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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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 1

Polyploidy and multinucleation in the liver and kidney of p27CK– mice.

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Polyploidy and multinucleation in the liver and kidney of p27CK– mice. ...
(A) p27 and p27CK– expression during the cell cycle. Primary MEFs (passage 2) derived from p27+/+ and p27CK– 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), p27CK– (n = 8), and p27S10A/S10A (n = 4) mice. Error bars represent SD. Examples of p27+/+ and p27CK– 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 p27CK– 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 p27CK– (n = 5) mice was determined. For each animal, 250–500 cells were counted. **P < 0.01, Mann-Whitney U test. Examples of p27+/+ and p27CK– H&E-stained kidney sections illustrating multinucleation are shown (arrows) (original magnification, ×600). Error bars in C and D represent SEM.