FIGURE 1.

ZAP is phosphorylated. A, treatment of ZAP with alkaline phosphatase changes the mobility of ZAP. A Myc-NZAP332-expressing vector was transiently expressed in HEK293 cells. The protein was immunoprecipitated with protein G resin and anti-Myc antibody and treated with alkaline phosphatase or mock-treated. Samples were analyzed by Western blotting. B, substitutions of the serines between amino acids 255 and 295 with alanines alter the mobility of ZAP. The sequence of amino acids 255–295 of rat ZAP (rZAP) is shown (upper), and the substitutions of serines with alanines are schematically presented (lower left). The Myc-tagged NZAP295 proteins indicated were transiently expressed in HEK293 cells and analyzed by Western blotting (lower right). The numbers above the sequence are the numbers used to identify the serines studied in this work.