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.
Highlights
The B cell antigen receptor (BCR) is a macromolecular complex consisting of a membrane-bound immunoglobulin and the heterodimeric co-receptors CD79A and CD79B [1]
We demonstrate that SYK dimerization does modulate BCR signaling and increase the retention of kinase-BCR complexes at the plasma membrane
T cells (NFAT)-driven luciferase reporter plasmid and with a plasmid for Factor of cells (NFAT)-driven luciferase reporter plasmid and with a plasmid for the expression the expression of Mycepitope tag (Myc)-SYK-E. coli dihydrofolate reductase (eDHFR) were activated by receptor crosslinking in the absence or presence of
Summary
The B cell antigen receptor (BCR) is a macromolecular complex consisting of a membrane-bound immunoglobulin and the heterodimeric co-receptors CD79A and CD79B [1]. The most widely accepted model is the BCR clustering model, where binding of polyvalent antigens induces clustering of BCR complexes [5,6], bringing signaling components into close enough proximity to activate their functions. More BCR crosslinking induces a greater response Both signaling and aggregation of antigen-bound receptors are enhanced by intracellular components. We demonstrate that SYK dimerization does modulate BCR signaling and increase the retention of kinase-BCR complexes at the plasma membrane.
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