Alteration of poly(ADP-ribose) glycohydrolase nucleocytoplasmic shuttling characteristics upon cleavage by apoptotic proteases

Biol Cell. 2003 Dec;95(9):635-44. doi: 10.1016/j.biolcel.2003.10.003.

Abstract

Poly(ADP-ribosyl)ation is an important post-translational modification which mostly affects nuclear proteins. The major roles of poly(ADP-ribose) synthesis are assigned to DNA damage signalling during base excision repair, apoptosis and excitotoxicity. The transient nature and modulation of poly(ADP-ribose) levels depend mainly on the activity of poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG), the key catabolic enzyme of poly(ADP-ribose). Given the fact that PARG substrate, poly(ADP-ribose), is found almost exclusively in the nucleus and that PARG is mainly localized in the cytoplasm, we wanted to have a closer look at PARG subcellular localization in order to better understand the mechanism by which PARG regulates intracellular poly(ADP-ribose) levels. We examined the subcellular distribution of PARG and of its two enzymatically active C-terminal apoptotic fragments both biochemically and by fluorescence microscopy. Green fluorescent protein (GFP) fusion proteins were constructed for PARG (GFP-PARG), its 74 kDa (GFP-74) and 85 kDa (GFP-85) apoptotic fragments and transiently expressed in COS-7 cells. Localization experiments reveal that all three fusion proteins localize predominantly to the cytoplasm and that a fraction also co-localizes with the Golgi marker FTCD. Moreover, leptomycin B, a drug that specifically inhibits nuclear export signal (NES)-dependent nuclear export, induces a redistribution of GFP-PARG from the cytoplasm to the nucleus and this nuclear accumulation is even more pronounced for the GFP-74 and GFP-85 apoptotic fragments. This observation confirms our hypothesis for the presence of important regions in the PARG sequence that would allow the protein to engage in CRM1-dependent nuclear export. Moreover, the altered nuclear import kinetics found for the apoptotic fragments highlights the importance of PARG N-terminal sequence in modulating PARG nucleocytoplasmic trafficking properties.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus / drug effects
  • Animals
  • COS Cells
  • Caspases / metabolism*
  • Cell Nucleus / metabolism*
  • Fatty Acids, Unsaturated / pharmacology
  • Glycoside Hydrolases / genetics
  • Glycoside Hydrolases / metabolism*
  • Golgi Apparatus / metabolism
  • Green Fluorescent Proteins
  • HeLa Cells
  • Humans
  • Immunoblotting
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Confocal
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Processing, Post-Translational
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Transfection

Substances

  • Fatty Acids, Unsaturated
  • Luminescent Proteins
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Glycoside Hydrolases
  • poly ADP-ribose glycohydrolase
  • Caspases
  • leptomycin B