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Polo kinase interacts with RacGAP50C and is required to localize the cytokinesis initiation complex.

ABSTRACT: The assembly and constriction of an actomyosin contractile ring in cytokinesis is dependent on the activation of Rho at the equatorial cortex by a complex, here termed the cytokinesis initiation complex, between a microtubule-associated kinesin-like protein (KLP), a member of the RacGAP family, and the RhoGEF Pebble. Recently, the activity of the mammalian Polo kinase ortholog Plk1 has been implicated in the formation of this complex. We show here that Polo kinase interacts directly with the cytokinesis initiation complex by binding RacGAP50C. We find that a new domain of Polo kinase, termed the intermediate domain, interacts directly with RacGAP50C and that Polo kinase is essential for localization of the KLP-RacGAP centralspindlin complex to the cell equator and spindle midzone. In the absence of Polo kinase, RacGAP50C and Pav-KLP fail to localize normally, instead decorating microtubules along their length. Our results indicate that Polo kinase directly binds the conserved cytokinesis initiation complex and is required to trigger centralspindlin localization as a first step in cytokinesis.

SUBMITTER: Ebrahimi S

PROVIDER: S-EPMC2937893 | biostudies-other | 2010 Sep

REPOSITORIES: biostudies-other

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Polo kinase interacts with RacGAP50C and is required to localize the cytokinesis initiation complex.

Ebrahimi Saman S Fraval Hamilton H Murray Michael M Saint Robert R Gregory Stephen L SL

The Journal of biological chemistry 20100713 37

The assembly and constriction of an actomyosin contractile ring in cytokinesis is dependent on the activation of Rho at the equatorial cortex by a complex, here termed the cytokinesis initiation complex, between a microtubule-associated kinesin-like protein (KLP), a member of the RacGAP family, and the RhoGEF Pebble. Recently, the activity of the mammalian Polo kinase ortholog Plk1 has been implicated in the formation of this complex. We show here that Polo kinase interacts directly with the cyt ...[more]

PMID: 20628062

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Project description:BackgroundPolo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation.MethodsTo assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites RESULTS: After incubating trophozoites with 5 μM GW, the percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis and flagella formation.ConclusionsThese data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.

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Polo kinase interacts with RacGAP50C and is required to localize the cytokinesis initiation complex.

Publications

Polo kinase interacts with RacGAP50C and is required to localize the cytokinesis initiation complex.

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