FIGURE 1.

Dok-3/Grb2 attenuates the BCR-induced activation of Syk. A, Dok-3-deficient and HA-Dok-3-reconstituted DT40 cells as well as syk^−/− DT40 cells were left untreated (-) or stimulated for 3 min via their BCR (+). Syk was affinity-purified (AP) from cleared cellular lysates (CCL) using a doubly phosphorylated immunoreceptor tyrosine-based activation motif (pITAM) peptide. Samples were analyzed by anti-phosphotyrosine (α-pTyr, upper panel) or anti-Syk (α-Syk, lower panel) immunoblotting. The ratio of phospho-Syk (pSyk) to Syk signal intensities was calculated and plotted (right panel). The statistical significance of four independent experiments was determined using Student's t test. **, p < 0.01). B, schematic work flow of mass spectrometric analysis of Syk phosphotyrosine residues. Briefly, Syk/Dok-3-double-deficient DT40 cells were transfected with constructs encoding STrEP-tagged Syk together with either Dok-3(YF) as a control or Dok-3 and incubated in media containing “light” isotopes (¹²C₆,¹⁴N₂-Lys; ¹²C₆,¹⁴N₄-Arg) or “heavy” isotopes (²D₄,¹²C₆,¹⁴N₂-Lys; ¹³C₆,¹⁴N₄-Arg), respectively. Cells were stimulated for 3 min via their BCR, and Syk was purified with a Streptactin matrix. Samples were pooled 1:1, separated by PAGE, and the gel band referring to Syk was digested and analyzed by LC-MS/MS. C, the abundance of phosphopeptides encompassing the indicated phosphotyrosine residues was determined, and heavy/light ratios ± S.D. were calculated and depicted as a bar plot. DT40 wild-type and transfectants described in A were treated and lysed as above and subjected to Western blot analysis using phosphospecific anti-Syk Tyr-352 and anti-Syk antibodies (D, upper and lower panel, respectively). Signal quantification of n = 10 experiments was calculated as described in A. *, p < 0.05; **, p > 0.01. E, alternatively, cells were stimulated via their BCR, intracellularly stained with Alexa Fluor 633-conjugated anti-Syk pTyr-352 antibodies, and analyzed by flow cytometry. F, DG75 human B cells were either transduced with control virus or virus encoding for GFP fusion proteins of either Dok-3 or Dok-3(YF). Cells were loaded with Indo-I, and Ca²⁺ flux was analyzed by flow cytometry. As indicated, cells were stimulated after 30 s with anti-IgM antibodies. G, DG75 transductants were exposed to anti-Syk pTyr-352 Western blot analysis as described in D, and signals from n = 8 experiments were quantified. **, p < 0.01).