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Modulation of BCR Signaling by the Induced Dimerization of Receptor-Associated SYK

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Modulation of BCR Signaling by the Induced Dimerization of Receptor-Associated SYK

Mark L Westbroek et al. Antibodies (Basel).

Abstract

Clustering of the B cell antigen receptor (BCR) by polyvalent antigens is transmitted through the SYK tyrosine kinase to the activation of multiple intracellular pathways that determine the physiological consequences of receptor engagement. To explore factors that modulate the quantity and quality of signals sent by the crosslinked BCR, we developed a novel chemical mediator of dimerization to induce clustering of receptor-associated SYK. To accomplish this, we fused SYK with E. coli dihydrofolate reductase (eDHFR), which binds the small molecule trimethoprim (TMP) with high affinity and selectivity and synthesized a dimer of TMP with a flexible linker. The TMP dimer is able to induce the aggregation of eDHFR-linked SYK in live cells. The induced dimerization of SYK bound to the BCR differentially regulates the activation of downstream transcription factors, promoting the activation of Nuclear Factor of Activated T cells (NFAT) without affecting the activation of NFκB. The dimerization of SYK enhances the duration but not the amplitude of calcium mobilization by enhancing the extent and duration of its interaction with the crosslinked BCR at the plasma membrane.

Keywords: BCR signaling; NFAT; SYK; calcium mobilization; chemical inducer of dimerization.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
eDHFR-linked fusion proteins are stable and bind trimethoprim (TMP). (a) HEK293T cells were transfected with either recombinant Myc-SYK-eDHFR or control Myc-eDHFR plasmids and stable transfectant cell lines were produced. Western blot (WB) analysis of lysates from control HEK293T cells (Crtl) or stable transfectant HEK293T cells using antibodies against SYK or the Myc-epitope tag (Myc); (b) Lysates from HEK293T cells expressing Myc-SYK-eDHFR or Myc-eDHFR were adsorbed to immobilized TMP, shown in Figure 2a. Proteins in lysates (Input) or bound to beads were detected by Western blotting using anti-Myc epitope (Myc) antibodies.
Figure 2
Figure 2
Preparation of derivatives of trimethoprim (TMP). (a) Reaction scheme for the preparation of TMP covalently coupled to agarose beads; (b) Reaction scheme for the preparation of a TMP dimer.
Figure 3
Figure 3
The size of cellular ribonuclear protein aggregates increased when SYK and trimethoprim (TMP) dimerizing agent were present. (a) MCF7-BD cells stably expressing Myc-eDHFR or Myc-SYK-eDHFR were treated with or without MG132 to induce the formation of SGs in the presence or absence of TMP dimer. Cells were fixed and stained with antibodies against G3BP (red) and stained with 4’,6-diamidino-2-phenylindole (DAPI) to visualize nuclei (blue). Bar, 50 μm; (b) Large stress granules (SGs) were counted in cells treated as described in panel a. The histogram represents average ± standard error of the mean (SEM) of 80–100 cells from experiments repeated three times. * p < 0.05. DMSO, dimethylsulfoxide.
Figure 4
Figure 4
The trimethoprim (TMP) dimer enhances B cell antigen receptor (BCR)-dependent activation of Nuclear Factor of Activated T cells (NFAT) but not NFκB. (a) SYK-deficient DT40 cells transiently transfected with an NFAT-driven luciferase reporter plasmid and with plasmids for the expression of either Myc-eDHFR or Myc-SYK-eDHFR were activated by receptor crosslinking with anti-IgM where indicated in the presence of increasing concentrations of TMP dimer (TMPd). Values were normalized to 1 for cells activated with anti-IgM in the absence of TMPd. Values represent means ± standard error of the mean (SEM) of triplicate assays repeated three times. ** p < 0.01; (b) SYK-deficient DT40 cells were transfected and treated as in panel a, except that an NFκB-driven luciferase reporter plasmid was used. Values represent means ± SEM of triplicate assays repeated three times ns, not significant. The expression of Myc-SYK-eDHFR was verified by Western blotting with antibodies against SYK. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected as a loading control.
