Identification of IkappaBalpha as a substrate of Fas-associated phosphatase-1.
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.
Nakai
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Tsukuba Life Science Center, RIKEN (The Institute of Physical and Chemical Research), Ibaraki, Japan.
Irie
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Sato
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D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D020672:I-kappa B Proteins; D000072000:NF-KappaB Inhibitor alpha; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D002352:Carrier Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha; D014443:Tyrosine; D054645:Protein Phosphatase 1; C516742:PTPN13 protein, human; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases