Proteomic analysis of human metaphase chromosomes reveals Topoisomerase II alpha as an Aurora B substrate

Ciaran Morrison, Alexander J Henzing, Ole Nørregaard Jensen, Neil Osheroff, Helen Dodson, Stefanie E Kandels-Lewis, Richard R Adams, William C Earnshaw

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Udgivelsesdato: 2002-Dec-1
OriginalsprogEngelsk
TidsskriftNucleic Acids Research
Vol/bind30
Udgave nummer23
Sider (fra-til)5318-27
Antal sider9
ISSN0305-1048
StatusUdgivet - 2002

Fingeraftryk

Metaphase
Proteins
HeLa Cells
Aurora Kinase B
Chromatids
Organism Cloning
DNA topoisomerase II alpha

Citer dette

Morrison, C., Henzing, A. J., Jensen, O. N., Osheroff, N., Dodson, H., Kandels-Lewis, S. E., ... Earnshaw, W. C. (2002). Proteomic analysis of human metaphase chromosomes reveals Topoisomerase II alpha as an Aurora B substrate. Nucleic Acids Research, 30(23), 5318-27.
Morrison, Ciaran ; Henzing, Alexander J ; Jensen, Ole Nørregaard ; Osheroff, Neil ; Dodson, Helen ; Kandels-Lewis, Stefanie E ; Adams, Richard R ; Earnshaw, William C. / Proteomic analysis of human metaphase chromosomes reveals Topoisomerase II alpha as an Aurora B substrate. I: Nucleic Acids Research. 2002 ; Bind 30, Nr. 23. s. 5318-27.
@article{532aaa206fcf11db81a9000ea68e967b,
title = "Proteomic analysis of human metaphase chromosomes reveals Topoisomerase II alpha as an Aurora B substrate",
abstract = "The essential Aurora B kinase is a chromosomal passenger protein that is required for mitotic chromosome alignment and segregation. Aurora B function is dependent on the chromosome passenger, INCENP. INCENP, in turn, requires sister chromatid cohesion for its appropriate behaviour. Relatively few substrates have been identified for Aurora B, so that the precise role it plays in controlling mitosis remains to be elucidated. To identify potential novel mitotic substrates of Aurora B, extracted chromosomes were prepared from mitotically-arrested HeLa S3 cells and incubated with recombinant human Aurora B in the presence of radioactive ATP. Immunoblot analysis confirmed the HeLa scaffold fraction to be enriched for known chromosomal proteins including CENP-A, CENP-B, CENP-C, ScII and INCENP. Mass spectrometry of bands excised from one-dimensional polyacrylamide gels further defined the protein composition of the extracted chromosome fraction. Cloning, fluorescent tagging and expression in HeLa cells of the putative GTP-binding protein NGB/CRFG demonstrated it to be a novel mitotic chromosome protein, with a perichromosomal localisation. Identi fication of the protein bands corresponding to those phosphorylated by Aurora B revealed topoisomerase II alpha (topo IIalpha) as a potential Aurora B substrate. Purified recombinant human topo IIalpha was phosphorylated by Aurora B in vitro, confirming this proteomic approach as a valid method for the initial definition of candidate substrates of key mitotic kinases.",
keywords = "Antigens, Neoplasm, Chromosomes, Human, DNA Topoisomerases, Type II, Eukaryotic, DNA-Binding Proteins, Fluorescent Antibody Technique, GTP-Binding Proteins, Hela Cells, Humans, Metaphase, Nuclear Proteins, Protein-Serine-Threonine Kinases, Proteomics, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization",
author = "Ciaran Morrison and Henzing, {Alexander J} and Jensen, {Ole N{\o}rregaard} and Neil Osheroff and Helen Dodson and Kandels-Lewis, {Stefanie E} and Adams, {Richard R} and Earnshaw, {William C}",
year = "2002",
language = "English",
volume = "30",
pages = "5318--27",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Heinemann",
number = "23",

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Morrison, C, Henzing, AJ, Jensen, ON, Osheroff, N, Dodson, H, Kandels-Lewis, SE, Adams, RR & Earnshaw, WC 2002, 'Proteomic analysis of human metaphase chromosomes reveals Topoisomerase II alpha as an Aurora B substrate', Nucleic Acids Research, bind 30, nr. 23, s. 5318-27.

