Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores

Mathijs Vleugel; Manja Omerzu; Vincent  Groenewold; Michael A  Hadders; Susanne M A Lens; Geert J P L Kops

doi:10.1016/j.molcel.2014.12.036

Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores

Mathijs Vleugel, Manja Omerzu, Vincent Groenewold, Michael A Hadders, Susanne M A Lens, Geert J P L Kops

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Regulated recruitment of the kinase-adaptor complex BUB1/BUB3 to kinetochores is crucial for correcting faulty chromosome-spindle attachments and for spindle assembly checkpoint (SAC) signaling. BUB1/BUB3 localizes to kinetochores by binding phosphorylated MELT motifs (MELpT) in the kinetochore scaffold KNL1. Human KNL1 has 19 repeats that contain a MELT-like sequence. The repeats are, however, larger than MELT, and repeat sequences can vary significantly. Using systematic screening, we show that only a limited number of repeats is "active." Repeat activity correlates with the presence of a vertebrate-specific SHT motif C-terminal to the MELT sequence. SHT motifs are phosphorylated by MPS1 in a manner that requires prior phosphorylation of MELT. Phospho-SHT (SHpT) synergizes with MELpT in BUB3/BUB1 binding in vitro and in cells, and human BUB3 mutated in a predicted SHpT-binding surface cannot localize to kinetochores. Our data show sequential multisite regulation of the KNL1-BUB1/BUB3 interaction and provide mechanistic insight into evolution of the KNL1-BUB3 interface.

Original language	English
Pages (from-to)	824-835
Number of pages	12
Journal	Molecular Cell
Volume	57
Issue number	5
DOIs	https://doi.org/10.1016/j.molcel.2014.12.036
Publication status	Published - 2015

Keywords

Amino Acid Motifs
Amino Acid Sequence
Cell Cycle Proteins
HeLa Cells
Humans
Immunoblotting
Kinetochores
M Phase Cell Cycle Checkpoints
Microtubule-Associated Proteins
Mitosis
Models, Molecular
Molecular Sequence Data
Mutation
Nocodazole
Phosphorylation
Protein Binding
Protein Structure, Tertiary
Protein-Serine-Threonine Kinases
Protein-Tyrosine Kinases
RNA Interference
Repetitive Sequences, Amino Acid
Sequence Homology, Amino Acid
Time-Lapse Imaging
Tubulin Modulators

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title = "Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores",

abstract = "Regulated recruitment of the kinase-adaptor complex BUB1/BUB3 to kinetochores is crucial for correcting faulty chromosome-spindle attachments and for spindle assembly checkpoint (SAC) signaling. BUB1/BUB3 localizes to kinetochores by binding phosphorylated MELT motifs (MELpT) in the kinetochore scaffold KNL1. Human KNL1 has 19 repeats that contain a MELT-like sequence. The repeats are, however, larger than MELT, and repeat sequences can vary significantly. Using systematic screening, we show that only a limited number of repeats is {"}active.{"} Repeat activity correlates with the presence of a vertebrate-specific SHT motif C-terminal to the MELT sequence. SHT motifs are phosphorylated by MPS1 in a manner that requires prior phosphorylation of MELT. Phospho-SHT (SHpT) synergizes with MELpT in BUB3/BUB1 binding in vitro and in cells, and human BUB3 mutated in a predicted SHpT-binding surface cannot localize to kinetochores. Our data show sequential multisite regulation of the KNL1-BUB1/BUB3 interaction and provide mechanistic insight into evolution of the KNL1-BUB3 interface.",

keywords = "Amino Acid Motifs, Amino Acid Sequence, Cell Cycle Proteins, HeLa Cells, Humans, Immunoblotting, Kinetochores, M Phase Cell Cycle Checkpoints, Microtubule-Associated Proteins, Mitosis, Models, Molecular, Molecular Sequence Data, Mutation, Nocodazole, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, RNA Interference, Repetitive Sequences, Amino Acid, Sequence Homology, Amino Acid, Time-Lapse Imaging, Tubulin Modulators",

author = "Mathijs Vleugel and Manja Omerzu and Vincent Groenewold and Hadders, {Michael A} and Lens, {Susanne M A} and Kops, {Geert J P L}",

year = "2015",

doi = "10.1016/j.molcel.2014.12.036",

language = "English",

volume = "57",

pages = "824--835",

journal = "Molecular Cell",

issn = "1097-2765",

publisher = "Cell Press",

number = "5",

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T1 - Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores

