Cloning and functional expression of a degradation-resistant novel isoform of p27Kip1

K. Hirano, M. Hirano, Y. Zeng, J. Nishimura, K. Hara, K. Muta, H. Nawata, H. Kanaide

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    Abstract

    p27Kip1 is an inhibitor of cyclin-dependent kinases. It has been implicated as having a role in the induction of growth arrest at the G1 phase of the cell cycle in response to anti-mitogenic signals such as cell contact and serum starvation. Proteasome-mediated degradation plays an important role in the rapid inactivation of p27Kip1, causing quiescent cells to re-enter the cell cycle. Although the existence of a second isoform has been suggested, no such isoform was isolated. Through screening of a cDNA library derived from growth-arrested confluent porcine endothelial cells, we obtained clones for a novel isoform of p27Kip1 in addition to the original isoform. The novel isoform differed from the original isoform at the C-terminus. The tissue-specific expression of the original and novel isoforms was demonstrated at the mRNA and protein levels. An in vitro degradation assay demonstrated this novel isoform to be resistant to proteasome-mediated destruction. The expression as a fusion protein with green fluorescent protein revealed this isoform to be targeted to the nucleus by a bipartite nuclear-localization signal with a C-terminal part different from that of the original isoform. The expression of the novel isoform caused the growth arrest of HeLa cells and an accumulation of cells in the G0/G1 phase, and this effect was similar to that seen with the original isoform. The present study suggests that the novel isoform functions as a negative regulator of the cell cycle, and may play a distinct role. The novel isoform was named p27Kip1R because of its resistance to degradation.

    Original languageEnglish
    Pages (from-to)51-57
    Number of pages7
    JournalBiochemical Journal
    Volume353
    Issue number1
    DOIs
    Publication statusPublished - Jan 1 2001

    All Science Journal Classification (ASJC) codes

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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    Hirano, K., Hirano, M., Zeng, Y., Nishimura, J., Hara, K., Muta, K., Nawata, H., & Kanaide, H. (2001). Cloning and functional expression of a degradation-resistant novel isoform of p27Kip1. Biochemical Journal, 353(1), 51-57. https://doi.org/10.1042/bj3530051