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Synthesis and Functional Analyses of Nuclear Clusterin, a Cell Death Protein
Open accessAbstract
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.
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Clusterin inhibits apoptosis by interacting with activated Bax
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Metadata
- Authors
- Leskov, K. S., Klokov, D. Y., Li, J., Kinsella, T. J., & Boothman, D. A.
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 2003-03
- Volume
- 278
- Issue
- 13
- Pages
- 11590-11600
- ISSN
- 0021-9258 (ISSN-L)
- OpenAlex
- W2049119567
- Cited by
- 395
- References
- 40
- FWCI
- 11.07
- Type
- journal article
- Language
- en
- Open access
- hybrid
- Licence
- www.elsevier.com/tdm/userlicense/1.0/ (tdm)
- creativecommons.org/licenses/by/4.0/ (vor, 6422 d)
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Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).
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