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 6

p27CK– interferes with citron-K function.

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p27CK– interferes with citron-K function. (A) p27CK– does not affect...
(A) p27CK– does not affect the multinucleation caused by citron-K depletion. HeLa cells were transfected with scrambled (scr) or citron-K siRNAs and plasmids expressing p27CK– 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 p27CK– overexpression. HeLa cells were transfected with p27CK– 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 p27CK– primary MEFs. p27CK– 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 p27CK–-induced multinucleation. HeLa cells were transfected with scrambled or RhoA siRNA and plasmids expressing p27CK– 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.