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Synthesis and functional analyses of nuclear clusterin, a cell death protein.

Nuclear clusterin (nCLU) is an ionizing radiation (IR)-inducible protein that binds Ku70, and triggers apoptosis when overexpressed in MCF-7 cells. We demonstrate that endogenous nCLU synthesis is a product of alternative splicing. Reverse transcriptase-PCR analyses revealed that exon II, containing the first AUG and encoding the endoplasmic reticulum-targeting peptide, was omitted. Exons I and III are spliced together placing a downstream AUG in exon III as the first available translation start site. This shorter mRNA produces the 49-kDa precursor nCLU protein. Ku70 binding activity was localized to the C-terminal coiled-coil domain of nCLU. Leucine residues 357, 358, and 361 of nCLU were necessary for Ku70-nCLU interaction. The N- and C-terminal coiled-coil domains of nCLU interacted with each other, suggesting that the protein could dimerize or fold. Mutation analyses indicate that the C-terminal NLS was functional in nCLU with the same contribution from N-terminal NLS. The C-terminal coiled-coil domain of nCLU was the minimal region required for Ku binding and apoptosis. MCF-7 cells show nuclear as well as cytoplasmic expression of GFP-nCLU in apoptotic cells. Cytosolic aggregation of GFP-nCLU was found in viable cells. These results indicate that an inactive precursor of nCLU exists in the cytoplasm of non-irradiated MCF-7 cells, translocates into the nucleus following IR, and induces apoptosis.

Pubmed ID: 12551933

Authors

  • Leskov KS
  • Klokov DY
  • Li J
  • Kinsella TJ
  • Boothman DA

Journal

The Journal of biological chemistry

Publication Data

March 28, 2003

Associated Grants

  • Agency: NCI NIH HHS, Id: CA78530
  • Agency: NCI NIH HHS, Id: CA84578
  • Agency: NCI NIH HHS, Id: P30 CA43703
  • Agency: NCI NIH HHS, Id: R01 CA078530

Mesh Terms

  • Amino Acid Sequence
  • Apoptosis
  • Base Sequence
  • Blotting, Western
  • Cell Line
  • Cell Nucleus
  • Clusterin
  • DNA, Complementary
  • Glycoproteins
  • Humans
  • Molecular Chaperones
  • Molecular Sequence Data
  • Protein Binding
  • RNA Splicing
  • RNA, Messenger
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Two-Hybrid System Techniques