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

Authors: Burkard, M. E.; Maciejowski, J.; Rodriguez Bravo, V.; Repka, M.; Lowery, D. M.; Clauser, K. R.; Zhang, C.; Shokat, K. M.; Carr, S. A.; Yaffe, M. B.; Jallepalli, P. V.
Article Title: Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells
Abstract: 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. © 2009 Burkard et al.
Keywords: signal transduction; controlled study; protein expression; protein phosphorylation; oncoprotein; human cell; genetics; proto-oncogene proteins; cell cycle protein; animal; metabolism; animals; cell cycle proteins; allele; cell division; serine; cell line; small interfering rna; protein binding; rna, small interfering; protein serine threonine kinase; phosphorylation; brca1 protein; physiology; animalia; regulatory mechanism; polo like kinase 1; polo-like kinase 1; protein-serine-threonine kinases; immunoprecipitation; immunoblotting; phosphospecific antibody; rhoa guanine nucleotide binding protein; ect2 protein, human; guanosine triphosphatase activating protein; mgcracgap; anaphase; binding site; chemical genetics; gene control; microtubule assembly; spindle cell; tandem mass spectrometry; armyworm; mitosis spindle; gtpase-activating proteins; mitotic spindle apparatus; spodoptera
Journal Title: PLoS Biology
Volume: 7
Issue: 5
ISSN: 1544-9173
Publisher: Public Library of Science  
Date Published: 2009-05-01
Start Page: e1000111
Language: English
DOI: 10.1371/journal.pbio.1000111
PUBMED: 19468302
PROVIDER: scopus
PMCID: PMC2680336
Notes: --- - "Cited By (since 1996): 13" - "Export Date: 30 November 2010" - "Art. No.: e1000111" - "CODEN: PBLIB" - "Source: Scopus"
Citation Impact