Role for adapter proteins in costimulatory signals of CD2 and IL-2 on NK cell activation.
Natural killer (NK) cells participate in both innate and adaptive immunity through the prompt secretion of cytokines and ability to lyse virally infected cells or tumor cells. Triggering of NK cells requires aggregation of surface receptors such as CD2 and CD16, and NK cell activity can be augmented in vitro by stimulation with IL-2. In this study, we examined the role of adapter proteins in the increased NK activation following CD2 crosslinking and IL-2 stimulation of NK3.3 cells. NK3.3 cells lysed NK-sensitive K562 cells in a CD2-dependent manner, and IL-2 markedly enhanced lytic activity in a 4h cytotoxic assay. IL-2 also enhanced spontaneous and CD2-mediated granule exocytosis from NK3.3 cells. CD2 crosslinking markedly induced tyrosine phosphorylation of Cbl associated with Grb2 or CrkL, Shc and LAT, compared with IL-2 stimulation. However, costimulation of IL-2 with CD2 crosslinking remarkably enhanced associations of Grb2-Shc and CrkL-Cbl, compared to IL-2 stimulation or CD2 crosslinking alone. In vitro binding studies using GST-fusion proteins revealed that interactions of Grb2-Shc and CrkL-Cbl were mediated through each SH2 domain in tyrosine phosphorylation-dependent manner. Furthermore, CD2 crosslinking, but not IL-2 stimulation, markedly induced tyrosine phosphorylation of LAT. Thus, tyrosine phosphorylation of different adapter proteins and consequent interactions between signaling molecules described here may explain the molecular mechanisms of the additive effects of IL-2 stimulation and CD2 crosslinking on NK cell activation.
Umehara
Hisanori
H
Department of Internal Medicine, Osaka Dental University, Hirakata, Osaka 573-1121, Japan. umehara@kuhp.kyoto-u.ac.jp
Inoue
Hiroshi
H
Huang
Jianyong
J
Kono
Takeshi
T
Minami
Yasuhiro
Y
Tanaka
Yoshiya
Y
Okazaki
Toshiro
T
Mimori
Tsuneyo
T
Bloom
Eda T
ET
Domae
Naochika
N
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D018801:CD2 Antigens / Q000378:metabolism*; D002352:Carrier Proteins / Q000502:physiology; D002460:Cell Line; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic; D005089:Exocytosis; D051380:GRB2 Adaptor Protein; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology*; D008213:Lymphocyte Activation; D008565:Membrane Proteins; D009687:Nuclear Proteins / Q000502:physiology; D010750:Phosphoproteins / Q000502:physiology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011506:Proteins / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000502:physiology; D050721:Proto-Oncogene Proteins c-cbl; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D044767:Ubiquitin-Protein Ligases
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D018801:CD2 Antigens; C082869:CRKL protein; D002352:Carrier Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D007376:Interleukin-2; C110810:LAT protein, human; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019000:Phosphotyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C067419:CBL protein, human