Proteomic analysis of human metaphase chromosomes reveals Topoisomerase II alpha as an Aurora B substrate. / Morrison, Ciaran; Henzing, Alexander J; Jensen, Ole Nørregaard; Osheroff, Neil; Dodson, Helen; Kandels-Lewis, Stefanie E; Adams, Richard R; Earnshaw, William C.

I: Nucleic Acids Research, Bind 30, Nr. 23, 2002, s. 5318-27.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Proteomic analysis of human metaphase chromosomes reveals Topoisomerase II alpha as an Aurora B substrate

AU - Morrison, Ciaran

AU - Henzing, Alexander J

AU - Jensen, Ole Nørregaard

AU - Osheroff, Neil

AU - Dodson, Helen

AU - Kandels-Lewis, Stefanie E

AU - Adams, Richard R

AU - Earnshaw, William C

PY - 2002

Y1 - 2002

N2 - The essential Aurora B kinase is a chromosomal passenger protein that is required for mitotic chromosome alignment and segregation. Aurora B function is dependent on the chromosome passenger, INCENP. INCENP, in turn, requires sister chromatid cohesion for its appropriate behaviour. Relatively few substrates have been identified for Aurora B, so that the precise role it plays in controlling mitosis remains to be elucidated. To identify potential novel mitotic substrates of Aurora B, extracted chromosomes were prepared from mitotically-arrested HeLa S3 cells and incubated with recombinant human Aurora B in the presence of radioactive ATP. Immunoblot analysis confirmed the HeLa scaffold fraction to be enriched for known chromosomal proteins including CENP-A, CENP-B, CENP-C, ScII and INCENP. Mass spectrometry of bands excised from one-dimensional polyacrylamide gels further defined the protein composition of the extracted chromosome fraction. Cloning, fluorescent tagging and expression in HeLa cells of the putative GTP-binding protein NGB/CRFG demonstrated it to be a novel mitotic chromosome protein, with a perichromosomal localisation. Identi fication of the protein bands corresponding to those phosphorylated by Aurora B revealed topoisomerase II alpha (topo IIalpha) as a potential Aurora B substrate. Purified recombinant human topo IIalpha was phosphorylated by Aurora B in vitro, confirming this proteomic approach as a valid method for the initial definition of candidate substrates of key mitotic kinases.

AB - The essential Aurora B kinase is a chromosomal passenger protein that is required for mitotic chromosome alignment and segregation. Aurora B function is dependent on the chromosome passenger, INCENP. INCENP, in turn, requires sister chromatid cohesion for its appropriate behaviour. Relatively few substrates have been identified for Aurora B, so that the precise role it plays in controlling mitosis remains to be elucidated. To identify potential novel mitotic substrates of Aurora B, extracted chromosomes were prepared from mitotically-arrested HeLa S3 cells and incubated with recombinant human Aurora B in the presence of radioactive ATP. Immunoblot analysis confirmed the HeLa scaffold fraction to be enriched for known chromosomal proteins including CENP-A, CENP-B, CENP-C, ScII and INCENP. Mass spectrometry of bands excised from one-dimensional polyacrylamide gels further defined the protein composition of the extracted chromosome fraction. Cloning, fluorescent tagging and expression in HeLa cells of the putative GTP-binding protein NGB/CRFG demonstrated it to be a novel mitotic chromosome protein, with a perichromosomal localisation. Identi fication of the protein bands corresponding to those phosphorylated by Aurora B revealed topoisomerase II alpha (topo IIalpha) as a potential Aurora B substrate. Purified recombinant human topo IIalpha was phosphorylated by Aurora B in vitro, confirming this proteomic approach as a valid method for the initial definition of candidate substrates of key mitotic kinases.

KW - Antigens, Neoplasm

KW - Chromosomes, Human

KW - DNA Topoisomerases, Type II, Eukaryotic

KW - DNA-Binding Proteins

KW - Fluorescent Antibody Technique

KW - GTP-Binding Proteins

KW - Hela Cells

KW - Humans

KW - Metaphase

KW - Nuclear Proteins

KW - Protein-Serine-Threonine Kinases

KW - Proteomics

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

M3 - Journal article

C2 - 12466558

VL - 30

SP - 5318

EP - 5327

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 23

ER -

Morrison C, Henzing AJ, Jensen ON, Osheroff N, Dodson H, Kandels-Lewis SE et al. Proteomic analysis of human metaphase chromosomes reveals Topoisomerase II alpha as an Aurora B substrate. Nucleic Acids Research. 2002;30(23):5318-27.