Abstract
The CDK inhibitor, p27kip1, encoded by the Cdkn1b gene can negatively modulate cell proliferation. The control of p27 activity during the cell cycle is regulated at multiple levels, including transcription, translation, and protein stability. The last residue of p27 (threonine 198 in human, threonine 197 in mouse) is involved in the control of protein stability. We have generated a murine knock-in model (Cdkn1b T197A) in which threonine 197 is replaced by alanine, which renders p27 protein highly unstable due to a high rate of proteasomal degradation. Expectedly, Cdkn1b T197A/T197A mice present with increased body size and weight, organomegaly, and multiple organ hyperplasia, similar to what is observed in Cdkn1b KO/KO mice. We investigated the effects exerted by the restoration of normal levels of p27 protein in the tissue of Cdkn1b T197A/T197A mice. We found that proteasome inhibition with bortezomib rescues the hyperplasia induced by the lack of p27 expression in Cdkn1b T197A/T197A but not in Cdkn1b KO/KO mice. However, BAY 11-7082, a proteasome inhibitor that stabilizes IκB but not p27, fails to rescue hyperplasia in Cdkn1b T197A/T197A mice. Bortezomib increases p27 half-life and reduces the proliferation in MEFs derived from Cdkn1b T197A/T197A but not from Cdkn1b WT/WT mice, whereas BAY 11-7082 had no effect on the protein levels of p27 and on the proliferation rate of Cdkn1b T197A/T197A MEFs.The results presented here demonstrate that Cdkn1b T197A/T197A mice represent an attractive in vivo model to investigate whether the targeting of p27 degradation machinery might prove beneficial in the treatment of a variety of human proliferative disorders caused by increased turnover of p27 protein.
Highlights
Cell-cycle progression is regulated by the activity of cyclindependent kinases (CDK) that induce progression towards S-phase and initiate mitosis
The average weight of the organs resected from homozygous Cdkn1bT197A/T197A mice was consistently higher compared with the organs resected from wild-type mice [intestine: 199.7 (Æ 29.5) mg vs. 332.5 (Æ 83.6) mg; uterus 100 (Æ 22) mg vs. 145.9 (Æ 28) mg; spleen: 103.8 (Æ 27.9) mg vs. 211.9 (Æ 64.8) mg]
Because stability of p27 protein is controlled by the rate of its degradation through the ubiquitine/proteasome pathway, we investigated whether BZB, a proteasome inhibitor currently used in multiple myeloma and other cancers [27], increased the halflife of p27T197A protein, restoring normal level in mouse tissues
Summary
Cell-cycle progression is regulated by the activity of cyclindependent kinases (CDK) that induce progression towards S-phase and initiate mitosis. CDKs are activated by cyclin binding and inhibited by CDK inhibitors Two families of CKI negatively regulate cell-cycle progression: inhibitors of CDK4 (INK4) and kinase inhibitor proteins The KIP inhibitor p27Kip (hereafter p27) is encoded by the Cdkn1b gene [3,4,5]. P27 can modulate the activities of most CDK complexes [1], regulating the progression of cells from G1 to. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Recent studies have shown that p27 is capable to regulate, motility, and apoptosis [6,7,8]
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