Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication

Abstract

Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr(199) by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr(199) remains at centrosomes. It has been shown that Thr(199) phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr(199). Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr(199)-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

FIG. 1.

Physical interaction between NPM/B23 and ROCK II in vitro and in vivo. (A) Identification of ROCK II as an NPM/B23-interacting centrosomal protein. Centrosomes isolated from NIH 3T3 cells were denatured in 9 M urea to dissociate centrosomal components, followed by renaturation. During the renaturing process, the samples were incubated with either GST-NPM/B23 (lane 1) or GST (lane 2) and pulled down with GST affinity beads. The protein complexes were resolved by SDS-PAGE and visualized by silver staining. The protein bands specific to GST-NPM/B23 were excised, subjected to mass spectrometric analysis, and identified as ROCK II and vimentin. (B) Physical interaction between NPM/B23 and ROCK II in vivo. The lysates prepared from NIH 3T3 cells were subjected to immunoprecipitation using (a) either anti-ROCK I (lane 1) or ROCK II (lane 2) antibodies (lane 3, a control IgG) and (b) anti-NPM/B23 antibody (lane 1) (lane 2, a control IgG). The immunoprecipitates were then immunoblotted with anti-NPM/B23, anti-ROCK I, and anti-ROCK II antibodies. The cell lysates (5% of the amount used for immunoprecipitation) were included in the analyses. (C) Diagram of ROCK II wild-type and deletion mutants. (D) Identification of the sequence of ROCK II critical for NPM/B23 binding. Bacterially purified His₆⁺-NPM/B23 proteins were mixed with bacterially purified GST-CAT (lane 1), -CAT/KD (lane 2), -kinase (lane 3), -coil (lane 4), -RB (lane 5), -PH (lane 6), and -CATΔ373-420 (lane 7). The reaction samples were subjected to precipitation using GST affinity beads and immunoblotted with anti-NPM/B23 antibody (upper panel) and anti-GST antibody (lower panel). (E) In vivo demonstration of the critical sequence of ROCK II for NPM/B23 binding. NIH 3T3 cells were transfected with either GFP-tagged full-length ROCK II or ROCK II with aa 373 to 420 deleted. A GFP vector was transfected as a control. Expression levels of transfected ROCK II proteins were determined by immunoblot (IB) analysis using anti-GFP antibody (top). The lysates were also subjected to coimmunoprecipitation (IP) assay using anti-GFP (middle) and anti-NPM/B23 (bottom) antibodies. The immunoprecipitates were immunoblotted with anti-NPM/B23 as well as anti-GFP antibodies. wt, wild type.

FIG. 2.

Localization of ROCK II at centrosomes. (A) Centrosomes were isolated from NIH 3T3 cells by discontinuous sucrose gradient centrifugation, and the resulting fractions were immunoblotted using anti-ROCK II (top) and anti-γ-tubulin (bottom) antibodies. (B) Cells were briefly extracted prior to fixation and coimmunostained with rabbit anti-ROCK II (green) and mouse anti-γ-tubulin (red) antibodies. Cells were also counterstained for DNA with DAPI (blue) and merged with the images of ROCK II and γ-tubulin immunostaining. (a to c) Cell with unduplicated centrosomes; (d to f) cell with duplicated centrosomes; (g to i) mitotic cell. The arrows point to the positions of centrosomes. Scale bar, 10 μm. (C) siRNA-mediated silencing of ROCK II. NIH 3T3 cells were cotransfected with a pSUPER plasmid that encodes siRNA specific for ROCK II (lane 1) and a plasmid encoding a puromycin-resistant gene at a 20:1 molar ratio. The vector (pSUPER with a random sequence of the same nucleotide composition with ROCK II siRNA) was transfected as a control (lane 2). Puromycin-resistant colonies were pooled, and the lysates from the transfectants were immunoblotted with anti-ROCK II (top) and anti-β-tubulin (bottom) antibodies. (D) The ROCK II-RNAi and vector control cells described above were coimmunostained with anti-ROCK II (red) and anti-γ-tubulin (green) antibodies. DAPI-stained images (blue) were merged with the images of γ-tubulin immunostaining. (c and f) Merged images of ROCK II, γ-tubulin, and DNA staining. The arrows point to the positions of centrosomes. Scale bar, 10 μm.

FIG. 3.

Characterization of the centrosome-binding activity of ROCK II. (A) Wild-type ROCK II and deletion mutants were tagged with GFP and transfected into NIH 3T3 and ROCK II-RNAi cells. A GFP vector was transfected as a control. At 36 h posttransfection, cells were briefly extracted, fixed and immunostained for γ-tubulin, and examined for the centrosomal localization of GFP-tagged ROCK II proteins, and results are summarized to the right of the diagram. (B) Representative images of the GFP-, GFP-CAT-, and GFP-PH-transfected NIH 3T3 cells. (C) GFP-CAT-, GFP-PH-, and GFP-Δ457-553-transfected ROCK II-RNAi cells. DAPI-stained images (blue) were laid over γ-tubulin- and GFP-immunostained images. The arrows point to the positions of centrosomes. The panel to the right of each image shows the magnified image of the area indicated by arrows. Scale bar, 10 μm.

FIG. 4.

ROCK II promotes centrosome reduplication. (A) NIH 3T3 cells were first arrested by Aph (2 μg/ml) treatment for 24 h. Cells were then transfected with either the GFP-CAT or the GFP vector in the presence of Aph. After the transfection period (12 h), either the Y-27632 ROCK inhibitor (100 μM) or dimethyl sulfoxide (DMSO) was added to the media in the duplicate GFP-CAT-transfected cell cultures. Cells were fixed at 24 h posttransfection, immunostained for γ-tubulin, and counterstained for DNA with DAPI, and the centrosome profiles of the GFP-positive cells were determined (>300 cells). The results are shown as averages ± standard errors from three independent experiments. (B) Representative immunostained images of vector-transfected cells (a and b) and GFP-CAT-transfected cells (c and d) as well as GFP-CAT-transfected cells in the presence of Y-27632 (e and f) are shown. The panels on the right show the magnified images of the areas indicated by arrows. Scale bar, 10 μm. It should be noted that in this experiment, the cells were directly fixed without preextraction to observe the membrane blebbing associated with the constitutive activation of ROCK II. However, without preextraction, due to the ubiquitous presence of GFP-CAT, the specific localization of GFP-CAT at the centrosomes is highly masked.

FIG. 5.

Centrosomal localization is required for ROCK II to promote centrosome reduplication. (A) NIH 3T3 cells were transfected with GFP-CAT, GFP-CATΔ457-533, or a GFP vector. The lysates were prepared from the transfectants at 18 h after transfection and immunoprecipitated with anti-GFP antibody. The immunoprecipitates were immunoblotted with anti-GFP antibody (top). The immunoprecipitates were also subjected to in vitro kinase assay using vimentin as a substrate as described previously (20) (middle). The substrate band in the Coomassie blue (CB) stain of the gel is shown in the bottom panel. IP, immunoprecipitation; IB, immunoblotting. (B) NIH 3T3 cells were transfected with GFP-CAT, GFP-CATΔ457-533, or a vector control. The lysates prepared at 24 h after transfection were immunoblotted with anti-GFP (top), anti-Ser¹⁹ phospho-MLC2 (middle), and anti-MLC2 (bottom) antibodies. Quantification of the levels of total and Ser¹⁹ phospho-MLC2 are shown in the graph at the bottom. (C) NIH 3T3 cells prearrested with Aph for 24 h were transfected with GFP-CAT, GFP-CATΔ457-533, or a GFP vector in the presence of Aph. The transfected cells were incubated for 36 h after transfection in the presence of Aph and immunostained for γ-tubulin, and the centrosome profiles of the GFP-positive cells were analyzed (>300 cells). The results are shown as averages ± standard errors from three independent experiments.

FIG. 6.

Role of ROCK II in the timely initiation of centrosome duplication. (A) ROCK II-RNAi and control cells as well as ROCK II-RNAi cells transiently transfected with GFP-CAT were subjected to centrosome reduplication assay. The cells were exposed to Aph for 60 h, and the centrosome profiles were determined by γ-tubulin immunostaining (>300 cells). The results are averages ± standard errors from three experiments. (B) Representative γ-tubulin-immunostained images of the vector control cells and ROCK II-RNAi cells after Aph treatment are shown. Scale bar, 10 μm. (C) MSFs were prepared from the abdominal skin of 8-week-old male mice. MSFs silenced for ROCK II expression and control MSFs were generated by the method used for the generation of NIH 3T3 ROCK II-RNAi cells and control NIH 3T3 cells described in the legend to Fig. 2. (a) Immunoblot analysis of ROCK II expression in ROCK II-RNAi cells and control MSFs. ROCK II-RNAi and control MSFs were serum starved for 30 h and serum stimulated in the presence of BrdU for 21 h. To determine the rates of centrosome duplication and BrdU incorporation, we carried out the experiment with a single cell culture as well as separately with duplicate cultures, which gave almost identical results. For a determination of centrosome duplication, antibody against centrin, a known centriole marker (36), was used. An example of the immunostained images for which anticentrin antibody was used to differentiate unduplicated and duplicated centrosomes is shown on the left. We repeated the experiment twice, and we obtained almost identical results. The averages from two experiments are plotted in the graph.

