Phosphorylation of dynamin II at serine-764 is associated with cytokinesis

Megan Chircop, Boris Sarcevic, Martin Røssel Larsen, Chandra S Malladi, Ngoc Chau, Michael Zavortink, Charlotte M Smith, Annie Quan, Victor Anggono, Peter G Hainsa, Mark E Graham, Phillip J Robinson

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and GST-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.
OriginalsprogEngelsk
TidsskriftB B A - Molecular Cell Research
Vol/bind1818
Udgave nummer10
Sider (fra-til)1689-1699
ISSN0167-4889
DOI
StatusUdgivet - 29. dec. 2010

Fingeraftryk

Dynamin II
Cytokinesis
Serine
Endocytosis
Calcineurin
CDC2 Protein Kinase
Clathrin
Peptides
HeLa Cells
Phosphoric Monoester Hydrolases
Alanine
Small Interfering RNA
Chromatography

Citer dette

Chircop, M., Sarcevic, B., Larsen, M. R., Malladi, C. S., Chau, N., Zavortink, M., ... Robinson, P. J. (2010). Phosphorylation of dynamin II at serine-764 is associated with cytokinesis. B B A - Molecular Cell Research, 1818(10), 1689-1699. https://doi.org/10.1016/j.bbamcr.2010.12.018
Chircop, Megan ; Sarcevic, Boris ; Larsen, Martin Røssel ; Malladi, Chandra S ; Chau, Ngoc ; Zavortink, Michael ; Smith, Charlotte M ; Quan, Annie ; Anggono, Victor ; Hainsa, Peter G ; Graham, Mark E ; Robinson, Phillip J. / Phosphorylation of dynamin II at serine-764 is associated with cytokinesis. I: B B A - Molecular Cell Research. 2010 ; Bind 1818, Nr. 10. s. 1689-1699.
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title = "Phosphorylation of dynamin II at serine-764 is associated with cytokinesis",
abstract = "Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and GST-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80{\%}, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.",
author = "Megan Chircop and Boris Sarcevic and Larsen, {Martin R{\o}ssel} and Malladi, {Chandra S} and Ngoc Chau and Michael Zavortink and Smith, {Charlotte M} and Annie Quan and Victor Anggono and Hainsa, {Peter G} and Graham, {Mark E} and Robinson, {Phillip J}",
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Chircop, M, Sarcevic, B, Larsen, MR, Malladi, CS, Chau, N, Zavortink, M, Smith, CM, Quan, A, Anggono, V, Hainsa, PG, Graham, ME & Robinson, PJ 2010, 'Phosphorylation of dynamin II at serine-764 is associated with cytokinesis', B B A - Molecular Cell Research, bind 1818, nr. 10, s. 1689-1699. https://doi.org/10.1016/j.bbamcr.2010.12.018

Phosphorylation of dynamin II at serine-764 is associated with cytokinesis. / Chircop, Megan; Sarcevic, Boris; Larsen, Martin Røssel; Malladi, Chandra S; Chau, Ngoc; Zavortink, Michael; Smith, Charlotte M; Quan, Annie; Anggono, Victor; Hainsa, Peter G; Graham, Mark E; Robinson, Phillip J.

I: B B A - Molecular Cell Research, Bind 1818, Nr. 10, 29.12.2010, s. 1689-1699.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Phosphorylation of dynamin II at serine-764 is associated with cytokinesis

AU - Chircop, Megan

AU - Sarcevic, Boris

AU - Larsen, Martin Røssel

AU - Malladi, Chandra S

AU - Chau, Ngoc

AU - Zavortink, Michael

AU - Smith, Charlotte M

AU - Quan, Annie

AU - Anggono, Victor

AU - Hainsa, Peter G

AU - Graham, Mark E

AU - Robinson, Phillip J

N1 - Copyright © 2010. Published by Elsevier B.V.

PY - 2010/12/29

Y1 - 2010/12/29

N2 - Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and GST-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.

AB - Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and GST-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.

U2 - 10.1016/j.bbamcr.2010.12.018

DO - 10.1016/j.bbamcr.2010.12.018

M3 - Journal article

VL - 1818

SP - 1689

EP - 1699

JO - B B A - Molecular Cell Research

JF - B B A - Molecular Cell Research

SN - 0167-4889

IS - 10

ER -