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. 2017 Aug 7;216(8):2339-2354.
doi: 10.1083/jcb.201512055. Epub 2017 Jun 19.

A Bifurcated Signaling Cascade of NIMA-related Kinases Controls Distinct Kinesins in Anaphase

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Free PMC article

A Bifurcated Signaling Cascade of NIMA-related Kinases Controls Distinct Kinesins in Anaphase

Sierra N Cullati et al. J Cell Biol. .
Free PMC article

Abstract

In mitosis, cells undergo a precisely orchestrated series of spatiotemporal changes in cytoskeletal structure to divide their genetic material. These changes are coordinated by a sophisticated network of protein-protein interactions and posttranslational modifications. In this study, we report a bifurcation in a signaling cascade of the NIMA-related kinases (Neks) Nek6, Nek7, and Nek9 that is required for the localization and function of two kinesins essential for cytokinesis, Mklp2 and Kif14. We demonstrate that a Nek9, Nek6, and Mklp2 signaling module controls the timely localization and bundling activity of Mklp2 at the anaphase central spindle. We further show that a separate Nek9, Nek7, and Kif14 signaling module is required for the recruitment of the Rho-interacting kinase citron to the anaphase midzone. Our findings uncover an anaphase-specific function for these effector kinesins that is controlled by specific Nek kinase signaling modules to properly coordinate cytokinesis.

