Myosin IIA phosphorylation at S1943 is required for NK-cell cytotoxicity, but not IFN-γ secretion. Cytotoxic activity (A-B) and IFN-γ secretion (C) of YTS cells expressing wild-type myosin IIA-GFP (YTS-MyoIIA-GFP), 1933x myosin IIA-GFP (YTS-1933x-GFP), or S1943A myosin IIA-GFP (YTS-S1943A-GFP) was measured against 721.221 target cells. Cytotoxicity was measured at 1 hour (A) and 4 hours (B) after conjugation by ⁵¹Cr-release assay. (C) IFN-γ secretion into the supernatant was measured 24 hours after conjugation by ELISA and calculated by subtracting IFN-γ secretion of NK cells alone. In all experiments, results are the mean of 3 independent assays and error bars indicate SD. (D) GFP-tagged proteins were immunoprecipitated from lysates from YTS cells or YTS cells expressing wild-type myosin IIA-GFP (MIIA), S1943A myosin IIA-GFP (S1943A), or 1933x myosin IIA-GFP (1933x). GFP immunoprecipitates were evaluated by gel staining for total phosphorylation (Pro-Q) and total protein (Sypro). The larger of the 2 bands represents the myosin IIA-GFP fusion, which is enriched in the immunoprecipitates, and the smaller the endogenous myosin IIA. Results are representative of 3 independent experiments.