Cloning and functional expression of a degradation-resistant novel isoform of p27Kip1.
p27(Kip1) is an inhibitor of cyclin-dependent kinases. It has been implicated 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 and serum starvation. Proteasome-mediated degradation plays an important role in the rapid inactivation of p27(Kip1), 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 p27(Kip1) 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 G(0)/G(1) 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 p27(Kip1R) because of its resistance to degradation.
Hirano
K
K
Department of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Hirano
M
M
Zeng
Y
Y
Nishimura
J
J
Hara
K
K
Muta
K
K
Nawata
H
H
Kanaide
H
H
D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000737:chemistry; D003001:Cloning, Molecular; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004727:Endothelium / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D019913:Nuclear Localization Signals; D009928:Organ Specificity; D046988:Proteasome Endopeptidase Complex; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013552:Swine / Q000235:genetics*; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D008869:Microtubule-Associated Proteins; D009097:Multienzyme Complexes; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex