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. 2017 Jan 12;292(8):3213–3223. doi: 10.1074/jbc.M116.759977

FIGURE 1.

FIGURE 1.

Cellular activation by NKp65 requires phosphotyrosinylation of the NKp65 hemITAM. A, degranulation of NK-92MI cells transduced with cDNA encoding for NKp65 or NKp65-Y7F or empty vector (mock) after 2-h co-culture with P815 loaded with the anti-NKp65 mAb OMAR1 or an irrelevant IgG1. For control, NK-92MI cells were treated with PMA plus ionomycin (P/I). The percentage of CD107a+ NK-92MI cells is shown. Viable (DAPI−) CD56+ cells were gated for analysis by flow cytometry. Depicted are means ± S.D. of triplicates from one representative of at least three independent experiments. B, the calcium flux of NK-92MI-NKp65 or NK-92MI-NKp65-Y7F cells was assessed by flow cytometry upon NKp65 cross-linking. OMAR1 (solid line) or an irrelevant IgG1 (dashed line) was added to NK-92MI cells and, after baseline recording for 30 s, cross-linked by addition of anti-mouse IgG antibodies (vertical dashed line). The ratio of median fluorescence intensities (fluo-4/fura red) was recorded by flow cytometry as a function of time. One representative of three independent experiments is depicted. C, amino acid alignment of the cytoplasmic domains of hemITAM-bearing human CTLR. Dashes indicate identical amino acids, and dots represent sequence gaps. The hemITAM comprises a triacidic sequence (shaded) preceding the strictly conserved YXXL module. D, NK-92MI-NKp65 cells were treated with or without pervanadate (PV) at 37 °C for 3 min. FLAG-tagged NKp65 was immunoprecipitated (IP) with OMAR1 and probed with the anti-phosphotyrosine mAb 4G10 (Tyr(P)) in immunoblotting. The membrane was reprobed with the anti-FLAG mAb M2 for control. E, NK-92MI-NKp65 or NK-92MI-NKp65-Y7F cells preloaded with OMAR1 were incubated for the indicated times at 37 °C with anti-mouse IgG antibodies to achieve NKp65 cross-linking (XL) and subsequently lysed. NKp65 immunoprecipitation and immunoblotting were performed as in D. For control, NK-92MI-NKp65 cells were preloaded with irrelevant IgG1. Depicted is one representative of four independent experiments. The position on NKp65 is marked by arrowheads.