FIGURE 3.

Arginine 6 of the NKp80 hemITAM impedes NKp80 phosphorylation by Src kinases. A, NKp80 phosphorylation is Src-family kinase-dependent. NK-92MI-NKp80 cells were pretreated for 20 min with various concentrations of the Src-family kinase inhibitor PP2, or the Syk kinase inhibitor piceatannol, respectively. Before cell lysis, cells were pervanadate-treated (2 min) and precipitated NKp80 assayed for tyrosine phosphorylation. B, impaired tyrosine phosphorylation of NKp80 can be reversed by substituting arginine for glycine at position 6. NK-92MI-NKp80, NK-92MI-NKp80/EGY, or NK-92MI-NKp80/DGY transductants were treated for various times with pervanadate before cell lysis, NKp80 precipitation, and assessment of NKp80 tyrosine phosphorylation. A and B, NKp80 was precipitated from lysates using mAb 5D12 and assayed for tyrosine phosphorylation by immunoblotting. Precipitated amounts of NKp80 were monitored with a polyclonal anti-NKp80 reagent. Comparable expression of NKp80 and NKp80 mutants by NK-92MI cells is shown in Fig. 5A.