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.
Leskov
Konstantin S
KS
Department of Radiation Oncology, Laboratory of Molecular Stress Responses, Ireland Comprehensive Cancer Center and Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4942, USA.
Klokov
Dmitry Y
DY
Li
Jing
J
Kinsella
Timothy J
TJ
Boothman
David A
DA
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D051152:Clusterin; D018076:DNA, Complementary; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D011485:Protein Binding; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
C495242:CLU protein, human; D051152:Clusterin; D018076:DNA, Complementary; D006023:Glycoproteins; D018832:Molecular Chaperones; D012333:RNA, Messenger