p27(Kip1) controls cytokinesis via the regulation of citron kinase activation

Abstract

p27(Kip1) (p27) acts as a tumor suppressor by inhibiting cyclin-cyclin-dependent kinase (cyclin-CDK) activity. However, mice expressing a form of p27 that is unable to bind or inhibit cyclin-CDK complexes (p27(CK-)) have increased incidence of tumor development as compared with wild-type and p27(-/-) mice, revealing an oncogenic role for p27. Here, we identified a phenotype of multinucleation and polyploidy in p27(CK-) mice not present in p27(-/-) animals, suggesting a role for p27 in G2/M that is independent of cyclin-CDK regulation. Further analysis revealed that p27(CK-) expression caused a cytokinesis and abscission defect in mouse embryonic fibroblasts. We identified the Rho effector citron kinase (citron-K) as a p27-interacting protein in vitro and in vivo and found that p27 and citron-K colocalized at the contractile ring and mid-body during telophase and cytokinesis. Moreover, overexpression of the minimal p27-binding domain of citron-K was sufficient to rescue the phenotype caused by p27(CK-). Conversely, expression of a mutant p27(CK-) unable to bind citron-K did not induce multinucleation. Finally, by binding to citron-K, p27 prevented the interaction of citron-K with its activator RhoA. Taken together, these data suggest a role for p27 during cytokinesis via the regulation of citron-K activity.

Figure 1. Polyploidy and multinucleation in the liver and kidney of p27^CK– mice.

(A) p27 and p27^CK– expression during the cell cycle. Primary MEFs (passage 2) derived from p27^+/+ and p27^CK– mice were serum starved for 72 hours in DMEM/0.1% serum and replated in 10% serum medium for the indicated time. Proteins (40 μg) were resolved on SDS-PAGE, and membranes were probed with anti-p27 (C19) antibody; Grb2 levels were used as loading control. The graph shows the corresponding flow cytometry analysis of propidium iodide–stained serum-stimulated MEFs (average of 3 independent experiments ± SEM). (B) Analysis of hepatocyte DNA content of Feulgen-stained liver sections in p27^+/+ (n = 11), p27^–/– (n = 5), p27^CK– (n = 8), and p27^S10A/S10A (n = 4) mice. Error bars represent SD. Examples of p27^+/+ and p27^CK– H&E-stained liver sections illustrating nuclear size (original magnification, ×1,000). (C) Multinucleated cells were counted on 3-μm-thick sections stained with β-catenin or H&E in a total of 1,000 hepatocytes from at least 3 different mice per genotype. The results were analyzed using 1-way ANOVA with the Tukey-Kramer multiple comparison test; ***P < 0.001. Examples of p27^+/+ and p27^CK– H&E-stained liver sections illustrating multinucleation are shown (original magnification, ×1,000). (D) The percentage of multinucleated cells in kidney sections of p27^+/+ (n = 4) and p27^CK– (n = 5) mice was determined. For each animal, 250–500 cells were counted. **P < 0.01, Mann-Whitney U test. Examples of p27^+/+ and p27^CK– H&E-stained kidney sections illustrating multinucleation are shown (arrows) (original magnification, ×600). Error bars in C and D represent SEM.

Figure 2. Multinucleation and centrosome amplification in p27^CK– MEFs.

(A) Primary MEFs (passages 2–3) derived from p27^+/+, p27^–/–, and p27^CK– mice were stained with anti–β-catenin and anti–lamin-A antibodies to visualize plasma membrane and nuclear envelope, respectively (original magnification, ×400). For each experiment, 500 cells were counted per genotype. The graph shows the mean of 3 independent experiments. Results were analyzed by 1-way ANOVA with the Tukey-Kramer multiple comparison test. p27^+/+ versus p27^CK–, ***P < 0.001. (B) The number of nuclei per cell was counted in two lines of immortalized MEFs per genotype. Cells were stained as in A, and 500 cells per line were counted (original magnification, ×400). The graph shows the average of 4 independent experiments. Statistical analyses were performed as in A. p27^+/+ versus p27^CK–, **P < 0.01. (C) Centrosomes were visualized by γ-tubulin staining and counted in 300 cells per genotype (original magnification, ×600). Graph shows the average of 2 different MEF lines per genotype in 4 independent experiments. Statistical analyses were performed as in A; p27^+/+ versus p27^CK–, ***P < 0.001. Error bars in A–C represent SEM.

Figure 3. p27^CK– causes a cytokinesis defect.

(A) Multinucleation was quantified (500 cells counted/line, n = 3) in two lines of immortalized p27^–/– MEFs infected or not with p27^CK– and stained for β-catenin and lamin-A (original magnification, ×400). p27^CK– levels following retroviral infection were measured by immunoblot for p27, and β-tubulin levels were used as loading control. Lower graph: For each cell line, the proportion of mitotic cells in telophase and cytokinesis was determined in 50–100 mitotic figures; results are mean of 5 independent experiments. (B) Overexpression of p27^CK– in HeLa cells causes multinucleation. HeLa cells were transfected with either empty vector or a vector encoding p27^CK–. Transfected cells were visualized by p27 staining and co-labeled for β-catenin, and 600 p27-positive cells were counted (original magnification, ×400). Graphs show the average number of multinucleated cells in 3 independent experiments. Right graph: number of mitotic cells in telophase and cytokinesis observed in 100 mitotic figures (n = 4). In A and B, *P < 0.05, **P < 0.01, Student’s t test. (C) Number of mitotic cells in cytokinesis in immortalized p27^+/+, p27^–/–, and p27^CK– MEFs. One hundred mitotic figures were counted per genotype in 3 independent experiments. Results were analyzed by 1-way ANOVA with the Tukey-Kramer multiple comparison test; p27^+/+ versus p27^CK–, **P < 0.01. Error bars in A–C represent SEM. (D–H) Videomicroscopy of p27^+/+, p27^–/–, and p27^CK– MEFs undergoing mitosis. (D) Incidence of multinucleation caused by cytokinetic regression relative to the total number of mitotic figures recorded (p27^+/+: n = 181; p27^–/–: n = 117; p27^CK–: n = 243). (E) Nature of the defect causing multinucleation: multinucleation caused by reopening of the intercellular bridge as a percentage of the total number of multinucleation was determined for each genotype. (F–H) Examples of phase contrast videomicroscopy of mitotic p27^+/+ (F) and p27^CK– MEFs (G and H). Arrows indicate the intercellular bridge. In p27^CK–, cells undergo mitosis normally until the formation of intercellular bridge, which regresses to form binucleated cells. Original magnification, ×200.

Figure 4. The C-terminal half of p27 interacts with the HR1 domain of citron-K.

(A) Schematic representation of citron-K (Cit-K) and deletion mutants. All constructs except the Cit-ΔN mutant contain an N-terminal Myc epitope tag. S/T kinase, Ser/Thr kinase domain; CC, CC domain; Zn-F, Zn finger; PH, pleckstrin homology; CNH, citron homology; Pro-rich, proline-rich region: PDZ, PDZ binding domain. (B–E) HEK293 cells transfected with citron-K or the indicated deletion mutant were subjected to pull-down assays using GST or GST-p27 beads. The amounts of citron-K or deletion mutant bound to the beads and of transfected protein present in the extracts were detected by immunoblot using the indicated antibodies (Mα, mouse anti-; Gα, goat anti-; Rα, rabbit anti-). The amount of GST or GST-p27 used in the assays was visualized by Coomassie staining. (B and C) p27 interacts with citron-K. (D) p27 interacts with the CC domain of citron-K and more weakly with the RBD domain. (E) The HR1 domain present in the CC domain is necessary for p27 interaction. (F–I) Co-immunoprecipitations using rabbit anti-p27 (C19) (F, H, and I) or two different goat anti-citron antibodies (C20 or S20) (G). Co-immunoprecipitated proteins were detected by immunoblot with the indicated antibodies. The immunoprecipitated proteins were visualized by reprobing for mouse anti-p27 (F8) (F, H, and I) or goat anti-citron (C20) (G). Immunoblots of extracts show the level of transfected proteins in each condition. Grb2 levels were used as loading control. (F and G) p27 interacts with citron-K in vivo. (H and I) p27 interacts with all mutants containing the HR1 domain. A weaker interaction is also seen with the RBD. (J) p27 interacts with citron-K in HeLa cells. Endogenous p27 was immunoprecipitated using rabbit anti-p27 (C19) in HeLa cells. The co-immunoprecipitated citron-K was visualized using mouse anti–citron-K antibody. Endogenous levels of each protein are shown in the lower panels. Noc, nocodazole; purvA, purvalanol A; C, control. (K) p27 interacts with citron-K directly. Different amounts of recombinant His-p27 were used in pull-down assays with GST or GST-HR1-RBD beads. Immunoblot show the amount of p27 bound to the beads. The amount of GST or GST-HR1-RBD used in the assay was visualized by Coomassie staining. (L) The C-terminal half of p27 interacts with citron-K. Pull-downs were realized as in B with GST-p27WT, GST-p27N_T (N-terminal, aa 1–86), and GST-p27C_T (C terminal, aa 87–198).

Figure 5. Colocalization of p27 and citron-K at the mid-body.