Figure 5
Figure 5
Dimerization of membrane-associated SYK is not sufficient to activate transcription factors. (a) SYK-deficient DT40 cells were transiently transfected to express NLyn-SYK-eDHFR or Myc-SYK-eDHFR. Cells were fixed and stained with antibodies against SYK. Nuclei were stained with DAPI. The expression of each protein was verified by Western blotting; (b,c) SYK-deficient DT40 cells were transiently transfected with plasmids coding for NLyn-SYK-eDHFR and luciferase reporter plasmids for detecting the activation of either Nuclear Factor of Activated T cells (NFAT) (b) or NFκB (c). Cells were treated with anti-IgM antibodies to crosslink the BCR or with increasing concentrations of trimethoprim (TMP) dimer (TMPd). Data represent the means ± standard error of the mean (SEM) of triplicate experiments repeated three times. The expression of NLyn-SYK-eDHFR was verified by Western blotting with antibodies against SYK.
Figure 6
Figure 6
Enhanced Nuclear Factor of Activated T cells (NFAT) activation by the trimethoprim (TMP) dimer requires SYK dimerization. (a) SYK-deficient DT40 cells transiently transfected with an NFAT-driven luciferase reporter plasmid and with a plasmid for the expression of SYK-EGFP were activated by receptor crosslinking with anti-IgM where indicated in the presence of increasing concentrations of TMP dimer (TMPd). Values represent means ± standard error of the mean (SEM) of triplicate assays repeated three times; (b) SYK-deficient DT40 cells transiently transfected with an NFAT-driven luciferase reporter plasmid and with a plasmid for the expression of Myc-SYK-eDHFR were activated by receptor crosslinking with anti-IgM in the presence of 10 μM TMP dimer (TMPd). Where indicated, an excess of TMP (100 μM) was added 2 h following activation. Values represent means ± SEM of triplicate assays repeated three times. ** p < 0.01. The expression of SYK-EGFP and Myc-SYK-eDHFR were verified by Western blotting.
Figure 7
Figure 7
The trimethoprim (TMP) dimer enhances the duration of calcium mobilization. SYK-deficient DT40 cells stably expressing Myc-SYK-eDHFR and labeled with Fluo-4 were activated by anti-IgM in the absence or presence of the TMP dimer (TMPd). Fluorescence readings were obtained every 5 s. Data represent means ± standard error of the mean (SEM) of triplicate experiments repeated three times.
Figure 8
Figure 8
The trimethoprim (TMP) dimer increases the retention of B cell antigen receptor (BCR) complexes. (a) DT40 cells stably expressing Myc-SYK-eDHFR were treated with Texas Red-labled anti-IgM (red) with or without TMP dimer for the indicated time points, then fixed, permeabilized and stained with antibodies against SYK (blue); (b) Images were analyzed by ImageJ for co-localization of anti-IgM and SYK; (c) SYK-dependent DT40 cells transiently transfected with an Nuclear Factor of Activated T cells (NFAT)-driven luciferase reporter plasmid and with a plasmid for the expression of Myc-SYK-eDHFR were activated by receptor crosslinking in the absence or presence of anti-IgM, LatB and/or TMP dimer. Values represent means ± standard error of the mean (SEM) of triplicate assays repeated three times. DMSO, dimethylsulfoxide.
Figure 8
Figure 8
The trimethoprim (TMP) dimer increases the retention of B cell antigen receptor (BCR) complexes. (a) DT40 cells stably expressing Myc-SYK-eDHFR were treated with Texas Red-labled anti-IgM (red) with or without TMP dimer for the indicated time points, then fixed, permeabilized and stained with antibodies against SYK (blue); (b) Images were analyzed by ImageJ for co-localization of anti-IgM and SYK; (c) SYK-dependent DT40 cells transiently transfected with an Nuclear Factor of Activated T cells (NFAT)-driven luciferase reporter plasmid and with a plasmid for the expression of Myc-SYK-eDHFR were activated by receptor crosslinking in the absence or presence of anti-IgM, LatB and/or TMP dimer. Values represent means ± standard error of the mean (SEM) of triplicate assays repeated three times. DMSO, dimethylsulfoxide.

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