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. 2017 Aug 7;216(8):2339–2354. doi: 10.1083/jcb.201512055

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© 2017 Cullati et al.

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Figure 7. — Nek6 phosphorylates Mklp2 at Ser244, which affects both kinesin localization and anaphase progression. (A) In vitro kinase assay reveals five Nek6-dependent phosphorylation sites on Mklp2 by mass spectrometry. CC, coiled-coil. (B) Mutation of Ser244 to unphosphorylatable Ala disrupts the central spindle localization of Mklp2. Detergent-extracted HeLa cells transiently transfected with EGFP-Mklp2-WT or -S244A. n > 100 anaphase cells per condition in more than three independent experiments. (C) Live-cell imaging of a stable EGFP-Mklp2-S244A HeLa cell line reveals a decrease in the time to cleavage furrow ingression that is dependent on Nek6. n ≥ 5 cells per condition. (D) Quantification of furrow ingression relative to the equatorial cell diameter at metaphase. At 4–6 min after anaphase onset, EGFP-Mklp2-S244A has ingressed to a greater extent than EGFP-Mklp2-WT. (E) GFP immunoprecipitation of EGFP-Mklp2-WT or S244A expressed in stable or transiently transfected HeLa cells. EGFP-Mklp2-S244A forms a heterodimer with endogenous Mklp2 when expressed at low levels in the stable cell line and a homodimer when overexpressed by transient transfection. Black arrows indicate EGFP-Mklp2; gray arrows indicate endogenous Mklp2. WB, Western blot. (F) Depletion of endogenous Mklp2 results in a high proportion of binucleated cells. Simultaneous expression of EGFP-Mklp2-WT but not EGFP-Mklp2-S244A rescues this phenotype. Expression of EGFP-Mklp2-S244E can partially rescue. Cotransfected cells were fixed and stained for immunofluorescence, and the percentage of binucleated cells in >500 interphase cells was counted in three independent experiments. *, P < 0.05 by Student’s t test; bar graphs show means ± SD.