Phylogenomics-guided discovery of a novel conserved cassette of short linear motifs in BubR1 essential for the spindle checkpoint

Abstract

The spindle assembly checkpoint (SAC) maintains genomic integrity by preventing progression of mitotic cell division until all chromosomes are stably attached to spindle microtubules. The SAC critically relies on the paralogues Bub1 and BubR1/Mad3, which integrate kinetochore-spindle attachment status with generation of the anaphase inhibitory complex MCC. We previously reported on the widespread occurrences of independent gene duplications of an ancestral 'MadBub' gene in eukaryotic evolution and the striking parallel subfunctionalization that lead to loss of kinase function in BubR1/Mad3-like paralogues. Here, we present an elaborate subfunctionalization analysis of the Bub1/BubR1 gene family and perform de novo sequence discovery in a comparative phylogenomics framework to trace the distribution of ancestral sequence features to extant paralogues throughout the eukaryotic tree of life. We show that known ancestral sequence features are consistently retained in the same functional paralogue: GLEBS/CMI/ CDII/kinase in the Bub1-like and KEN1/KEN2/D-Box in the BubR1/ Mad3-like. The recently described ABBA motif can be found in either or both paralogues. We however discovered two additional ABBA motifs that flank KEN2. This cassette of ABBA1-KEN2-ABBA2 forms a strictly conserved module in all ancestral and BubR1/Mad3-like proteins, suggestive of a specific and crucial SAC function. Indeed, deletion of the ABBA motifs in human BUBR1 abrogates the SAC and affects APC/C-Cdc20 interactions. Our detailed comparative genomics analyses thus enabled discovery of a conserved cassette of motifs essential for the SAC and shows how this approach can be used to uncover hitherto unrecognized functional protein features.

Dynamic Kinetochore Size Regulation Promotes Microtubule Capture and Chromosome Biorientation in Mitosis

“…Spindly in turn recruits dynein/dynactin through two motifs: the Spindly box (SB), and the recently identified CC1 box (Figure 1b and S2a) 35,43,51,52 . Our ConFeaX pipeline, which identifies conserved motifs in orthologous protein families 56,57 , additionally identified a Qxx[HY] motif close to the CC1 box, which we here named the CC2 box ( Figure 1b). CC2-like boxes can be found in other dynein/dynactin adaptors (BICD1/2, BICDR1/2, TRAK1/2, and HAP1, Figure S2a), suggesting they are involved in dynein/dynactin binding.…”

The BUBR1 pseudokinase domain promotes efficient kinetochore PP2A-B56 recruitment to regulate spindle checkpoint silencing and chromosome alignment

“…In an attempt to identify potential functions for the BUBR1 pseudokinase domain, we sought to determine whether there is a co-evolutionary relationship between the pseudokinase and additional domains of BUBR1. To do this, we analyzed sequences from Ensembl Orthologs as well as previously collated sequences 39, 50 . We measured co-evolution between domains for both BUB1 and BUBR1 by looking at the mutual information between residues and applying normalizations to correct for background noise 51 and intrinsic differences in variability between different domains (see methods).…”

“…A number of early studies suggested that characteristic BUBR1 mitotic hyperphosphorylation occurs through kinase activation and autophosphorylation resulting from a direct association of the kinase domain with the kinesin motor CENP-E 41–43 . More recently, it has been proposed that vertebrate BUBR1 is in fact an unusual pseudokinase that has retained the catalytic triad, but is inactive as a result of the degeneration of the Gly-rich loop, together with an activation loop phosphorylation that is incompatible with catalysis 38, 39, 44, 45 . Instead, the BUBR1 kinase domain has been shown to confer stability, and mutations in and around the C-terminal domain reduce total BUBR1 protein levels and are associated with the cancer predisposition disease Mosaic Variegated Aneuploidy (MVA) 46–49 .…”

“…In most vertebrates a third scenario occurred where the KEN box-containing gene retained a kinase domain lacking catalytic function (domain in purple). Some exceptions to this exist and are discussed in detail in Suijkerbuijk et al 38 and Vleugel et al 40 and Tromer et al 39 .…”

Mitotic Regulators and the SHP2-MAPK Pathway Promote Insulin Receptor Endocytosis and Feedback Regulation of Insulin Signaling

“…1c). BUBR1 interacts with CDC20 through multiple binding motifs 22, [33][34][35] . We constructed BUBR1 mutants targeting KEN1 (ΔKEN1) or KEN2 (ΔKEN2) boxes in the N-terminal region, and Phe (ΔPhe, also known as ABBA3) and D boxes (ΔD2) in the middle region ( Fig.…”