
D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D050257:Tubulin Modulators; C577488:CDC20 protein, human; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D015739:Nocodazole