FIGURE 3.

Differential roles of the activation loop tyrosines in ITK-SYK and SYK. Western blot analysis of COS7 cells expressing ITK-SYK activation loop tyrosine mutants alone (A) or with SYK (B). (For details regarding constructs, see Table 1.) C, SLP-76 is a direct substrate of ITK-SYK. Western blot analysis of COS7 cells co-transfected with plasmid constructs encoding SLP-76 and ITK-SYK activation loop mutants. D, SYK activation loop mutants were co-transfected with ITK-SYK, and total and phosphorylated proteins were detected as described in Fig. 1B. The effect of Y525E substitution did not differ from wild-type SYK (data not shown). E, schematic representation of phosphorylation status of ITK-SYK activation loop mutants. Phenylalanine (upper panel in each graph) is a nonphosphorylatable substitution, whereas glutamic acid (lower panel in each graph) is a phosphomimetic with regard to the altered charge. Bottom panel illustrates negative regulation of kinase activity by Y386E mutant. The circled P represents no change, and circled P with × corresponds to undetectable phosphorylation. F, FACS histogram indicates means ± 1 S.D. (n = 6) of CD69 expression of Jurkat cells with the corresponding ITK-SYK activation loop plasmids. Compared with ITK-SYK, all mutants displayed significant statistical difference at p value of <0.01. ★ denotes two of the least statistically significant differences at p value of <0.01. G, mean values ± 1 S.D. (n = 6), of IL-2 secretion (picograms/ml) in Jurkat cells. Compared with ITK-SYK, all mutants showed significant statistical differences at p value of <0.01. ★ denotes the least statistically significant difference at p value <0.01. n.s., statistically non-significant.