Figure 5A shows that the S297A variant translocated to the plasma membrane more efficiently than the WT counterpart. Quantification of the microscopic images using ImageJ (lower panel) confirmed that the enhanced recruitment of 14-3-3γ-bindingless Syk remained constant for at least 15 min after BCR ligation. The data are consistent with the results from our reverse Nivolumab order interactome analysis of

the S297A mutant (see above) and strongly suggest that 14-3-3γ inhibits stimulation-dependent membrane recruitment of Syk. To address whether 14-3-3γ also controls the degree and kinetics of Syk activation we immunoprecipitated WT and mutant Syk from resting and stimulated B cells and subjected the obtained proteins to anti-phosphotyrosine

immunoblotting Protein Tyrosine Kinase inhibitor (Fig. 5B). Inactivation of the 14-3-3γ-binding site caused a marked increase in Syk phosphorylation 2 and 5 min after BCR ligation (compare lanes 3–4 with 8–9). Quantification of the signal intensities revealed an approx. 40% amplification of Syk phosphorylation at these time points of BCR stimulation (lower panel). In summary, phosphorylation of S297 and the accompanied recruitment of 14-3-3γ dampen the efficiency with which Syk translocates to the plasma membrane upon BCR activation, thereby limiting phosphorylation-induced Syk activation and subsequent triggering of downstream effector pathways. These findings are not restricted to DT40 B cells as Syk also co-immunoprecipitated with 14-3-3γ in BCR-activated DG75 human B cells, which showed robust L-gulonolactone oxidase phosphorylation of the mode 1 binding motif (Fig. 6A, upper and middle panels, respectively). Note that maximal association between Syk and 14-3-3γ is observed in both cell lines after 5 min of BCR stimulation, which is consistent with the phosphorylation kinetics of S297. Similarly, we confirmed the increased membrane translocation of S297A mutant Syk in DG75 B cells (Fig. 6B). Owing to the endogenously expressed Syk in those

transfectants, their BCR-induced Ca2+ mobilization was normal as expected (data not shown). Taken together, the inhibitory complex between Syk and 14-3-3γ operates in chicken and human B cells. Understanding the diverse functions of Syk during development, activation and neoplastic transformation of hematopoietic cells requires comprehensive knowledge about its regulation by phosphorylation and the identity of Syk ligands. We have now determined the phosphorylation profile and the interactome of Syk in B cells. This was accomplished by affinity purification of Syk from SILAC-labeled resting or activated B cells followed by quantitative LC-MS/MS analysis of Syk phosphopeptides and Syk ligands. The B-lymphoid Syk phosphotome encompasses 32 acceptor sites with a strong prevalence for tyrosine residues (15) followed by serine (11) and threonine (6). More than 25 distinct Syk ligands were identified and most of these interactors required BCR activation.