Complementary phosphorylation sites in the adaptor protein SLP-76 promote synergistic activation of natural killer cells.
The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require synergistic signals from specific pairs of co-activation receptors, such as CD314 (also known as NKG2D) and CD244 (2B4), which bind to distinct ligands present on target cells. These signals are required to overcome inhibition mediated by the E3 ubiquitin ligase c-Cbl of the guanine nucleotide exchange factor Vav1, which promotes activation of NK cells. Here, we showed that the adaptor protein SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons) was required for this synergy and that distinct tyrosine residues in SLP-76 were phosphorylated by each member of a pair of synergistic receptors. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76 enabled binding of SLP-76 to Vav1. Selective phosphorylation of SLP-76 at these residues was restricted to receptors that stimulated ligand-dependent target cell killing; antibody-dependent stimulation of the Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 mutant proteins showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that combined phosphorylation of separate tyrosine residues in SLP-76 forms the basis of synergistic NK cell activation.
Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA. hunkim@amc.seoul.kr
Hun Sik
HS
Kim
Eric O
EO
Long
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000704:Analysis of Variance; D015703:Antigens, CD / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D006801:Humans; D047468:Immunoprecipitation; D007371:Interferon-gamma / Q000378:metabolism; D007694:Killer Cells, Natural / Q000378:metabolism / Q000502:physiology*; D008213:Lymphocyte Activation / Q000502:physiology*; D008954:Models, Biological; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051567:Proto-Oncogene Proteins c-vav / Q000378:metabolism; D034622:RNA Interference; D011971:Receptors, Immunologic / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D000071176:Signaling Lymphocytic Activation Molecule Family; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; C084093:CD244 protein, human; D017931:DNA Primers; D055655:NK Cell Lectin-Like Receptor Subfamily K; D010750:Phosphoproteins; D051567:Proto-Oncogene Proteins c-vav; D011971:Receptors, Immunologic; C092947:SLP-76 signal Transducing adaptor proteins; D000071176:Signaling Lymphocytic Activation Molecule Family; C496618:VAV1 protein, human; D014443:Tyrosine; D007371:Interferon-gamma