Phosphorylation by SYK occurs on tyrosine 517 of SWAP-70. (A) Schematic representation of SWAP-70 showing its known domains and the bacterial expression constructs used to identify the phosphorylation site. (B) SWAP-70 is phosphorylated at its C-terminal. Different domains of SWAP-70, the full-length protein, or BSA were incubated with a recombinant SYK-IgH fusion protein in the presence of γ-³²P-ATP. After SDS-PAGE the phosphorylation was detected by autoradiography. Arrows indicate the position of the different constructs, the full-length protein, and BSA. Only the C-terminal of SWAP-70 and the full-length protein are phosphorylated. Figure is representative of 5 independent experiments. (C) Comparison of the murine Swap-70 with other orthologues showing the overall conservation of the phosphorylation site and surrounding sequence. (D) Phoshorylation of the C-terminal is inhibited by the presence of the middle construct. Kinase reactions involving the C-terminus of SWAP-70 in the presence and absence of the N-terminal or middle-region proteins were analyzed by SDS-PAGE followed by autoradiography. The image shows the phosphorylated C-terminus. The accompanying bar graph shows the densitometric analysis of the C-terminus band with SD from 4 experiments. Only the 1:2 mixture of C-terminus with the middle of SWAP-70 showed significant inhibition of phosphorylation as indicated.