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. 2014 Oct 27;207(2):201–211. doi: 10.1083/jcb.201408081

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© 2014 Kachaner

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 3. — Map205 sequesters unphosphorylated Polo on microtubules and inhibits its kinase activity. (A) Silencing of Map205 rescues the midbody localization of Polo^T182A-GFP. Cells were treated for 3 d with dsRNA against Map205 or the bacterial kanamycin resistance gene (control). (A, left) Immunofluorescence showing the localization of Polo^WT-GFP or Polo^T182A-GFP during cytokinesis. Arrowheads, midbody. Bar, 5 µm. (A, top right) RNAi depletion of Map205 assessed by Western blots. (A, bottom right) Percentage of cytokinetic cells where Polo^WT-GFP or Polo^T182A-GFP were clearly localized at the midbody after RNAi treatment (n = 40, repeated three times). Error bars indicate SD. (B) A fragment of Map205 interacts with Polo in vitro. GST-Map205_254-416 or GST-bound Sepharose were incubated with lysates of cells transfected with Polo^WT-Myc, Polo^T182A-Myc, or Polo^T182D-Myc. Pull-down products were analyzed by Western blots. (C) In vitro kinase assays with Polo (100 ng) and casein as a substrate. Increasing amounts of GST-Map205_254-416 were added. Reactions were analyzed by autoradiography, Western blots, and amido black (total protein). Note that Polo is capable of autophosphorylation in vitro (Fenton and Glover, 1993). (D) Kinase assays using immunoprecipitated Polo-Myc WT and mutants. Myc-tagged proteins were immunoprecipitated and used in kinase reactions in the presence of purified GST-Map205_254-416 or the Polo inhibitor BI 2536 (300 nM) as indicated. (E) Model for the spatial regulation of the Polo in cytokinesis. Aurora B phosphorylates and activates Polo during cytokinesis. This event allows Polo to dissociate from Map205 and to relocalize to the midbody.