Cloning and Functional Expression of a Degradation-Resistant Novel Isoform of p27Kip1

Biochem J. 2001 Jan 1;353(Pt 1):51-57.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing / genetics
  • Amino Acid Sequence
  • Animals
  • Apoptosis
  • Cell Cycle
  • Cell Cycle Proteins*
  • Cell Nucleus / chemistry
  • Cloning, Molecular
  • Cyclin-Dependent Kinase Inhibitor p27
  • Cysteine Endopeptidases / metabolism
  • Endothelium / cytology
  • Endothelium / metabolism
  • Flow Cytometry
  • Gene Expression Profiling
  • HeLa Cells
  • Humans
  • Microtubule-Associated Proteins / chemistry
  • Microtubule-Associated Proteins / genetics*
  • Microtubule-Associated Proteins / metabolism*
  • Molecular Sequence Data
  • Multienzyme Complexes / metabolism
  • Nuclear Localization Signals
  • Organ Specificity
  • Proteasome Endopeptidase Complex
  • Protein Isoforms / chemistry
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • RNA, Messenger / analysis
  • RNA, Messenger / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Alignment
  • Swine / genetics*
  • Tumor Suppressor Proteins*

Substances

  • Cell Cycle Proteins
  • Microtubule-Associated Proteins
  • Multienzyme Complexes
  • Nuclear Localization Signals
  • Protein Isoforms
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Tumor Suppressor Proteins
  • Cyclin-Dependent Kinase Inhibitor p27
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex

Associated data

  • GENBANK/AB031955
  • GENBANK/AB031956
  • GENBANK/AB031958