(A and B) After 24 hours in culture, HeLa cells were permeabilized with digitonin (40 μg/ml) before fixation. Mid-bodies were visualized with citron-K (goat anti–citron C20) staining in red (A) or green (B); p27 was detected with rabbit anti-p27 (C19) antibody in green (A) or red (B); and microtubules were stained with mouse anti–β-tubulin (purple) (original magnification, ×600). (C) HeLa cells were permeabilized with digitonin prior to fixation and stained with mouse anti-RhoA (26C4) antibody (green), rabbit anti-p27 (C19) (red), and goat anti-citron (C20) (purple) (original magnification, ×600). (D) Primary MEFs derived from p27^+/+, p27^–/–, and p27^CK– mice were stained with anti-p27 (C19), anti-citron (C20), and anti–β-tubulin after digitonin permeabilization and fixation (original magnification, ×600).

Figure 6. p27^CK– interferes with citron-K function.

(A) p27^CK– does not affect the multinucleation caused by citron-K depletion. HeLa cells were transfected with scrambled (scr) or citron-K siRNAs and plasmids expressing p27^CK– or empty vector (pQP). Citron-K knockdown was controlled by immunoblotting with goat anti-citron antibodies; β-tubulin levels were used as loading control. Multinucleation was quantified in cells (500 cells/condition, n = 3) stained with β-catenin and p27 antibodies and Hoechst (original magnification, ×400). Results were analyzed using 1-way ANOVA with the Tukey-Kramer multiple comparison test; *P < 0.05. (B) The minimal p27-binding domain of citron-K (HR1) rescues the multinucleation caused by p27^CK– overexpression. HeLa cells were transfected with p27^CK– and/or different domains of citron-K. Multinucleation was quantified (500 cells/condition, n = 3). Results were analyzed by 1-way ANOVA with the Newman-Keuls test; ***P < 0.001. (C) The HR1 domain rescues the multinucleation in p27^CK– primary MEFs. p27^CK– primary MEFs were transfected with GFP or with the HR1 domain, and multinucleation was evaluated 48 hours later (350–600 cells/condition, n = 3). Results were analyzed using Student’s t test; *P < 0.05. (D) RhoA depletion has no effect on p27^CK–-induced multinucleation. HeLa cells were transfected with scrambled or RhoA siRNA and plasmids expressing p27^CK– or empty vector (pQP). RhoA knockdown and p27 expression were controlled by immunoblotting with anti-RhoA and anti-p27 antibodies; β-tubulin levels were used as loading control. Multinucleation was quantified (500 cells/condition, n = 3). Results were analyzed as in A; *P < 0.05, **P < 0.01. (A–D) Error bars represent SEM.

Figure 7. p27^CK– interferes with Rho–citron-K interaction.

(A) HEK293 cells were transfected with p27, Myc-RhoA63L, and/or Cit-ΔN. RhoA63L was immunoprecipitated with mouse anti-Myc (9E10) antibodies. The amount of p27 co-immunoprecipitated was determined by probing with rabbit anti-p27 (C19). Immunoprecipitated RhoA63L was visualized by reprobing with rabbit anti-Myc (A14). The amount of transfected proteins present in cell extracts is shown in the lower panels. (B) HEK293 cells were transfected with p27 or p27^CK–, RhoB, and/or Myc–citron-K. Citron-K was immunoprecipitated using mouse anti-Myc (9E10). The amount of RhoB bound to citron-K was determined by probing with anti-RhoB (119) antibodies. After stripping, membranes were reprobed with goat anti-citron (C20). Amounts of transfected proteins are shown in the lower panels. (C and D) HEK293 cells were transfected or not with p27, eGFP-RhoA63L, and the HR1-RBD domain of citron-K (C) or with p27, eGFP-RhoA, and the RBD domain of citron-K (D). Citron-K domains were immunoprecipitated with rabbit anti-Myc (A14) antibodies. Membranes were probed successively using mouse anti-p27 (F8), mouse anti-RhoA (26C4), and mouse anti-Myc (9E10) antibodies. The amount of each transfected protein is shown in the lower panels. (E) The last 8 aa of p27 are required for binding to citron-K, and phosphorylation of p27 on Thr198 prevents citron-K binding. Pull-down experiments were performed with HEK293 cells transfected with citron-K, Cit-ΔN, or HR1-RBD using the indicated GST-p27 beads. The amounts of proteins bound to the beads or transfected in the input extracts were determined by immunoblot using the indicated antibodies. The amount of GST beads used in the assays was visualized by Coomassie staining. (F) The ability to bind citron-K is required for p27^CK– to cause multinucleation. HeLa cells were transfected with empty vector or vectors encoding the indicated p27^CK– mutants. Immunoblot shows the expression level of each p27mutant. In this exposure, endogenous p27 is not visible. Multinucleated cells were counted (200–500 positive cells/condition, n = 5) using β-catenin and p27 staining to visualize transfected cells. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA with the Tukey-Kramer multiple comparison test; error bars represent SEM. (G) Immortalized p27^–/– MEFs were infected with p27^CK– or different p27^CK– mutants. Multinucleated cells were quantified (500 cells/condition, n = 3). The level of p27^CK– or p27^CK– mutants was measured by immunoblot against p27. Results were analyzed as in F.