Molecular analysis of NTB-A signaling: a role for EAT-2 in NTB-A-mediated activation of human NK cells.
Engagement of NTB-A on human NK cells by homophilic interaction with NTB-A-expressing target cells can trigger NK cell cytotoxicity, cytokine production, and proliferation. To better understand how NTB-A can activate NK cells, we analyzed the molecular mechanisms of NTB-A signaling. We show that NTB-A is tyrosine phosphorylated in unstimulated human NK cells and associates with SLAM-associated protein (SAP) and EAT-2. This phosphorylation of NTB-A is mediated by Src family kinases and is most likely a result of the homophilic interaction of NTB-A among neighboring NK cells. Stimulation of NK cells by NTB-A-positive targets results in increased NTB-A phosphorylation. The cytoplasmic tail of NTB-A contains three tyrosines, two of which are embedded within an immunoreceptor tyrosine-based switch motif. We generated a NTB-A-negative NK cell line, in which we expressed different mutants of NTB-A. Functional studies showed that the second tyrosine is sufficient and essential for NTB-A-mediated cytotoxicity. EAT-2, but not SAP, is recruited to this second tyrosine, indicating that SAP may be dispensable for this NTB-A function. To further investigate this, we silenced SAP expression in NK cell lines. Functional analysis of these cells showed that NTB-A can mediate NK cell cytotoxicity in the absence of SAP, probably via EAT-2. In contrast, NTB-A-mediated IFN-gamma production was greatly reduced in the absence of SAP, demonstrating that cytokine production and cytotoxicity are differentially dependent on SAP and possibly EAT-2.
Eissmann
Philipp
P
Institute for Immunology, University Heidelberg, Heidelberg, Germany.
Watzl
Carsten
C
D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000276:immunology*; D010766:Phosphorylation; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D000071179:Signaling Lymphocytic Activation Molecule Associated Protein; D000071176:Signaling Lymphocytic Activation Molecule Family; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D015703:Antigens, CD; D047908:Intracellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; C114953:SH2D1A protein, human; C103572:SH2D1B protein, human; C000604060:SLAMF6 protein, human; D000071179:Signaling Lymphocytic Activation Molecule Associated Protein; D000071176:Signaling Lymphocytic Activation Molecule Family; D014157:Transcription Factors; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1; D014443:Tyrosine; D007371:Interferon-gamma