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Identification of ubiquitination sites on the X-linked inhibitor of apoptosis protein

Hwain Shin1, Kazuya Okada, John C Wilkinson

  • 1The Program in Apoptosis and Cell Death Research, Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

The Biochemical Journal
|May 16, 2003
PubMed

Insights

The X-linked inhibitor of apoptosis (XIAP) protein

Area of Science:

  • Cellular biology
  • Molecular biology
  • Biochemistry

Background:

  • Members of the inhibitor of apoptosis (IAP) protein family regulate apoptosis execution.
  • The C-terminal RING domain in some IAPs is thought to control ubiquitination and apoptosis thresholds.

Purpose of the Study:

  • To identify the auto-ubiquitination sites of X-linked inhibitor of apoptosis (XIAP).
  • To determine the role of XIAP auto-ubiquitination in regulating apoptosis.

Main Methods:

  • Site-directed mutagenesis was used to alter specific lysine residues (Lys322 and Lys328) in XIAP to arginine.
  • Ubiquitination levels of XIAP were assessed.
  • The ability of XIAP to inhibit apoptosis was tested using two independent apoptotic inducers in cell-based assays.

Main Results:

  • The auto-ubiquitination sites of XIAP were identified as Lys322 and Lys328 within the third baculovirus IAP repeat domain.
  • Mutating these sites significantly reduced XIAP auto-ubiquitination.
  • XIAP's anti-apoptotic function remained unaffected despite the reduction in auto-ubiquitination.

Conclusions:

  • The identified sites are the primary auto-ubiquitination sites for XIAP.
  • XIAP auto-ubiquitination is not essential for its function in inhibiting apoptosis.
  • The role of XIAP auto-ubiquitination in regulating XIAP protein levels is questionable.

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