AU - Vleugel, Mathijs

AU - Omerzu, Manja

AU - Groenewold, Vincent

AU - Hadders, Michael A

AU - Lens, Susanne M A

AU - Kops, Geert J P L

PY - 2015

Y1 - 2015

N2 - Regulated recruitment of the kinase-adaptor complex BUB1/BUB3 to kinetochores is crucial for correcting faulty chromosome-spindle attachments and for spindle assembly checkpoint (SAC) signaling. BUB1/BUB3 localizes to kinetochores by binding phosphorylated MELT motifs (MELpT) in the kinetochore scaffold KNL1. Human KNL1 has 19 repeats that contain a MELT-like sequence. The repeats are, however, larger than MELT, and repeat sequences can vary significantly. Using systematic screening, we show that only a limited number of repeats is "active." Repeat activity correlates with the presence of a vertebrate-specific SHT motif C-terminal to the MELT sequence. SHT motifs are phosphorylated by MPS1 in a manner that requires prior phosphorylation of MELT. Phospho-SHT (SHpT) synergizes with MELpT in BUB3/BUB1 binding in vitro and in cells, and human BUB3 mutated in a predicted SHpT-binding surface cannot localize to kinetochores. Our data show sequential multisite regulation of the KNL1-BUB1/BUB3 interaction and provide mechanistic insight into evolution of the KNL1-BUB3 interface.

AB - Regulated recruitment of the kinase-adaptor complex BUB1/BUB3 to kinetochores is crucial for correcting faulty chromosome-spindle attachments and for spindle assembly checkpoint (SAC) signaling. BUB1/BUB3 localizes to kinetochores by binding phosphorylated MELT motifs (MELpT) in the kinetochore scaffold KNL1. Human KNL1 has 19 repeats that contain a MELT-like sequence. The repeats are, however, larger than MELT, and repeat sequences can vary significantly. Using systematic screening, we show that only a limited number of repeats is "active." Repeat activity correlates with the presence of a vertebrate-specific SHT motif C-terminal to the MELT sequence. SHT motifs are phosphorylated by MPS1 in a manner that requires prior phosphorylation of MELT. Phospho-SHT (SHpT) synergizes with MELpT in BUB3/BUB1 binding in vitro and in cells, and human BUB3 mutated in a predicted SHpT-binding surface cannot localize to kinetochores. Our data show sequential multisite regulation of the KNL1-BUB1/BUB3 interaction and provide mechanistic insight into evolution of the KNL1-BUB3 interface.

KW - Amino Acid Motifs

KW - Amino Acid Sequence

KW - Cell Cycle Proteins

KW - HeLa Cells

KW - Humans

KW - Immunoblotting

KW - Kinetochores

KW - M Phase Cell Cycle Checkpoints

KW - Microtubule-Associated Proteins

KW - Mitosis

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Mutation

KW - Nocodazole

KW - Phosphorylation

KW - Protein Binding

KW - Protein Structure, Tertiary

KW - Protein-Serine-Threonine Kinases

KW - Protein-Tyrosine Kinases

KW - RNA Interference

KW - Repetitive Sequences, Amino Acid

KW - Sequence Homology, Amino Acid

KW - Time-Lapse Imaging

KW - Tubulin Modulators

U2 - 10.1016/j.molcel.2014.12.036

DO - 10.1016/j.molcel.2014.12.036

M3 - Article

C2 - 25661489

SN - 1097-2765

VL - 57

SP - 824

EP - 835

JO - Molecular Cell

JF - Molecular Cell

IS - 5

ER -

Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores

Abstract

Keywords

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