Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Dec 10;10(12):e0144673.
doi: 10.1371/journal.pone.0144673. eCollection 2015.

Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic Checkpoint Kinase Bub1

Claudia Breit  1 , Tanja Bange  1 , Arsen Petrovic  1 , John R Weir  1 , Franziska Müller  1 , Doro Vogt  1 , Andrea Musacchio  1   2
Affiliations

Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic Checkpoint Kinase Bub1

Claudia Breit et al. PLoS One. .

Abstract

The spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Reconstitution of Bub1 kinase and BubR1 pseudo-kinase.
(A) Bub1 and BubR1 share a similar domain organization. Schematic description of the domains and constructs used in this manuscript: TPR–tetratricopeptide repeat, BD–binding domain, WD40 –an approximately 40-residue sequence repeat often terminating with a tryptophan (W) and aspartate (D) dipeptide. (B) Purified Bub1kinase, Bub1:Bub3, BubR1kinase, and BubR1:Bub3 were separated by SDS-PAGE. Their respective expected molecular mass is indicated. (C) Both Bub1kinase and BubR1kinase bind to mant-ATP. The change in fluorescence emission at 450 nm is plotted as a function of total mant-ATP concentration. The data were fitted with a one site binding equation using Origin 9.0, with R2 = 0.99 for both curves. Error bars represent SD of a mean of at least 2 independent experiments. a.u.–arbitrary units. (D-E) ESI-MS spectra of purified Bub1kinase (D) or BubR1kinase (E) before and after incubation with ATP for auto-phosphorylation. Theoretical calculated masses are given in brackets under the measured masses. (F) BubR1 is not an active kinase. Maltose binding protein (MBP), H1, and the Borealin:Survivin complex (Bor:Sur) were incubated with 50 nM BubR1 constructs and ATP and analyzed on SDS PAGE visualizing phosphates using the Pro-Q® Diamond Phosphoprotein Gel Stain. 10 nM Bub1:Bub3 was used as a positive control. BR1kinase–BubR1kinase, BR1B3 –BubR1:Bub3.
Fig 2
Fig 2. Kinetic characterization of Bub1 complexes.
(A-C) Bub1kinase and Bub1:Bub3 complex exhibit similar catalytic activity toward H2A (A) and Bor:Sur substrates (B) and hydrolyze ATP at similar rates (C). The mutation D917N abrogates ATPase activity (C). The kinase activity was determined using the ADP-GloTM Kinase Assay and is plotted as a function of substrate concentration to allow fitting according to the Michaelis-Menten equation with R2 = 0.99 (Bub1kinase on Bor:Sur R2 = 0.97). KD–kinase dead. Error bars represent SD of a mean of at least 2 independent experiments. (D) Kinetic parameters of the Michaelis-Menten fits as determined in (A-C). (E) H2A contained in H3- or CENP-A nucleosomes is efficiently phosphorylated by Bub1kinase and Bub1:Bub3. (F) The kinase activity, plotted as a function of substrate concentration, allows fitting with the Michaelis-Menten equation with R2 = 0.95 (H3) and 0.99 (CENP-A). Error bars represent SD of a mean of at least two independent experiments. (G-H) EMSA assays showing DNA and nucleosome binding of Bub1kinase and H3- or CENP-A nucleosomes. MN–mononucleosomes.
Fig 3
Fig 3. S969 is a major phosphorylation site in Bub1kinase.
(A) Ribbon diagram showing the model of phosphorylated Bub1kinase. N and C indicate the N- and C-terminus, respectively. The P+1 loop harboring the phosphorylated S969 is highlighted in green, the activation loop is in blue, and the catalytic loop in red. Side chains of residues D946, D917, S969 and ADP are shown as sticks, an Mg2+ atom is represented by a gray sphere. (B) Detailed view of the active site of Bub1 showing P-S969 pointing towards the catalytic aspartate at position 917 (D917) and ADP. The electron density around the P+1 loop represents a 2Fo-Fc map contoured at 1.5σ as a blue mesh. (C-D) Overlay of phosphorylated Bub1 with unphosphorylated Bub1 [4R8Q, (C)] and phosphorylated Bub1 [4QPM, (D)]. The P+1 loop of 4R8Q and 4QPM is colored in shades of blue. All images were created with CCP4MG [53]. (E) Amino acid sequence of the Bub1 P+1 loop with the mutations of S969 highlighted in red (S969D) and blue (S969A). (F) Bub1kinase wild-type (WT) and S969A, S969D, S969E were incubated with ATP or λ-phosphatase (λ-pp) and analyzed on SDS PAGE using Phos-tag to detect a phosphorylation-specific shift. (G) P-S969 does not interfere with ATP binding of Bub1kinase. The change in fluorescence emission at 450 nm is plotted as a function of total mant-ATPγS concentration. The data were fitted with a one site binding equation using Origin 9.0 and R2 = 0.99. Error bars represent SD of a mean of at least two independent experiments. a.u.-arbitrary units. (H) The phosphomimetic S969D is able to restore kinase activity while S969A is catalytically inactive. The kinase activity is plotted as a function of substrate concentration to allow fitting according to Michaelis-Menten kinetics with R2 = 0.99 (WT), R2 = 0.97 (S969D). Error bars represent SD of a mean of at least 2 independent experiments.
