title: Functional and Structural Characterization of Bub3·BubR1 Interactions Required for Spindle Assembly Checkpoint Signaling in Human Cells.
abstract: The spindle assembly checkpoint (SAC) is an essential safeguarding mechanism devised to ensure equal chromosome distribution in daughter cells upon mitosis. The proteins Bub3 and BubR1 are key components of the mitotic checkpoint complex, an essential part of the molecular machinery on which the SAC relies. In the present work we have performed a detailed functional and biochemical characterization of the interaction between human Bub3 and BubR1 in cells and in vitro Our results demonstrate that genetic knockdown of Bub3 abrogates the SAC, promotes apoptosis, and inhibits the proliferation of human cancer cells. We also show that the integrity of the human mitotic checkpoint complex depends on the specific recognition between BubR1 and Bub3, for which the BubR1 Gle2 binding sequence motif is essential. This 1:1 binding event is high affinity, enthalpy-driven and with slow dissociation kinetics. The affinity, kinetics, and thermodynamic parameters of the interaction are differentially modulated by small regions in the N and C termini of the Gle2 binding domain sequence, suggesting the existence of "hotspots" for this protein-protein interaction. Furthermore, we show that specific disruption of endogenous BubR1·Bub3 complexes in human cancer cells phenocopies the effects observed in gene targeting experiments. Our work enhances the current understanding of key members of the SAC and paves the road for the pursuit of novel targeted cancer therapies based on SAC inhibition.
authors.affiliation: TRG Oncology, Bayer Pharma AG, Global Drug Discovery, 13353 Berlin, Germany florian.prinz@bayer.com.
authors.forename: Florian
authors.initials: F
authors.lastname: Prinz
authors.affiliation: From Lead Discovery.
authors.forename: Vera
authors.initials: V
authors.lastname: Puetter
authors.affiliation: From Lead Discovery.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Holton
authors.affiliation: From Lead Discovery.
authors.forename: Dorothee
authors.initials: D
authors.lastname: Andres
authors.affiliation: From Lead Discovery.
authors.forename: Christian M
authors.initials: CM
authors.lastname: Stegmann
authors.affiliation: From Lead Discovery.
authors.forename: Dennis
authors.initials: D
authors.lastname: Kwiatkowski
authors.affiliation: From Lead Discovery.
authors.forename: Stefan
authors.initials: S
authors.lastname: Prechtl
authors.affiliation: TRG Oncology, Bayer Pharma AG, Global Drug Discovery, 13353 Berlin, Germany.
authors.forename: Kirstin
authors.initials: K
authors.lastname: Petersen
authors.affiliation: Target Discovery, and.
authors.forename: Georg
authors.initials: G
authors.lastname: Beckmann
authors.affiliation: TRG Oncology, Bayer Pharma AG, Global Drug Discovery, 13353 Berlin, Germany.
authors.forename: Bertolt
authors.initials: B
authors.lastname: Kreft
authors.affiliation: TRG Oncology, Bayer Pharma AG, Global Drug Discovery, 13353 Berlin, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Mumberg
authors.affiliation: From Lead Discovery, amaury.fernandez@bayer.com.
authors.forename: Amaury
authors.initials: A
authors.lastname: Fernández-Montalván
mesh_terms: D017209:Apoptosis; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D059566:M Phase Cell Cycle Checkpoints / Q000235:genetics / Q000502:physiology*; D061986:MCF-7 Cells; D008958:Models, Molecular; D000075223:Poly-ADP-Ribose Binding Proteins; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: C489444:BUB3 protein, human; D018797:Cell Cycle Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D012333:RNA, Messenger; C584277:BUB1 protein, human; D017346:Protein Serine-Threonine Kinases
keywords: Bub3; BubR1; WD40; kinetics; mitosis; protein-protein interaction; spindle assembly checkpoint; structure-function; thermodynamics