Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells.

Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but...

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Autores principales:	Mark E Burkard, John Maciejowski, Verónica Rodriguez-Bravo, Michael Repka, Drew M Lowery, Karl R Clauser, Chao Zhang, Kevan M Shokat, Steven A Carr, Michael B Yaffe, Prasad V Jallepalli
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2009
Materias:	Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/efcc32a4efcb4dc7bae4cafa4776e364
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spelling	oai:doaj.org-article:efcc32a4efcb4dc7bae4cafa4776e3642021-11-25T05:34:10ZPlk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells.1544-91731545-788510.1371/journal.pbio.1000111https://doaj.org/article/efcc32a4efcb4dc7bae4cafa4776e3642009-05-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19468302/pdf/?tool=EBIhttps://doaj.org/toc/1544-9173https://doaj.org/toc/1545-7885Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but how such cues are generated remains unclear. Using chemical genetics to achieve both temporal and spatial control, we show that the self-organized delivery of Polo-like kinase 1 (Plk1) to the midzone and its local phosphorylation of a MT-bound substrate are critical for generating this furrow-inducing signal. When Plk1 was active but unable to target itself to this equatorial landmark, both cortical RhoA recruitment and furrow induction failed to occur, thus recapitulating the effects of anaphase-specific Plk1 inhibition. Using tandem mass spectrometry and phosphospecific antibodies, we found that Plk1 binds and directly phosphorylates the HsCYK-4 subunit of centralspindlin (also known as MgcRacGAP) at the midzone. At serine 157, this modification creates a major docking site for the tandem BRCT repeats of the Rho GTP exchange factor Ect2. Cells expressing only a nonphosphorylatable form of HsCYK-4 failed to localize Ect2 at the midzone and were severely impaired in cleavage furrow formation, implying that HsCYK-4 is Plk1's rate-limiting target upstream of RhoA. Conversely, tethering an inhibitor-resistant allele of Plk1 to HsCYK-4 allowed furrows to form despite global inhibition of all other Plk1 molecules in the cell. Our findings illuminate two key mechanisms governing the initiation of cytokinesis in human cells and illustrate the power of chemical genetics to probe such regulation both in time and space.Mark E BurkardJohn MaciejowskiVerónica Rodriguez-BravoMichael RepkaDrew M LoweryKarl R ClauserChao ZhangKevan M ShokatSteven A CarrMichael B YaffePrasad V JallepalliPublic Library of Science (PLoS)articleBiology (General)QH301-705.5ENPLoS Biology, Vol 7, Iss 5, p e1000111 (2009)
institution	DOAJ
collection	DOAJ
language	EN
topic	Biology (General) QH301-705.5
spellingShingle	Biology (General) QH301-705.5 Mark E Burkard John Maciejowski Verónica Rodriguez-Bravo Michael Repka Drew M Lowery Karl R Clauser Chao Zhang Kevan M Shokat Steven A Carr Michael B Yaffe Prasad V Jallepalli Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells.
description	Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but how such cues are generated remains unclear. Using chemical genetics to achieve both temporal and spatial control, we show that the self-organized delivery of Polo-like kinase 1 (Plk1) to the midzone and its local phosphorylation of a MT-bound substrate are critical for generating this furrow-inducing signal. When Plk1 was active but unable to target itself to this equatorial landmark, both cortical RhoA recruitment and furrow induction failed to occur, thus recapitulating the effects of anaphase-specific Plk1 inhibition. Using tandem mass spectrometry and phosphospecific antibodies, we found that Plk1 binds and directly phosphorylates the HsCYK-4 subunit of centralspindlin (also known as MgcRacGAP) at the midzone. At serine 157, this modification creates a major docking site for the tandem BRCT repeats of the Rho GTP exchange factor Ect2. Cells expressing only a nonphosphorylatable form of HsCYK-4 failed to localize Ect2 at the midzone and were severely impaired in cleavage furrow formation, implying that HsCYK-4 is Plk1's rate-limiting target upstream of RhoA. Conversely, tethering an inhibitor-resistant allele of Plk1 to HsCYK-4 allowed furrows to form despite global inhibition of all other Plk1 molecules in the cell. Our findings illuminate two key mechanisms governing the initiation of cytokinesis in human cells and illustrate the power of chemical genetics to probe such regulation both in time and space.
format	article
author	Mark E Burkard John Maciejowski Verónica Rodriguez-Bravo Michael Repka Drew M Lowery Karl R Clauser Chao Zhang Kevan M Shokat Steven A Carr Michael B Yaffe Prasad V Jallepalli
author_facet	Mark E Burkard John Maciejowski Verónica Rodriguez-Bravo Michael Repka Drew M Lowery Karl R Clauser Chao Zhang Kevan M Shokat Steven A Carr Michael B Yaffe Prasad V Jallepalli
author_sort	Mark E Burkard
title	Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells.
title_short	Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells.
title_full	Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells.
title_fullStr	Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells.
title_full_unstemmed	Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells.
title_sort	plk1 self-organization and priming phosphorylation of hscyk-4 at the spindle midzone regulate the onset of division in human cells.
publisher	Public Library of Science (PLoS)
publishDate	2009
url	https://doaj.org/article/efcc32a4efcb4dc7bae4cafa4776e364
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Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells.

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