The phosphorylation of S291 on Syk and its role in B cell signal transduction

Leela Louise Paris, Purdue University

Abstract

Spleen tyrosine kinase (Syk), a 72 kD non-receptor tyrosine kinase, is expressed in most hematopoietic cells including B-cells, mast cells, immature T-cells, and platelets. In B cells, Syk is required for signal propagation after the engagement of the B-cell receptor (BCR) with antigens. Syk is autophosphorylated on multiple tyrosine residues following BCR engagement. Metabolic labeling and phosphoamino acid analyses indicate that Syk also is phosphorylated on serine. A tryptic digest of Syk yielded four phosphoserine-containing peptides. The phosphorylation of one of these was enhanced following receptor ligation or the treatment of cells with phorbol myristate acetate. Phosphopeptide mapping and mass spectroscopy studies identified the site of phosphorylation as serine 291 (S291). The kinase responsible for the phosphorylation of S291 was identified to be a PKC based on metabolic labeling and phosphopeptide mapping studies in the prescence or absence of protein kinase inhibitors. This site of phosphorylation was shown to be important for interacting with prohibitin, a novel binding partner of Syk identified using mass spectrometry studies. Syk also was found to bind 14-3-3 in a manner dependent on the linker B region of Syk. As compared to wild-type Syk, Syk(S291A) exhibited a reduced ability to couple the BCR to the activation of nuclear factor of activated T cells (NFAT) while a Syk(S291D) exhibited enhanced activity. The BCR-dependent activation of an Elk-1 driven luciferase reporter also was decreased in Syk-deficient DT40 cells expressing Syk(S291A) as compared to cells expressing wild-type Syk. This decrease in Elk-1 transcriptional activity correlated with a deficiency in the activation of the extracellular signal-regulated kinase (ERK). To characterize this pathway further, we investigated the activation of Ras by monitoring its ability to bind to a GST-RBD (glutathions S-transferase fused to the Ras binding domain of Raf-1) in a pull-down assay. As compared to wild-type Syk, the expression of Syk(S291A) resulted in a reduced Ras activity after anti-IgM stimulation. The defect in ERK activation was not due to a change in the intrinsic catalytic activity of Syk as revealed by an in vitro kinase assay. Taken together, these results indicate that the phosphorylation of Syk on S291 is important for proper BCR-dependent signaling to both the NFAT and ERK signaling pathways.

Degree

Ph.D.

Advisors

Geahlen, Purdue University.

Subject Area

Molecular biology

COinS