The University of Southampton
University of Southampton Institutional Repository

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: a reappraisal of translation initiation during mitosis

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: a reappraisal of translation initiation during mitosis
Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: a reappraisal of translation initiation during mitosis
Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G 1/S boundary) or the Cdk1 inhibitor, RO3306 (G 2/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2? phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.
1538-4101
1-14
Coldwell, Mark J.
a3432799-ed45-4948-9f7a-2a284d3ec65c
Cowan, Joanne L.
a34fc26a-e7a3-435f-85c8-4f196b1c8d63
Vlasak, Markete
9bb9fec4-2d64-42b9-84e7-f92be380d5b5
Mead, Abbie
93c092b9-d3d3-4763-b020-04d8ac48f503
Willett, Mark
dfa36c04-719a-4884-87cd-2ae13a3d2b67
Perry, Lisa S.
54503067-f509-4deb-9c54-6f656787894b
Morley, Simon J.
059a6269-c9e5-4165-8498-238153537ceb
Coldwell, Mark J.
a3432799-ed45-4948-9f7a-2a284d3ec65c
Cowan, Joanne L.
a34fc26a-e7a3-435f-85c8-4f196b1c8d63
Vlasak, Markete
9bb9fec4-2d64-42b9-84e7-f92be380d5b5
Mead, Abbie
93c092b9-d3d3-4763-b020-04d8ac48f503
Willett, Mark
dfa36c04-719a-4884-87cd-2ae13a3d2b67
Perry, Lisa S.
54503067-f509-4deb-9c54-6f656787894b
Morley, Simon J.
059a6269-c9e5-4165-8498-238153537ceb

Coldwell, Mark J., Cowan, Joanne L., Vlasak, Markete, Mead, Abbie, Willett, Mark, Perry, Lisa S. and Morley, Simon J. (2013) Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: a reappraisal of translation initiation during mitosis. Cell Cycle, 12 (23), 1-14. (doi:10.4161/cc.26588). (PMID:24091728)

Record type: Article

Abstract

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G 1/S boundary) or the Cdk1 inhibitor, RO3306 (G 2/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2? phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.

Text
359385COLDWELL5.pdf - Version of Record
Restricted to Repository staff only
Request a copy

More information

e-pub ahead of print date: 1 October 2013
Organisations: Molecular and Cellular

Identifiers

Local EPrints ID: 359385
URI: http://eprints.soton.ac.uk/id/eprint/359385
ISSN: 1538-4101
PURE UUID: a04ca3f9-8b16-4fd3-83dd-b8ad29b355f5
ORCID for Mark J. Coldwell: ORCID iD orcid.org/0000-0002-6243-3886

Catalogue record

Date deposited: 29 Oct 2013 14:40
Last modified: 14 Mar 2024 15:21

Export record

Altmetrics

Contributors

Author: Mark J. Coldwell ORCID iD
Author: Joanne L. Cowan
Author: Markete Vlasak
Author: Abbie Mead
Author: Mark Willett
Author: Lisa S. Perry
Author: Simon J. Morley

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×