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1.
Figure 3

Figure 3. HsCYK-4 is an anaphase-specific Plk1 substrate at the midzone.. From: Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells.

(A) Validation of a pS170-specific antibody. Known amounts of pS170 or S170 (unphosphorylated) peptides were spotted on PVDF and immunoblotted with anti-pS170 antibody to assess phosphoselectivity. (B) Phosphorylation of S170 peaks during anaphase and telophase. HCT116 cells were synchronized by nocodazole block and release and either immunoblotted directly (top panels) or subjected to immunoprecipitation of total HsCYK-4, followed by immunoblot detection of pS170. Note that the securin destruction and mitotic exit data were recently reported [69] and are reproduced here to provide essential context. (C) Phosphorylation of S170 occurs on the spindle midzone and requires both the catalytic activity and self-primed association of Plk1 with the spindle midzone. RPE cells of the indicated genotypes were synchronized with monastrol, treated with 3-MB-PP1 in anaphase, and processed for immunofluorescence microscopy to visualize Plk1as, total HsCYK-4, and pS170. Scale bar, 10 µm.

Mark E. Burkard, et al. PLoS Biol. 2009 May;7(5):e1000111.
2.
Figure 6

Figure 6. Recruiting Plk1 to the midzone via a PBD-independent mechanism reactivates the Ect2-RhoA network and induces equatorial cleavage furrows.. From: Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells.

(A) Plk1as cells were transduced with retroviruses expressing either the catalytic domain of Plk1 alone (Plk1cat) or the same domain fused to HsCYK-4 (Plk1catC4) as FLAG-tagged proteins. (B) Plk1catC4 is recruited to the midzone during anaphase. Immunofluorescence microscopy was performed as in Figure 1B, except that anti-FLAG antibodies were used. Scale bar, 10 µm. (C) Plk1catC4 can activate the Ect2-RhoA network. Cells of each genotype were synchronized with monastrol, released, and treated with 3-MB-PP1 for 20 min. Phosphorylation of pS170, Ect2 recruitment, cortical RhoA recruitment, and incorporation of Citron kinase into the contractile ring were assessed by immunofluorescence microscopy. Scale bar, 10 µm. (D) The percentage of cells with equatorially localized proteins or ingressing furrows was quantified for each genotype. (E) Plk1catC4 confers cytokinesis-specific rescue of Polo kinase function. Cells were cultured in the presence of 3-MB-PP1 for 8 d and stained with crystal violet to assess growth.

Mark E. Burkard, et al. PLoS Biol. 2009 May;7(5):e1000111.
3.
Figure 5

Figure 5. Phosphorylation of HsCYK-4 on S157 creates a docking site for the BRCT repeats on Ect2.. From: Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells.

(A) HeLa cells were cotransfected with plasmids expressing wild-type or non-phosphorylatable versions of FLAG-HsCYK-4 and a myc-tagged N-terminal fragment of Ect2 containing both BRCT repeats but lacking an inhibitory Cdk1 phosphorylation site (1-352 T342A [14]; denoted here as myc-BRCT*). Mitotic cell extracts were immunoprecipitated with anti-myc antibodies, then analyzed by SDS-PAGE and Western blotting with anti-FLAG and anti-myc antibodies to assess binding. 3A denotes the S157A/S164A/S170A triple mutant; 2A indicates the S214A/T260A double mutant. To ensure specificity, control immunoprecipitations were performed in the presence of myc peptide as a competitor. (B) Plk1 phosphorylates S157 in vivo. A pS157-specific antibody was generated (see Materials and Methods and Figure S5) and used to stain 3-MB-PP1-treated Plk1wt and Plk1as cells. (C) Alignment of amino acid sequences surrounding S157 in HsCYK-4 orthologs. Note that a Plk1 phosphorylation consensus site (D/E-x-S/T-ϕ) is conserved from Drosophila to humans.

Mark E. Burkard, et al. PLoS Biol. 2009 May;7(5):e1000111.
4.
Figure 2

Figure 2. The RhoGAP subunit of the centralspindlin complex, HsCYK-4, is a substrate and binding partner of Plk1 in vitro.. From: Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells.

(A) The N terminus of HsCYK-4 contains multiple Plk1 phosphorylation sites. MS/MS spectra of Plk1-phosphorylated HsCYK-4 (1–288) are presented in Figure S2. (B) HsCYK-4 is a Plk1 substrate in vitro. Recombinant HsCYK-4 fragments were used as substrates for Plk1 kinase assays. Where indicated, BTO-1 [25] was added to inhibit Plk1 activity. (C) Plk1 associates with the Ect2/HsCYK-4 complex. HeLa cells were released from a nocodazole block and sampled at the indicated timepoints. DSP-treated whole-cell extracts were immunoprecipitated with anti-HsCYK-4 or control goat IgG and immunoblotted for Plk1 and Ect2. (D) Plk1 phosphorylation of HsCYK-4 promotes PBD docking. In vitro kinase reactions were performed as in (B), except that unlabeled ATP was used. The reaction products were resolved by SDS-PAGE and subjected to Far Western blotting using a soluble GST-PBD fusion protein as the probe. Comparable loading was verified by immunoblotting for HsCYK-4N via its chitin-binding domain (CBD) tag.

Mark E. Burkard, et al. PLoS Biol. 2009 May;7(5):e1000111.
5.
Figure 8

Figure 8. A model for Plk1-mediated generation of the midzone-derived signal for cell division.. From: Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells.

In prometaphase, high Cdk1 levels maintain Ect2, centralspindlin, and PRC1 in a functionally inactive state. Upon Cdk1 inactivation at anaphase onset, the MKLP1 subunit of centralspindlin and PRC1 are dephosphorylated and thus able to bind and bundle antiparallel MT arrays to form the spindle midzone [12],[13]. At the same time, Plk1 is released from its early mitotic substrates by dephosphorylation of Cdk1-primed PBD docking sites, and inhibitory phosphorylation of Ect2 at Cdk1 sites is also reversed [14]. Plk1 then rapidly self-organizes onto the midzone via PBD- and activity-dependent positive-feedback loops involving multiple substrates, including PRC1 [36], MKLP2 [56], and HsCYK-4 (this study). Furthermore, in the case of HsCYK-4, Plk1 phosphorylation at S157 also creates a docking site for the tandem BRCT repeats of Ect2, thereby targeting the RhoGEF to the midzone and perhaps stimulating its activity [48]. The resulting increase in RhoA-GTP at the adjacent equatorial cortex promotes focal recruitment and activation of RhoA effectors needed for actin polymerization and myosin II motor activity within the contractile ring [2],[3].

Mark E. Burkard, et al. PLoS Biol. 2009 May;7(5):e1000111.
6.
Figure 1

Figure 1. Self-assembly of Plk1 at the spindle midzone is required to activate the Ect2-RhoA network and induce formation of cleavage furrows.. From: Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells.

(A) Human PLK1Δ/Δ cells were reconstituted with an EGFP-tagged analog-sensitive Plk1 allele (Plk1as) and mCherry-tagged wild-type (Plk1wt) or pincer-defective (Plk1AA) alleles that are analog-resistant. (B) Plk1 immunoprecipitates were incubated with [γ-32P]ATP and casein in the presence of 3-MB-PP1 with or without BI 2536. (C) Plk1AA cannot associate with the spindle midzone. Plk1as/wt and Plk1as/AA cells were stained with EGFP- and mCherry-specific antibodies and processed for immunofluorescence as outlined in the Materials and Methods section. Scale bar, 10 µm. (D) A chemical genetic assay for cytokinesis initiation. Plk1as, Plk1as/wt, and Plk1as/AA cells were synchronously released from a monastrol block and treated with 3-MB-PP1 to acutely inhibit Plk1as activity in early anaphase. Midzone localization of the Ect2/HsCYK-4 complex, cortical generation of active RhoA, and furrow ingression were analyzed by immunofluorescence microscopy. Scale bar, 10 µm. (E) Quantification of cytokinesis onset. Ect2 recruitment, cortical RhoA activation, and furrow ingression were determined by immunofluorescence microscopy (n = 100 anaphase cells per genotype). (F) Cells were cultured in the presence of various concentrations of 3-MB-PP1 for 8 d and stained with crystal violet to assess growth.

Mark E. Burkard, et al. PLoS Biol. 2009 May;7(5):e1000111.
7.
Figure 7

Figure 7. Tethering Plk1 to the centralspindlin complex rescues the initiation but not the completion of cytokinesis.. From: Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells.

(A) Monastrol-synchronized cells were released for 60 min into drug-free medium, followed by addition of 3-MB-PP1 and initiation of timelapse imaging (time 00). Examples of cells that traversed anaphase (A) and either formed equatorial furrows (F) or failed to do so (X) are indicated (see also Videos S1 and S2). Scale bar, 10 µm. (B) Biphasic furrow dynamics in Plk1as/catC4 cells. At least 18 cells of each genotype were imaged as above, and the average depth of the furrow relative to the initial equatorial diameter at anaphase onset was calculated. Furrow trajectories in individual cells are provided in Figure S6. (C) Cytokinesis ultimately fails in Plk1as/catC4 cells. Cells were synchronized in prometaphase by monastrol block and release as above. Thirty minutes after monastrol release, 3-MB-PP1 was added to 10 µM for an additional 90 min to inhibit Plk1as activity during mitotic exit. Adherent cells were collected by trypsinization, fixed and stained with Hoechst 33352, and analyzed microscopically to determine the fraction of binucleated cells in each population (n = 3 determinations, 100 cells/sample; values are reported as means ± SEM). Scale bar, 10 µm.

Mark E. Burkard, et al. PLoS Biol. 2009 May;7(5):e1000111.
8.
Figure 4

Figure 4. Plk1 phosphorylation of HsCYK-4 localizes Ect2 at the midzone and stimulates RhoA-dependent contractile ring assembly at the equatorial cortex.. From: Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells.

(A) Human RPE cells were stably transduced with GFP-tagged siRNA-resistant HsCYK-4 alleles, or with the empty GFP vector as a negative control, then transfected with HsCYK-4 siRNA. Samples were analyzed by SDS-PAGE and Western blotting 48 h after transfection. We observed increased levels of both GFP-tagged polypeptides after depletion of endogenous HsCYK-4, possibly due to reduced competition for MKLP1 or other stabilizing factors. Tubulin was used to confirm equal loading. (B) Cells were transfected as above, synchronized with monastrol, and fixed 60 min after release from the monastrol block. The fraction of anaphase/telophase cells with pS170 staining at the midzone is indicated (green numbers, second column) and was used to establish the efficiency of RNAi depletion and/or transgenic rescue in individual cells. The percentage of pS170-positive and -negative cells with equatorially localized proteins or ingressing furrows was quantified for each genotype. Scale bar, 10 µm. (C and D) Abrogation of HsCYK-4 phosphorylation prevents Ect2 recruitment to the spindle midzone and inhibits RhoA activation and contractile ring assembly. The percentage of anaphase/telophase cells with accumulation of Ect2 at the midzone, incorporation of active RhoA or Citron-kinase into the presumptive contractile ring, and furrow ingression was determined from four replicates in two separate experiments (n = 100 cells/sample); error bars denote SEM.

Mark E. Burkard, et al. PLoS Biol. 2009 May;7(5):e1000111.

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