Two wobble-splicing events affect ING4 protein subnuclear localization and degradation

Exp Cell Res. 2008 Oct 15;314(17):3130-41. doi: 10.1016/j.yexcr.2008.08.002. Epub 2008 Aug 15.

Abstract

ING4 (inhibitor of growth 4) is a candidate tumor suppressor gene that is implicated as a repressor of cell growth, angiogenesis, cell spreading and cell migration and can suppress loss of contact inhibition in vitro. Another group and we identified four wobble-splicing isoforms of ING4 generated by alternative splicing at two tandem splice sites, GC(N)(7)GT and NAGNAG, which caused canonical (GT-AG) and non-canonical (GC-AG) splice site wobbling selection. Expression of the four ING4 wobble-splicing isoforms did not vary significantly in any of the cell lines examined. Here we show that ING4_v1 is translocated to the nucleolus, indicating that ING4 contains an intrinsic nucleolar localization signal. We further demonstrate that the subcellular localization of ING4 is modulated by two wobble-splicing events at the exon 4-5 boundary, causing displacement from the nucleolus to the nucleus. We also observed that ING4 is degraded through the ubiquitin-proteasome pathway and that it is subjected to N-terminal ubiquitination. We demonstrate that nucleolar accumulation of ING4 prolongs its half-life, but lack of nucleolar targeting potentially increases ING4 degradation. Taken together, our data suggest that the two wobble-splicing events at the exon 4-5 boundary influence subnuclear localization and degradation of ING4.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Cell Line
  • Cell Nucleolus / metabolism
  • Cycloheximide / metabolism
  • Exons
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism*
  • Humans
  • Mice
  • Molecular Sequence Data
  • Nuclear Localization Signals / genetics
  • Nuclear Localization Signals / metabolism
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism*
  • Protein Synthesis Inhibitors / metabolism
  • Protein Transport
  • RNA Splicing*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Alignment
  • Tumor Suppressor Protein p14ARF / genetics
  • Tumor Suppressor Protein p14ARF / metabolism
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism*
  • Ubiquitin / metabolism

Substances

  • Cell Cycle Proteins
  • Homeodomain Proteins
  • ING4 protein, human
  • Nuclear Localization Signals
  • Protein Isoforms
  • Protein Synthesis Inhibitors
  • Recombinant Fusion Proteins
  • Tumor Suppressor Protein p14ARF
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • Ubiquitin
  • Cycloheximide
  • Proteasome Endopeptidase Complex