Molecular Mechanism of the Activation-Induced Cell Death Inhibition Mediated by a p70 Inhibitory Killer Cell Ig-Like Receptor in Jurkat T Cells

Diglycerides/pharmacology Cytoplasm Apoptosis Ligands Lymphocyte Activation Jurkat Cells Apoptosis/genetics Receptors Membrane Proteins/immunology Killer Cells Isoenzymes/metabolism Recombinant Proteins/pharmacology Lymphocyte Activation*/drug effects Protein Kinase C 0303 health sciences Membrane Glycoproteins Cytoplasm/immunology Ionomycin Isoenzymes/antagonists & inhibitors Phytohemagglutinins/pharmacology KIR Isoenzymes Killer Cells, Natural Immunologic/biosynthesis Cytoplasm/chemistry Peptide Fragments/immunology T-Lymphocytes/cytology 570 Fas Ligand Protein Protein Kinase C-alpha Protein Kinase C/antagonists & inhibitors Molecular Sequence Data Membrane Glycoproteins/antagonists & inhibitors Protein Kinase C/metabolism Phosphatidylserines Diglycerides Apoptosis/immunology* 03 medical and health sciences T-Lymphocytes/drug effects Cytoplasm/enzymology KIR3DL1 Humans Amino Acid Sequence Membrane Proteins/genetics Peptide Fragments/genetics Phytohemagglutinins Phosphatidylserines/pharmacology Peptide Fragments/pharmacology Membrane Proteins/pharmacology Immunologic/genetics Apoptosis/drug effects Ionomycin/pharmacology Membrane Proteins Immunologic/physiology* Cytoplasm/genetics Natural/metabolism Lymphocyte Activation*/genetics Peptide Fragments Natural/immunology Protein Kinase C-theta Membrane Glycoproteins/biosynthesis
DOI: 10.4049/jimmunol.169.7.3726 Publication Date: 2014-04-21T23:18:01Z
ABSTRACT
AbstractIn this study we investigated the molecular mechanism of the activation-induced cell death (AICD) inhibition mediated by a p70 inhibitory killer cell Ig-like receptor (KIR3DL1, also called NKB1) in Jurkat T cells. Using stable Jurkat transfectants that express KIR or CD8-KIR fusion proteins we have shown for the first time that KIR inhibits, in a ligation-independent manner, the AICD induced by PHA, PMA/ionomycin, or anti-CD3 Ab. The AICD inhibition mediated by KIR appears to result from the blockade of Fas ligand induction upon activation of the Jurkat transfectants. Moreover, the membrane-proximal 20 aa of the KIR cytoplasmic tail were determined to play a crucial role in this process. Since the membrane-proximal portion of the KIR cytoplasmic tail contains a putative protein kinase C (PKC) substrate site, we investigated the molecular interaction between KIR and PKC. Immunoprecipitation analysis demonstrated that KIR constitutively bound both to PKCα, a conventional Ca2+-dependent PKC, and to PKCθ, a novel Ca2+-independent PKC. Furthermore, an in vitro kinase assay revealed that PKC activation was blocked after PHA stimulation in Jurkat transfectants expressing KIR. These observations were supported by the finding that a recombinant KIR cytoplasmic tail also appeared to inhibit PKCα activation in vitro. Taken together these data strongly suggest that KIR inhibits the AICD of T cells by blocking Fas ligand induction upon stimulation, in a process that seems to be accomplished by PKC recruitment to the membrane-proximal PKC binding site and subsequent inhibition of PKC activation against the activating stimuli.
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