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

Konstantin S. Leskov, Dmitry Y. Klokov, Jing Li, Timothy J. Kinsella, David A. Boothman

Research output: Contribution to journalArticle

308 Citations (Scopus)

Abstract

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 49kDa 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.

Original languageEnglish (US)
Pages (from-to)11590-11600
Number of pages11
JournalJournal of Biological Chemistry
Volume278
Issue number13
DOIs
StatePublished - Mar 28 2003

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Clusterin
Cell death
Cell Death
Proteins
MCF-7 Cells
Exons
Ionizing radiation
Apoptosis
Ionizing Radiation
RNA-Directed DNA Polymerase
Alternative Splicing
Nuclear Proteins
Reverse Transcriptase Polymerase Chain Reaction
Leucine
Endoplasmic Reticulum

ASJC Scopus subject areas

  • Biochemistry

Cite this

Leskov, K. S., Klokov, D. Y., Li, J., Kinsella, T. J., & Boothman, D. A. (2003). Synthesis and functional analyses of nuclear clusterin, a cell death protein. Journal of Biological Chemistry, 278(13), 11590-11600. https://doi.org/10.1074/jbc.M209233200

Synthesis and functional analyses of nuclear clusterin, a cell death protein. / Leskov, Konstantin S.; Klokov, Dmitry Y.; Li, Jing; Kinsella, Timothy J.; Boothman, David A.

In: Journal of Biological Chemistry, Vol. 278, No. 13, 28.03.2003, p. 11590-11600.

Research output: Contribution to journalArticle

Leskov, KS, Klokov, DY, Li, J, Kinsella, TJ & Boothman, DA 2003, 'Synthesis and functional analyses of nuclear clusterin, a cell death protein', Journal of Biological Chemistry, vol. 278, no. 13, pp. 11590-11600. https://doi.org/10.1074/jbc.M209233200
Leskov, Konstantin S. ; Klokov, Dmitry Y. ; Li, Jing ; Kinsella, Timothy J. ; Boothman, David A. / Synthesis and functional analyses of nuclear clusterin, a cell death protein. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 13. pp. 11590-11600.
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