Syk is a dual-specificity kinase that self-regulates the signal output from the B-cell antigen receptor

Abstract

Upon B-cell activation, the signaling subunits Ig-α and Ig-β of the B-cell antigen receptor become phosphorylated not only on tyrosines but also on serine residues. Using a specific antibody, we show that serine 197 (S197) in the cytoplasmic tail of Ig-α is phosphorylated upon B-cell antigen receptor activation, and that this modification inhibits the signal output of the B-cell antigen receptor. Surprisingly, we found that the well-known protein tyrosine kinase Syk (spleen tyrosine kinase) phosphorylates S197 on Ig-α, thus not only activating but also inhibiting signaling from the B-cell antigen receptor. This finding identifies Syk as a dual-specificity kinase and establishes a previously unexplored paradigm for the self-regulation of biological signaling processes.

Fig. 1.

Ig-α serine and threonine mutants exhibit increased BCR signaling compared with Ig-αWT. (A) The amino acid sequence of part of the mouse Ig-α cytoplasmic tail. The asterisks indicate the position of serine 191, serine 197, and threonine 203. The ITAM tyrosines are numbered (Y182 and Y193). The mutated amino acids are shown in red. The ITAM sequence is over-lined. (B and C) Following BCR antigen stimulation with NIP-BSA (1 and 10 ng/mL, as indicated), intracellular Ca²⁺ release was assessed in B cells carrying WT or mutant Ig-α. The control corresponds to Ig-α KO cells. Small arrows indicate the time points of NIP-BSA addition. (D and E) B cells containing Ig-α WT, Ig-αA or Ig-αAAV were activated with NIP-BSA (20 ng/mL) for the indicated time points. Western blot analysis of whole-cell lysates is shown with (D) an anti-pSyk (Y630) or with (E) an anti-pPKB (S473) antibody. Equal loading is shown with an antiactin antibody. Similar results were obtained in three independent experiments.

Fig. 2.

Phosphorylation of S197 in the Ig-α cytoplasmic tail plays a negative role in BCR activation. B cells expressing Ig-αWT or Ig-αA were stimulated with 20 ng/mL NIP-BSA for the indicated times. Western blot analysis of total cellular lysates is shown with (A) antiphospho-tyrosine (4G10) and antiactin antibodies; (B) anti-pERK (T202/Y204), anti-ERK2 and anti-actin antibodies. (C) Ig-α KO B cells expressing Ig-αWT, Ig-αA or Ig-αD were activated with 20 ng/mL NIP-BSA for the indicated times and the whole-cell lysates were analyzed by immunoblot with antiphospho-S197 Ig-α (pS197), anti-Flag and antiactin antibodies. Blots are representative of three independent experiments.

Fig. 3.

Syk mediates Ig-α serine phosphorylation. (A) Whole-cell lysates from S2 Drosophila cells transiently expressing scδm together with Ig-β, SLP-65, Syk, and either Ig-αWT (lanes 1 and 2) or Ig-αA (lanes 3 and 4) were blotted with different antibodies as indicated. (B) S2 cells were transiently transfected with scδm, Ig-β, SLP-65, Syk (lanes 3 and 4), SykKD (kinase death) (lanes 5 and 6), and either Ig-α WT (lanes 1, 3, and 5) or Ig-αA (lanes 2, 4, and 6). Total-cellular lysates were immunoblotted with 4G10, anti-pS197, anti-SLP-65, anti-Syk and anti-Flag antibodies. (C) S2 cells containing BCR components (μ, λ, Ig-β, and SLP-65) together with Syk (lanes 4–6) and either Ig-αWT (lanes 1 and 4), Ig-αFF (lanes 2 and 5), or Ig-αDD (lanes 3 and 6) were lysed and blotted with the following antibodies: 4G10, anti-pS197, anti-SLP-65, anti-Syk, and anti-Flag antibodies. Comparable results were achieved in three independent experiments.

Fig. 4.