FIG. 7.

Superactivation of ROCK II by NPM/B23 in vitro and in vivo and the functional interaction of ROCK II and NPM/B23 to promote centrosome reduplication. (A) Baculovirally prepared GST-CAT and GST-ROCK II were subjected to in vitro kinase assay either in the absence or in the presence of bacterially purified His₆⁺-NPM/B23 (top) using vimentin as a substrate. His₆⁺-tagged mortalin (Mot330) bacterially prepared by exactly the same method was used as a negative control. GST was also tested as a control. The bottom three panels show the immunoblots (IB) of the kinase reaction samples with antivimentin (second panel), anti-His₆⁺ (third panel), and anti-GST (fourth panel) antibodies. wt, wild type. (B) Cells were transfected with GFP-CAT plus the GFP vector, GFP-CAT plus GFP-NPM/B23, GFP-NPM/B23 plus the GFP vector, or the GFP vector alone. The lysates were prepared at 24 h posttransfection and immunoblotted with anti-GFP antibody (top), anti-phospho-Ser¹⁹ MLC2 antibody (middle), and anti-MLC2 antibody (bottom). The quantifications of the levels of total and phospho-Ser¹⁹ MLC2 are shown in the graph. (C) Either a pSuper plasmid encoding the RNAi sequence targeted for NPM/B23 or a control plasmid with a randomized sequence with the same nucleotide composition was cotransfected with a plasmid encoding a puromycin resistance gene as a rapid selection marker into NIH 3T3 cells. After 3 days of puromycin selection, the drug-resistant cells were pooled and transfected with GFP-CAT. The lysates were prepared from the transfectants at 24 h posttransfection and subjected to immunoblot analysis using anti-NPM/B23 (first panel), anti-GFP (second and third panels), anti-phospho-Ser¹⁹ MLC2 (fourth panel), and anti-MLC2 (fifth panel) antibodies. Quantifications of the levels of total and phospho-Ser¹⁹ MLC2 are shown in the graph. (D) NIH 3T3 cells prearrested by Aph treatment for 24 h were transfected with GFP-NPM/B23 plus the GFP vector, GFP-CAT plus the GFP vector, GFP-CAT plus GFP-NPM/B23, or the GFP vector alone. After transfection, cells were exposed to Aph for 36 h, and the centrosome profiles of the GFP-positive cells were determined (>300 cells). The results shown are averages ± standard errors from three independent experiments.

FIG. 8.

Thr¹⁹⁹ phosphorylation is important for NPM/B23 to superactivate ROCK II and to enhance the activity of ROCK II to promote centrosome reduplication. (A) NIH 3T3 cells prearrested by Aph treatment for 24 h were cotransfected with FLAG-tagged wild-type (wt) NPM/B23, the T199A mutant, the T199D mutant, or a vector control with GFP-CAT. After transfection, cells were incubated in the presence of Aph for 18 h, and the centrosome profiles of the GFP-positive cells were determined (>200 cells). The results shown are averages ± standard errors from three independent experiments. (B) NIH 3T3 cells were transfected with FLAG-tagged wild-type NPM/B23, the FLAG-tagged T199A mutant, the FLAG-tagged T199D mutant, or a vector plasmid. The lysates were prepared at 36 h posttransfection and immunoblotted with anti-FLAG (top blot), anti-phospho-Ser¹⁹ MLC2 (middle blot), and anti-MLC2 antibodies (bottom blot). Quantification of the levels of total and phospho-Ser¹⁹ MLC2 are shown in the graph at the bottom. (C) ROCK II-RNAi cells (NIH 3T3 origin) and the vector control cells were transfected with the FLAG-tagged T199D mutant. The lysates were prepared at 24 h posttransfection and immunoblotted with anti-ROCK II (first blot), anti-FLAG (second blot), anti-phospho-Ser¹⁹ MLC2 (third blot), and anti-MLC2 (fourth blot) antibodies. Quantifications of the levels of total and phospho-Ser¹⁹ MLC2 are shown in the graph at the bottom.

