Abstract
The protein p27Kip1 is an inhibitor of cell division1. An increase in p27 causes proliferating cells to exit from the cell cycle, and a decrease in p27 is necessary for quiescent cells to resume division2,3. Abnormally low amounts of p27 are associated with pathological states of excessive cell proliferation, especially cancers4,5,6,7,8. In normal and tumour cells, p27 is regulated primarily at the level of translation9,10,11 and protein turnover. Phosphorylation of p27 on threonine 187 (T187) by cyclin-dependent kinase 2 (Cdk2) is thought to initiate the major pathway for p27 proteolysis12,13,14,15. To critically test the importance of this pathway in vivo, we replaced the murine p27 gene with one that encoded alanine instead of threonine at position 187 (p27T187A). Here we show that cells expressing p27T187A were unable to downregulate p27 during the S and G2 phases of the cell cycle, but that this had a surprisingly modest effect on cell proliferation both in vitro and in vivo. Our efforts to explain this unexpected result led to the discovery of a second proteolytic pathway for controlling p27, one that is activated by mitogens and degrades p27 exclusively during G1.
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Acknowledgements
We thank B. Clurman, R. Sheaff, K.-I. Nakayama, B. Carter, M. Groudine, B. Luscher and members of the Roberts laboratory for advice, suggestions and reagents. M. Black and N. Jiang provided expert technical assistance with flow cytometry and with the generation of mutant mouse strains. We thank P. Porter for help with TUNEL staining. N.P.M. was supported by a grant from the Deutche Forschungsgemeinschaft. T.R.K. was supported by The National Science Foundation through the University of Washington Engineered Biomaterials Engineering Research Center. H.S. was supported by a grant from the National Institutes of Health. This work was supported by a grant from the National Institutes of Health to J.M.R. and by a George M. O'Brien Kidney Research Center award. J.M.R. is an Investigator of the Howard Hughes Medical Institute.
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Figure 1
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The same samples described in Figure 1B were immuneprecipitated with antibodies against cyclin E, and cyclin E-associated histone H1 kinase activity was measured. For convenience, the p27 immunoblots are reproduced here so that the pattern of p27 protein expression can be directly compared to the pattern of cyclin E activation
Figure 2
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Two examples of punch wounds of the skin 4.5 days after wounding. The leading edges of the new epithelial layer in the p27T187A mouse are indicated by arrows. The wound in this control mouse is completely covered by new epithelial cells. Magnification = 100x.
Figure 3
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Mean sizes of thymocytes from 8 week old mice of the indicated genotypes were determined byflow cytometric measurements of forward angle light scatter.
Figure 4
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Cells transfected with a p27 expression plasmid express predominantly the exogenous p27. 293 cells were transfected with the indicated plasmidsand cell extracts immunoblotted for expression of p27 protein. When compared to the expression level of the exogenous p27 protein (lanes 1,2 5,6), the amount of endogenous p27 is far less (lanes 3,4). Moreover, the amount of endogenous p27 is not detectably increased in cells expressing high amounts of exogenous p27.
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Malek, N., Sundberg, H., McGrew, S. et al. A mouse knock-in model exposes sequential proteolytic pathways that regulate p27Kip1 in G1 and S phase. Nature 413, 323–327 (2001). https://doi.org/10.1038/35095083
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DOI: https://doi.org/10.1038/35095083
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