Identification of IkappaBalpha as a substrate of Fas-associated phosphatase-1

Eur J Biochem. 2000 Dec;267(24):7170-5. doi: 10.1046/j.1432-1327.2000.01818.x.

Abstract

Fas (APO-1/CD95), a member of the tumor necrosis factor receptor (TNFR)/nerve growth factor receptor (NGFR) superfamily, is a cell-surface molecule that induces apoptosis upon activation. Fas-associated phosphatase-1 (FAP-1) is a 250-kDa protein tyrosine phosphatase (PTP) that is associated with the negative regulatory domain of Fas (C-terminal 15 amino acids). Human tumor cell lines become resistant to Fas-mediated apoptosis when transfected with FAP-1, indicating that FAP-1 functions as a negative regulator in Fas-mediated death signaling. However, the mechanisms by which FAP-1 inhibits apoptosis are still unclear. In order to determine how FAP-1 affects the signaling mediated by Fas, we set out to identify substrates of FAP-1. Toward this end, we prepared synthetic proteins with either the catalytic domain of FAP-1 (C-terminal 399 amino acids) or its inactive form (Cys2408-->Ser) fused to glutathione-S-transferase (GST). Using an in vitro dephosphorylation reaction, we found that FAP-1 dephosphorylates IkappaBalpha. Furthermore, a substrate trapping mutant was found to bind tyrosine-phosphorylated IkappaBalpha. Taken together, our data confirm that IkappaBalpha is a substrate of FAP-1.

Publication types

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

MeSH terms

  • Base Sequence
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • DNA Primers
  • DNA-Binding Proteins / metabolism*
  • Humans
  • I-kappa B Proteins*
  • NF-KappaB Inhibitor alpha
  • Phosphorylation
  • Protein Phosphatase 1
  • Protein Tyrosine Phosphatase, Non-Receptor Type 13
  • Protein Tyrosine Phosphatases / genetics
  • Protein Tyrosine Phosphatases / metabolism*
  • Substrate Specificity
  • Tumor Cells, Cultured
  • Tyrosine / metabolism

Substances

  • Carrier Proteins
  • DNA Primers
  • DNA-Binding Proteins
  • I-kappa B Proteins
  • NFKBIA protein, human
  • NF-KappaB Inhibitor alpha
  • Tyrosine
  • Protein Phosphatase 1
  • PTPN13 protein, human
  • Protein Tyrosine Phosphatase, Non-Receptor Type 13
  • Protein Tyrosine Phosphatases