FIG. 9.

Thr¹⁹⁹ phosphorylation of NPM/B23 is critical for NPM/B23-ROCK II complex formation in vivo. (A) NIH 3T3 cells were transfected with either the FLAG-tagged T199D mutant or the FLAG-tagged T199A mutant. The lysates were prepared at 24 h posttransfection and subjected to immunoprecipitation (IP) with either anti-ROCK II (a) or anti-FLAG (b) antibodies. The immunoprecipitates were then immunoblotted (IB) with anti-FLAG and anti-ROCK II antibodies. (B) Baculovirally purified GST-ROCK II was subjected to in vitro kinase assay in the presence of various concentrations of either the His₆⁺-tagged T199A mutant or the His₆⁺-tagged T199D mutant with vimentin as a substrate (top panel). Aliquots of the kinase reaction mixtures were immunoblotted with antivimentin, anti-NPM/B23, and anti-GST antibodies. We also tested GST-CAT, and we obtained similar results (data not shown). (C) NIH 3T3 cells were serum starved for 24 h and serum stimulated. Every 3 h for a period of 18 h, lysates were prepared. The lysates were immunoprecipitated with anti-cyclin E antibody, and the immunoprecipitates were subjected to in vitro histone H1 kinase assay (first panel). The lysates were also immunoblotted with anti-NPM/B23 (second panel), anti-ROCK II (third panel), and anti-phospho-Thr¹⁹⁹ NPM/B23 (fourth panel) antibodies. The lysates were also immunoprecipitated with anti-ROCK II antibody, and the immunoprecipitates were immunoblotted with anti-NPM/B23 antibodies (fifth panel) as well as anti-phospho-Thr¹⁹⁹ NPM/B23 antibodies (bottom panel).

FIG. 10.

Centrosome localization is critical for NPM/B23 to augment ROCK II activity to promote centrosome reduplication. (A) NIH 3T3 cells were transfected with FLAG-tagged wild-type (Wt) NPM/B23, the K263R mutant, or a vector plasmid. At 24 h posttransfection, the lysates were prepared and subjected to immunoprecipitation (IP) using either anti-FLAG or anti-ROCK II antibody. The immunoprecipitates were then immunoblotted (IB) with ant-FLAG as well as anti-ROCK II antibodies. (B) Baculovirally purified GST-CAT was subjected to in vitro kinase assay in the presence of His₆⁺-tagged wild-type NPM/B23, the T199D mutant, the K263R mutant, or the T199D K263R mutant at two different concentrations with vimentin as a substrate (first panel). Aliquots of the kinase reaction mixtures were immunoblotted with antivimentin, anti-NPM/B23, and anti-GST antibodies. We also tested GST-full-length ROCK II, and we obtained similar results (data not shown). (C) NIH 3T3 cells prearrested by Aph treatment for 24 h were cotransfected with GFP-CAT plus FLAG-tagged wild-type NPM/B23, GFP-CAT plus the T199D mutant, GFP-CAT plus the K263R mutant, or GFP-CAT plus the T199D/K263R mutant. For controls, cells were cotransfected with the GFP vector (GFP-vec) plus the FLAG vector (FLAG-vec), the GFP vector plus FLAG-tagged wild-type NPM/B23, or GFP-CAT plus the FLAG vector. After transfection, cells were incubated in the presence of Aph for 18 h, and the centrosome profiles of the GFP-positive cells were determined (>200 cells). The results shown are averages ± standard errors from three independent experiments.

FIG. 11.

Model of the ROCK II-NPM/B23-mediated regulation of centrosome duplication. During the early to mid-G₁ phase of the cell cycle, NPM/B23 localizes between the paired centrioles of unduplicated centrosomes, likely functioning in pairing the centrioles. In late G₁, CDK2/cyclin E becomes activated and phosphorylates NPM/B23 on Thr¹⁹⁹. Upon Thr¹⁹⁹ phosphorylation, the majority of NPM/B23 proteins dissociate from centrosomes, but some remain at centrosomes and translocate toward the mother centriole of the pair. These Thr¹⁹⁹-phosphorylated NPM/B23 proteins have high binding affinities to ROCK II and bind to ROCK II present at centrosomes. ROCK II is superactivated by NPM/B23 binding and rapidly targets the protein (shown as “X”) which plays a key role in the initiation of centrosome duplication.