Reconstitution of killer cell inhibitory receptor-mediated signal transduction machinery in a cell-free model system.
Recognition of class I MHC molecules on target cells by killer cell inhibitory receptors (KIRs) blocks natural cytotoxicity and antibody-dependent cell cytotoxicity of NK cells and CD3/TCR dependent cytotoxicity of T cells. The inhibitory effect of KIR ligation requires phosphorylation of the cytoplasmic tail of KIR and subsequent recruitment of an SH2-containing protein tyrosine phosphatase, SHP-1. To better understand the molecular mechanism of the KIR-mediated inhibitory signal transduction, we developed an in vitro assay system using a purified His-tag fusion protein of KIR cytoplasmic tail (His-CytKIR) and Jurkat T cell lysates. We identified a target molecule of SHP-1 by comparing the phosphorylation of major cellular substrates following in vitro phosphorylation of Jurkat cell lysates in the presence and absence of the His-CytKIR in this cell-free model system. The His-CytKIR was tyrosine phosphorylated by Lck in vitro, and the phosphorylated His-CytKIR recruited SHP-1. Interestingly, we observed that among major substrates phosphorylated in vitro, PLC-gamma exhibited a dramatic decrease in phosphorylation when the His-CytKIR was mixed with Jurkat T cell lysates. However, PLC-gamma exhibited no decrease in phosphorylation when SHP-1 or Lck was depleted or deficient in this reaction mixture, suggesting that the SHP-1 recruited by the phosphorylated His-CytKIR directly mediate the dephosphorylation of PLC-gamma. The cell-free model system could be used to reveal the detailed molecular interactions in the KIR-mediated signal transduction.
Cho
H I
HI
Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemoon-gu, Seoul, 120-752, Korea.
Park
C G
CG
Kim
J
J
D000595:Amino Acid Sequence; D001681:Biological Assay; D002474:Cell-Free System; D003602:Cytotoxicity, Immunologic; D015395:Histocompatibility Antigens Class I / Q000276:immunology / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000276:immunology / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D054340:Receptors, KIR; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*
D015395:Histocompatibility Antigens Class I; D047908:Intracellular Signaling Peptides and Proteins; D011971:Receptors, Immunologic; D054340:Receptors, KIR; D011993:Recombinant Fusion Proteins; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases