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

Mark L Westbroek, Purdue University

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, I developed a novel chemical mediator of dimerization to induce clustering of receptor-associated SYK. To accomplish this, I 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 binding of TMP derivatives to eDHFR containing fusion proteins was verified by affinity capture chromatography using a resin containing immobilized TMP. The TMP dimer is able to induce the aggregation of eDHFR-linked SYK in live cells, as evidenced by an increase in size of SYK-associated stress granules (SGs). As determined by a mass spectrometric analysis, the sodium arsenite-induced association of SYK with SGs induces a unique SYK-dependent tyrosine phosphorylation profile as compared to cells treated with the tyrosine phosphatase inhibitor, pervanadate, which does not induce SG formation. SYK tagged with eDHFR is able to support BCR signaling in SYK-deficient B cells. The induced dimerization of SYK bound to the BCR differentially regulates the activation of downstream transcription factors, promoting the activation of NFAT without affecting the activation of NFκB. This effect requires the association of SYK with the receptor as cytoplasmic or membrane-targeted SYK fusion proteins do not signal following dimerization in the absence of BCR crosslinking. 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. These results are consistent with a model whereby interactions between neighboring SYK-BCR complexes during aggregation promote the retention of these kinase-receptor complexes at the membrane leading to prolonged calcium mobilization and increased NFAT activation.

Degree

Ph.D.

Advisors

Geahlen, Purdue University.

Subject Area

Molecular biology|Chemistry|Immunology

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