Molecular basis for positive and negative signaling by the natural killer cell receptor 2B4 (CD244).
Triggering of 2B4 (CD244) can induce natural killer (NK)-cell activation, costimulation, or even inhibition of NK-cell activity. Here, we investigate the molecular basis for the different signals generated by 2B4. We show that the first immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of 2B4 is sufficient for 2B4-mediated NK-cell activation, whereas the third ITSM can negatively influence 2B4 signaling. We further identify signaling molecules that associate with 2B4. Signaling lymphocyte activation molecule-associated protein (SAP) can bind to all 4 ITSMs of 2B4 in a phosphorylation-dependent manner. The phosphorylated third ITSM can additionally recruit the phosphatases SHP-1, SHP-2, SHIP, and the inhibitory kinase Csk. SAP acts as an inhibitor of interactions between 2B4 and these negative regulatory molecules, explaining how 2B4 inhibits NK-cell activation in the absence of functional SAP, as occurs in cells from patients with X-linked lymphoproliferative syndrome (XLP). Recently, another function for SAP was proposed: SAP can recruit the kinase Fyn to the SLAM (CD150) immune receptor. We now show that Fyn can also associate with phosphorylated 2B4. Finally, we demonstrate that Fyn and Csk can both phosphorylate 2B4, suggesting a possible mechanism of 2B4 phosphorylation.
Eissmann
Philipp
P
Institute for Immunology, University Heidelberg, Germany.
Beauchamp
Lisa
L
Wooters
Joe
J
Tilton
John C
JC
Long
Eric O
EO
Watzl
Carsten
C
D020816:Amino Acid Motifs; D015703:Antigens, CD / Q000096:biosynthesis*; D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D002469:Cell Separation; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007136:Immunoglobulins / Q000378:metabolism / Q000502:physiology; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D007694:Killer Cells, Natural / Q000378:metabolism*; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000378:metabolism; D008232:Lymphoproliferative Disorders / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D011485:Protein Binding; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012190:Retroviridae / Q000235:genetics; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D015398:Signal Transduction; D000071176:Signaling Lymphocytic Activation Molecule Family; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism
D015703:Antigens, CD; C084093:CD244 protein, human; D018076:DNA, Complementary; D006023:Glycoproteins; D007136:Immunoglobulins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D010455:Peptides; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; C000604059:SLAMF1 protein, human; D000071176:Signaling Lymphocytic Activation Molecule Family; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1; D014443:Tyrosine; D005982:Glutathione Transferase; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; C000631346:CSK protein, human; D010744:Phosphoric Monoester Hydrolases; 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; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases