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 inhibit ory receptors (KIRs) blocks natural cytotoxicity and antibody-dependent cel l cytotoxicity of NR cells and CD3/TCR dependent cytotoxicity of T cells. T he inhibitory effect of KIR ligation requires phosphorylation of the cytopl asmic tail of KIR and subsequent recruitment of an SH2-containing protein t yrosine 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 tai l (His-CytKIR) and Jurkat T cell lysates. We identified a target molecule o f SHP-1 by comparing the phosphorylation of major cellular substrates follo wing in vitro phosphorylation of Jurkat cell lysates in the presence and ab sence 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 pho sphorylated in vitro, PLC-gamma exhibited a dramatic decrease in phosphoryl ation when the His-CytKIR was mixed with Jurkat T cell lysates. However, PL C-gamma exhibited no decrease in phosphorylation when SHP-1 or Lck was depl eted or deficient in this reaction mixture, suggesting that the SHP-1 recru ited by the phosphorylated His-CytKIR directly mediate the dephosphorylatio n of PLC-gamma. The cell-free model system could be used to reveal the deta iled molecular interactions in the KIR-mediated signal transduction. (C) 19 99 Academic Press.