Cloning and functional expression of a degradation-resistant novel isoformof p27(Kip1)

p27(Kip1) is an inhibitor of cyclin-dependent kinases. It has been implicat ed as having a role in the induction of growth arrest at the G(1) phase of the cell cycle in response to anti-mitogenic signals such as cell contact a nd serum starvation. Proteasome-mediated degradation plays an important rol e in the rapid inactivation of p27(Kip1), causing quiescent cells to re-ent er the cell cycle. Although the existence of a second isoform has been sugg ested, no such isoform was isolated. Through screening of a cDNA library de rived from growth-arrested confluent porcine endothelial cells, we obtained clones for a novel isoform of p27(Kip1) in addition to the original isofor m, The novel isoform differed from the original isoform at the C-terminus, The tissue-specific expression of the original and novel isoforms was demon strated at the mRNA and protein levels. An in vitro degradation assay demon strated this novel isoform to be resistant to proteasome-mediated destructi on. The expression as a fusion protein with green fluorescent protein revea led this isoform to be targeted to the nucleus by a bipartite nuclear-local ization signal with a C-terminal part different from that of the original i soform. The expression of the novel isoform caused the growth arrest of HeL a cells and an accumulation of cells in the G(0)/G(1) phase, and this effec t was similar to that seen with the original isoform. The present study sug gests that the novel isoform functions as a negative regulator of the cell cycle, and may play a distinct role. The novel isoform was named p27(Kip1R) because of its resistance to degradation.