p27Kip1 controls cytokinesis via the regulation of citron kinase activation
Murielle P. Serres, … , Nisar P. Malek, Arnaud Besson
Murielle P. Serres, … , Nisar P. Malek, Arnaud Besson
Published February 1, 2012
Citation Information: J Clin Invest. 2012;122(3):844-858. https://doi.org/10.1172/JCI60376.
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Research Article Oncology

p27Kip1 controls cytokinesis via the regulation of citron kinase activation

Abstract

p27Kip1 (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 (p27CK–) 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 p27CK– 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 p27CK– 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 p27CK–. Conversely, expression of a mutant p27CK– 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.

Authors

Murielle P. Serres, Uta Kossatz, Yong Chi, James M. Roberts, Nisar P. Malek, Arnaud Besson

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Figure 7

p27CK– interferes with Rho–citron-K interaction.

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p27CK– interferes with Rho–citron-K interaction. (A) HEK293 cells we...
(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 p27CK–, 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 p27CK– to cause multinucleation. HeLa cells were transfected with empty vector or vectors encoding the indicated p27CK– 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 p27CK– or different p27CK– mutants. Multinucleated cells were quantified (500 cells/condition, n = 3). The level of p27CK– or p27CK– mutants was measured by immunoblot against p27. Results were analyzed as in F.