BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1.
The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores.
Chen
Rey-Huei
RH
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA. rc70@cornell.edu
D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D018816:Genes, cdc / Q000502:physiology*; D018386:Kinetochores / Q000378:metabolism* / Q000648:ultrastructure; D064246:Mad2 Proteins; D008970:Molecular Weight; D009687:Nuclear Proteins; D009865:Oocytes; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification*; D017346:Protein Serine-Threonine Kinases; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D008941:Spindle Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D029867:Xenopus Proteins / Q000235:genetics / Q000302:isolation & purification*; D014982:Xenopus laevis
C577495:CDC20 protein, S cerevisiae; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D005656:Fungal Proteins; C501382:MAD1 protein, S cerevisiae; C501384:MAD1L1 protein, human; C577695:MAD2 protein, S cerevisiae; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D029867:Xenopus Proteins; C068688:centromere protein E; D011494:Protein Kinases; C584277:BUB1 protein, human; C524954:BUB1B protein, Xenopus; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases