The functional interplay between Syk phosphorylation and inducible binding of Syk ligands has been worked out to a large extent for phosphotyrosine/SH2 interactions 7. However, a high

density of phosphoserine/threonine residues was found in the regulatory interdomain B (see Fig. 1). To explore the impact of serine/threonine phosphorylation on the ability of Syk to interact with other proteins we focused on a phosphorylation motif with the consensus sequence R/KXXpS/T. Human Syk encompasses seven copies of that motif but only www.selleckchem.com/products/KU-60019.html five of which are evolutionary conserved (see Fig. 3A) and according to our phosphotome analysis four of these motifs undergo inducible phosphorylation, i.e. T256, S295, S297 and T530 (see Fig. 1). They all resemble canonical docking sites for the 14-3-3 family of phosphoserine/threonine-binding proteins 41, 42. Indeed, buy SCH 900776 the γ-isoform of 14-3-3 co-immunoprecipitated with WT Syk (Fig. 3B, lanes 2–5). Exchange of serine 297 within the insert region of interdomain B

for alanine (S297A) was sufficient to abolish Syk/14-3-3γ binding (lanes 6–9). Hence, phospho-S297 is indispensible for complex formation between Syk and 14-3-3γ. Far Western analysis of anti-Syk immunoprecipitates with recombinantly expressed GST-14-3-3γ fusion proteins showed that the interaction between WT Syk and 14-3-3γ is direct (Fig. 3C, lanes 2–6). A weak interaction between GST-14-3-3γ and S297A mutant Syk (lanes 7–11) suggested that additional phosphosites can be recognized by 14-3-3γ to some extent in vitro. However, individual inactivation of all other canonical 14-3-3γ-binding motifs only marginally affected the enzyme/adaptor interaction (Fig. 3D). Taken together,

phospho-S297 identified in our phosphotome analysis as the dominant phosphoacceptor of Syk serves as docking site Fossariinae for 14-3-3γ. In fact, the amino acid sequence environment of S297 perfectly matches a so-called mode 1 motif for 14-3-3 binding (R/KSXpSxP) 41, 42. In accordance with these findings, antibodies specific for phosphorylated mode 1 motifs recognized WT Syk from activated B cells but neither the S297A mutant nor Syk immunoprecipitated from unstimulated B cells (Fig. 3E). To independently confirm the association between Syk and 14-3-3 proteins and to elucidate the global impact of the S297A exchange on the composition of the Syk interactome we quantitatively compared the signaling networks of WT and mutant Syk by SILAC-based “reverse proteomics”. Therefore, DT40 B cells expressing OneStrep-tagged versions of WT Syk or the S297A mutant were labeled in light or heavy SILAC medium, respectively. Following BCR stimulation for 5 min, Syk proteins were affinity-purified and Syk signalosomes were identified as well as quantified by LC-MS/MS analysis as described above. Complete quantification as performed by MaxQuant software is shown in Supporting Information Table 3.