Alterations in the Post-Translational Modification and Intracellular Trafficking of Clusterin in MCF-7 Cells During Apoptosis

Cell Death Differ. 2003 Aug;10(8):914-27. doi: 10.1038/sj.cdd.4401254.

Abstract

Clusterin is a heterodimeric, disulfide-linked 70-80 kDa glycoprotein that is induced during regression of most, if not all, hormone-dependent epithelial tissues. These studies describe the biogenesis and intracellular trafficking of clusterin in MCF-7 cells before and after the initiation of apoptosis with antiestrogens and TNF alpha. Under physiological conditions, clusterin is modified in the endoplasmic reticulum (ER), and proteolytically cleaved in the Golgi to generate discrete alpha and beta chains prior to secretion. Treatment with TNFalpha or the antiestrogen, ICI 182,780, induces apoptosis in MCF-7 cells and leads to substantial changes in the activity of Golgi-resident enzymes, significantly altering the biogenesis of clusterin. This leads to the appearance of a 50-53 kDa uncleaved, nonglycosylated, disulfide-linked isoform of clusterin that accumulates in the nucleus. While clusterin contains a cryptic SV-40-like nuclear localization signal, mutation of this sequence does not affect the nuclear accumulation of the disulfide-linked nuclear isoform. Confocal microscopy demonstrates that the nuclear accumulation of clusterin is coincident with DNA fragmentation. These data suggest that, at least in secretory epithelial cells, retrograde transport from the Golgi to the ER of a nonglycosylated, uncleaved isoform and the subsequent translocation of clusterin to the nucleus occur in dying cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Apoptosis / physiology*
  • Base Sequence
  • Blotting, Western
  • Brefeldin A / pharmacology
  • Cell Line, Tumor / drug effects
  • Cell Line, Tumor / metabolism
  • Cell Nucleus / metabolism
  • Clusterin
  • DNA Fragmentation / drug effects
  • Dose-Response Relationship, Drug
  • Endoplasmic Reticulum / metabolism
  • Estradiol / analogs & derivatives*
  • Estradiol / pharmacology
  • Fulvestrant
  • Galactosyltransferases / metabolism
  • Glycoproteins / chemistry
  • Glycoproteins / genetics
  • Glycoproteins / metabolism*
  • Glycosylation / drug effects
  • Golgi Apparatus / metabolism
  • Humans
  • In Situ Nick-End Labeling
  • Mannosidases / metabolism
  • Microscopy, Fluorescence
  • Molecular Chaperones / chemistry
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism*
  • Mutagenesis, Site-Directed
  • Nuclear Localization Signals / genetics
  • Protein Processing, Post-Translational*
  • Protein Transport / drug effects
  • Transfection
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • CLU protein, human
  • Clusterin
  • Glycoproteins
  • Molecular Chaperones
  • Nuclear Localization Signals
  • Tumor Necrosis Factor-alpha
  • Brefeldin A
  • Fulvestrant
  • Estradiol
  • Galactosyltransferases
  • Mannosidases
  • mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase