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Reconstitution of killer cell inhibitory receptor-mediated signal transduction machinery in a cell-free model system

Authors
 Cho HI  ;  Park CG  ;  Kim J 
Citation
 ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, Vol.368(2) : 221-231, 1999 
Journal Title
 ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 
ISSN
 0003-9861 
Issue Date
1999
MeSH
Amino Acid Sequence ; Biological Assay ; Cell-Free System ; Cytotoxicity, Immunologic ; Histocompatibility Antigens Class I/immunology ; Histocompatibility Antigens Class I/metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; Jurkat Cells ; Killer Cells, Natural/immunology ; Killer Cells, Natural/metabolism* ; Molecular Sequence Data ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; Protein Tyrosine Phosphatases/immunology ; Protein Tyrosine Phosphatases/metabolism* ; Receptors, Immunologic/genetics ; Receptors, Immunologic/immunology ; Receptors, Immunologic/metabolism* ; Receptors, KIR ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/immunology ; Recombinant Fusion Proteins/metabolism ; Signal Transduction/immunology*
Abstract
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.
Full Text
https://www.sciencedirect.com/science/article/pii/S0003986199913346
DOI
10.1006/abbi.1999.1334
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Microbiology (미생물학교실) > 1. Journal Papers
Yonsei Authors
Kim, Jong Sun(김종선) ORCID logo https://orcid.org/0000-0002-3149-669X
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/172915
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