The S197 of Ig-α is specifically phosphorylated by Syk. In vitro kinase assay. (A) Purified GST, GST-Ig-α (WT), or GST-Ig-αA (A) were incubated without or with recombinant GST-Syk. Ig-α phosphorylation was detected by immunoblot with 4G10 and anti-pS197 antibodies. (B) GST or GST-Ig-α were incubated either with GST-Lck (lanes 2 and 5) or with GST-Syk. No kinases were added in the controls (-). Phosphorylation was identified by immunoblotting with 4G10 and with anti-pS197 antibodies. The total amount of GST and GST-Ig-α proteins is shown with an anti-GST antibody. Comparable results were achieved in three independent experiments.

Fig. 5.

Ig-α serine phosphorylation in primary B cells. (A) Splenic B cells were treated as indicated with 50 nM calyculin in the absence (c) or presence of 20 μg/mL anti-IgM (IgM+c). Total cellular lysates were analyzed by Western blot with 4G10, anti-pS197, anti-Ig-α and anti-actin antibodies. (B) Splenic B cells treated or not with Syk inhibitor piceatannol (50 μM) were activated with 50 nM calyculin and 20 μg/mL anti-IgM (IgM+c). Whole-cell lysates were immunoblotted with 4G10, anti-pS197, anti-pSyk (Y630), anti-Ig-α and anti-Syk antibodies. Blots are representative of three independent experiments.

Fig. 6.

Syk is a dual-specificity kinase. (A) Comparison of the activation loop from different kinases. The structural alignment for Syk (1xbc, blue), ZAP-70 (1u59, red), B-Raf (2fb8, yellow), and PKC (1xjd, gray) was done with PDB files using PyMOL software (39). The black circle indicates the activation loop kink. Y539 of Syk is depicted in the frame together with the corresponding amino acids of the indicated kinases. Shaded amino acids represent conserved residues inside the kinase activation loop. (B) Whole-cell lysates from S2 cells transiently transfected with CD8-Ig-α and either with SykWT or SykY/A were immunoblotted as indicated. Equal loading is shown with an anti-actin antibody. (C) SykWT, SykKD, SykY/A, and SykKDY/A proteins were expressed in S2 cells. After immunoprecipitation, the different kinases were used for the in vitro kinase assay by incubating them together with GST-Ig-α. Phosphorylation was detected by Western blot with the indicated antibodies (Upper and second panels). A loading control is shown with anti-Syk and anti-GST antibodies. (D–F) Syk deficient pre-B cells were transduced with SykWT or SykY/A and (D) analyzed for Syk expression by Western blot using anti-Syk antibody. Equal loading is shown with antiactin antibody. (E) Pre-B cells expressing either SykWT or SykY/A were activated with anti-IgM (20 μg/mL) for the indicated times. Western blot analysis of total cellular lysates was performed with 4G10, anti-pS197, anti-Ig-α, and anti-actin antibodies. (F) Following pre-BCR stimulation (anti-IgM 20 μg/mL), intracellular Ca²⁺ release was measured in Syk KO pre B-cells expressing either SykWT or SykY/A mutant. Syk KO pre B-cells were used as a control. Similar results were obtained in three independent experiments.

Fig. 7.

B-cell development defect of pre-B cells expressing SykY/A. Syk KO pre-B cells transduced with either control vector (GFP), SykWT, or SykY/A were injected i.v. into RAG-2^−/−/γ_c^−/− mice. Nine days after injection, cells from spleen (A) and BM (B) were analyzed by flow cytometry. (A) B cells are shown in the dot plots with anti-CD19 and anti-IgM antibodies. The cells were gated on the GFP⁺ population. The number represents the percentage of CD19⁺IgM⁺ cells. (B) The histogram shows IgM expression of the GFP⁺CD19⁺ gated cells. (C) Histograms indicate the number of the GFP⁺CD19⁺ cells found in the spleen (Left) and in the BM (Right). Error bars show the means + SEM (each with five mice; *, P < 0.01). The cell injection was done two times using three mice per group per experiment. In these two experiments, the cells were independently infected and sorted for GFP expression.