Figures

Figure 1.
Figure 1.
Nek depletion promotes cytokinesis failure. (A–C) HeLa (A), U2OS (B), and RPE1 (C) cells were treated with 10 nM of the indicated siRNAs for 48 and 72 h. Cells were fixed and stained for immunofluorescence, and the percentage of binucleated cells in >500 interphase cells was counted in three independent experiments. (D) Under conditions of Nek9 depletion, cytokinetic midbodies display disorganized PRC1 and tubulin staining that is consistent with depletion of citron kinase. Detergent-extracted HeLa cells stained with endogenous antibodies. n > 100 cytokinesis cells per condition in more than three independent experiments. *, P < 0.05 by Student’s t test; bar graphs show means ± SD. Bars: (main images) 20 µm; (insets) 5 µm.
Figure 2.
Figure 2.
Nek9 interacts with proteins involved in cytokinesis. (A) Nek9 signaling modules receive inputs from master mitotic regulators such as Cdk1 and Plk1 and either relay those signal to helper Nek6 and Nek7 kinases or signal directly to other targets. The integration of these signals is facilitated by protein–protein interaction domains on Nek9. CC, coiled-coil. (B) Proteins known to be involved in cytokinesis stably interact with Nek9. The coverage, peptide count, and intensity-based absolute quantification (iBAQ) area for proteins identified in Nek9 immunoprecipitates (IPs) and the iBAQ ratio (log2 ratio of iBAQ area in Nek9 immunoprecipitate over iBAQ area in control immunoprecipitates) represent the mean values from three independent AP-MS experiments. (C) Putative Nek9 interactors identified by mass spectrometry are confirmed by Western blot (WB). For the complete list of Nek9 interactors and corresponding quantification data, see Table S2.
Figure 3.
Figure 3.
Nek9 protein is required to localize Kif14 and Mklp2 to the anaphase midzone. (A and B) siRNA-mediated depletion of Nek9 results in mislocalization of Mklp2 (A) and Kif14 (B) in anaphase but not in telophase. Detergent-extracted HeLa cells stained with antibodies against endogenous proteins. (C) Quantification of localization behavior in A and B. n > 100 anaphase cells per condition in more than three independent experiments. (D) Live-cell imaging of a stable EGFP-Mklp2 HeLa cell line indicates that under conditions of Nek9 depletion, EGFP-Mklp2 localizes to the cell midzone 4–6 min later than in control cells. n ≥ 5 cells per condition. Bars: (A and B) 20 µm; (D) 10 µm. (E) Depletion of Mklp2 or Kif14 by siRNA significantly increases the proportion of binucleated cells observed after 48 h. 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; points and bar graphs show means ± SD.
Figure 4.
Figure 4.
Nek9 kinase activity is dispensable for Mklp2 and Kif14 localization. (A) Recombinant Nek9 WT and D176A (kinase-dead) were incubated with peptide substrate for 16 h at 28°C, and ATPase activity was measured. Graphs are normalized to mean WT kinase activity from three independent experiments. *, P < 0.05 by Student’s t test; bar graphs show means ± SD. (B and C) Stable HeLa cell lines containing either WT or D176A Nek9 rendered siRNA resistant by silent mutations were alternatively treated with Nek9 siRNA or transfection reagent alone, and then detergent was extracted, fixed, and stained for Mklp2 (B) or Kif14 (C) in anaphase and telophase cells. Note that both kinesins localize normally with or without Nek9 kinase activity in both mitotic phases. Bars, 20 µm.
Figure 5.
Figure 5.
Nek6 or Nek7 kinase activity is required to localize Mklp2 or Kif14 to the central spindle. (A–D) siRNA-mediated depletion of Nek6 but not Nek7 results in mislocalization of Mklp2 in anaphase, whereas depletion of Nek7 but not Nek6 results in a similar phase-specific mislocalization of Kif14. Green indicates Mklp2 (A and C) or Kif14 (B and D); red indicates tubulin; blue indicates DNA. n > 100 anaphase cells per condition in more than three independent experiments. *, P < 0.05 by Student’s t test; bar graphs show means ± SD. (C) Mklp2 localization was analyzed in siRNA-resistant WT or kinase-dead 6×Myc-Nek6 cell lines plus or minus Nek6 siRNA to deplete endogenous protein. Central spindle localization is dependent on Nek6 kinase activity. (D) Kif14 localization was analyzed in siRNA-resistant WT or kinase-dead Nek7 stable cell lines with or without Nek7 siRNA to deplete endogenous protein. Note that Kif14 anaphase central spindle localization is dependent on Nek7 kinase activity. All panels depict detergent-extracted HeLa cells stained with antibodies against endogenous proteins. Bars, 20 µm. KD, knockdown.
Figure 6.
Figure 6.
A requirement for cellular Nek7 kinase activity and direct phosphorylation of Kif14 in anaphase is also linked to its cargo, citron kinase. (A) In vitro kinase assay reveals five Nek7-dependent phosphorylation sites on Kif14 by mass spectrometry. CC, coiled-coil; FHA, forkhead-associated domain. (B) Mutation of individual Nek7 phosphorylation sites to alanine partially disrupts the central spindle localization of Kif14 in HeLa cells transiently transfected with various EGFP-Kif14 WT or mutant constructs. n > 50 anaphase cells per condition in more than three independent experiments. *, P < 0.05 by Student’s t test; bar graphs show means ± SD. (C) Collective mutation of all Nek7 sites fully disrupts the central spindle localization of Kif14, but only in anaphase. HeLa cells transiently transfected with EGFP-Kif14 constructs and mCherry as a positive control for transfection. (D) Nek9- or Nek7-dependent Kif14 mislocalization results in a corresponding delay in the localization of a known Kif14 cargo protein, the Rho-dependent kinase citron. Detergent-extracted HeLa cells stained with antibodies against endogenous proteins. n > 100 anaphase cells per condition in more than three independent experiments. Bars, 20 µm. Green, EGFP-Kif14-5A (C) or Kif14 (D); red, mCherry (C) or citron (D); blue, DNA.
Figure 7.
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.
Figure 8.
Figure 8.
Ser244 phosphorylation inhibits microtubule bundling but not microtubule binding or ATPase activity. (A) Western blot (WB) of pellets from microtubule (MT) pelleting assays performed in 25 mM KCl. Ser244 mutation to Ala or Glu does not affect the ability of Mklp2 to bind microtubules. (B) Mklp2 WT, S244A, or S244E was incubated with or without Nek6 followed by addition of taxol-stabilized microtubules and centrifugation onto coverslips. Microtubule bundling was observed by immunofluorescence. Nek6 inhibits bundling of Mklp2 WT, but Mklp2 S244A is refractory to Nek6 inhibition, whereas Mklp2 S244E is significantly attenuated in bundling activity in both conditions. Representative images of three independent experiments are shown. Bar, 20 µm. (C) Ser244 mutation to Ala or Glu does not affect the microtubule-dependent ATPase activity of Mklp2. Recombinant Mklp2 WT, S244A, and S244E were incubated with or without taxol-stabilized microtubules for 30 min at 28°C, and then ATPase activity was measured. Graphs are normalized to mean WT ATPase activity from three independent experiments. *, P < 0.05 by Student’s t test; bar graphs show means ± SD.
Figure 9.
Figure 9.
Model for Nek kinase-dependent signaling modules that specify anaphase behavior of Mklp2 and Kif14. For Mklp2, preanaphase cells contain high Cdk1 and Nek activity, rendering Mklp2 cytosolic and inactive. Dephosphorylation at anaphase onset by loss of Cdk1 activity and/or gain of phosphatase activity while maintaining Nek-dependent phosphorylation status licenses Mklp2 for deposition to the anaphase central spindle. Residence at the anaphase central spindle then registers it via an unknown mechanism for functions in telophase and beyond; analogous mechanisms are proposed to exist for Kif14. Pptase, phosphatase.

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