Fig 4
Fig 4. Bub1kinase possesses conserved residues that recognize a substrate consensus sequence.
(A) Alignment of phosphorylation sites found in Bub1-dependent phosphorylation reactions using the ClustalX algorithm in Jalview [54]. Numbers indicate boundaries of the protein segments shown; phosphorylated residues are denoted with an asterisk, the conservation of residues is highlighted using the ClustalX coloring scheme. (B) The structure of Bub1kinase (PDB ID 4QPM) was superimposed onto the structure of PKA bound to a pseudo-substrate inhibitor [PDB ID 1APM, reference [55]]. The image shows only Bub1 and the position of the pseudo-substrate peptide (yellow) after alignment of PKA to Bub1. Bub1 residues putatively involved in substrate recognition are depicted as sticks. (C) Bub1kinase sequence and structure conservation. The same residues as in (B) are shown in sticks. Conservation was determined by aligning Bub1kinase from 14 organisms with ConSurf [56], the scoring legend is depicted on the upper left. Conservation scores obtained for positions in the alignment that had less than 6 un-gapped amino acids were considered to be unreliable and colored light yellow in the graphic visualization output. Images were created with CCP4MG [53] and Pymol (Schrödinger LLC, Portland, OR). (D) Bub1kinase-dependent H2A phosphorylation can be reduced by mutating V115D, L116N, L117N on H2A. GST-H2A constructs were incubated with Bub1kinase and ATP, then analyzed by SDS PAGE, and phosphorylated proteins were specifically stained using Pro-Q® Diamond Phosphoprotein Gel Stain.
Fig 5
Fig 5. Biochemical characterization of Bub1 complexes.
(A) Bub1:Bub3 (green) and BubR1:Bub3 (blue) co-elute in a stable complex from a size exclusion chromatography column (black) and can be assembled as a complex onto phosphorylated MBP-Knl1138-225 (purple). A bar of the respective color indicates fractions analyzed on SDS PAGE (below). (B) Analytical ultracentrifugation. Normalized c(s) distribution curves for Bub1:Bub3 (green) and BubR1:Bub3 (blue). A predominant peak at 4.5 S (s20,w = 4.7) is apparent for Bub1:Bub3 and at 4.3 S (s20,w = 4.5) for BubR1:Bub3 indicating a single dominant sedimentation species that corresponds to a theoretical molecular weight of 150 kDa and 144 kDa, respectively. The theoretical mass of a 1:1 complex is indicated for both in brackets. Frictional ratios were determined as 2.16 for Bub1:Bub3 and 2.21 for BubR1:Bub3. (C) Complexes of Bub1 exhibit similar kinase activity toward a GST-H2A substrate. The kinase activity is plotted as a function of substrate concentration to allow fitting according to the Michaelis-Menten equation with R2 = 0.99. Error bars represent SD of a mean of at least two independent experiments. (D) Kinetic parameters of the Michaelis-Menten fits as determined in (C). a.u.-arbitrary units.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Foley EA, Kapoor TM. Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore. Nature reviews Molecular cell biology. 2013;14(1):25–37. 10.1038/nrm3494 - DOI - PMC - PubMed
    1. Lara-Gonzalez P, Westhorpe FG, Taylor SS. The spindle assembly checkpoint. Current biology: CB. 2012;22(22):R966–80. Epub 2012/11/24. 10.1016/j.cub.2012.10.006 . - DOI - PubMed
    1. London N, Biggins S. Signalling dynamics in the spindle checkpoint response. Nature reviews Molecular cell biology. 2014;15(11):736–47. 10.1038/nrm3888 - DOI - PMC - PubMed
    1. Suijkerbuijk SJ, van Dam TJ, Karagoz GE, von Castelmur E, Hubner NC, Duarte AM, et al. The vertebrate mitotic checkpoint protein BUBR1 is an unusual pseudokinase. Developmental cell. 2012;22(6):1321–9. 10.1016/j.devcel.2012.03.009 . - DOI - PubMed
    1. Vleugel M, Hoogendoorn E, Snel B, Kops GJ. Evolution and function of the mitotic checkpoint. Developmental cell. 2012;23(2):239–50. 10.1016/j.devcel.2012.06.013 . - DOI - PubMed

Publication types

MeSH terms

Grants and funding

A.M. gratefully acknowledges funding by the European Research Council (ERC) Advanced Investigator Grant RECEPIANCE (proposal number n° 669686).
-