Considerations for studying phosphorylation of the mitotic checkpoint pseudokinase BUBR1

Budding uninhibited by benzimidazole 1-related protein 1 (BUBR1) is a mitotic checkpoint (better known as the spindle assembly checkpoint) protein that forms part of an inhibitory complex required to delay mitosis when errors occur in the attachment between chromosomes and the mitotic spindle. If these errors remain uncorrected, it could result in unequal distribution of genetic material to each of the nascent daughter cells, leading to potentially disastrous consequences at both the cellular and organismal level. In some higher eukaryotes including vertebrates, BUBR1 has a C-terminal kinase fold that is largely thought to be inactive, whereas in many species this domain has been lost through evolution and the truncated protein is known as mitotic arrest deficient 3 (MAD3). Here we present advice and practical considerations for the design of experiments, their analysis and interpretation to study the functions of the vertebrate BUBR1 during mitosis with emphasis on analysis implicating the pseudokinase domain.