Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome congression.
During mitosis, the recruitment of spindle-checkpoint-associated proteins to the kinetochore occurs in a defined order. The protein kinase Bub1 localizes to the kinetochore very early during mitosis, followed by Cenp-F, BubR1, Cenp-E and finally Mad2. Using RNA interference, we have investigated whether this order of binding reflects a level of dependency in human somatic cells. Specifically, we show that Bub1 plays a key role in the assembly of checkpoint proteins at the kinetochore, being required for the subsequent localization of Cenp-F, BubR1, Cenp-E and Mad2. In contrast to studies in Xenopus, we also show that BubR1 is not required for kinetochore localization of Bub1. Repression of Bub1 increases the number of cells with lagging chromosomes at metaphase, suggesting that Bub1 plays a role in chromosome congression. However, repression of Bub1 does not appear to compromise spindle checkpoint function either during normal mitosis or in response to spindle damage. This raises the possibility that, in the absence of Bub1, other mechanisms contribute to spindle checkpoint function.
Johnson
Victoria L
VL
School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK.
Scott
Maria I F
MI
Holt
Sarah V
SV
Hussein
Deema
D
Taylor
Stephen S
SS
D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D002877:Chromosomes, Human / Q000502:physiology*; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D018386:Kinetochores / Q000378:metabolism*; D064246:Mad2 Proteins; D008856:Microscopy, Fluorescence; D008938:Mitosis; D008954:Models, Biological; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D034622:RNA Interference; D012097:Repressor Proteins
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D012097:Repressor Proteins; C068688:centromere protein E; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases