Abstract
The Syk protein-tyrosine kinase is phosphorylated on multiple tyrosines after the aggregation of the B cell antigen receptor. However, metabolic labeling experiments indicate that Syk is inducibly phosphorylated to an even greater extent on serine after receptor ligation. A combination of phosphopeptide mapping and mass spectrometric analyses indicates that serine 291 is a major site of phosphorylation. Serine 291 lies within a 23-amino acid insert located within the linker B region that distinguishes Syk from SykB and Zap-70. The phosphorylation of serine-291 by protein kinase C enhances the ability of Syk to couple the antigen receptor to the activation of the transcription factors NFAT and Elk-1. Protein interaction studies indicate a role for the phosphorylated linker insert in promoting an interaction between Syk and the chaperone protein, prohibitin.
Highlights
To its phosphorylation on tyrosines that regulate its activity and interactions with downstream effectors that bear Src homology 2 or other phosphotyrosine binding domains
Replacement of Tyr-290 with Phenylalanine—The replacement of Tyr-290 with phenylalanine was reported not to affect the ability of Syk to signal downstream of either Fc⑀RI or the T cell antigen receptor [8]. To determine whether this was true for the BCR, we expressed in Syk-deficient DT40 B cells Myc-tagged forms of wild-type Syk or a mutant in which Tyr-290 was replaced by phenylalanine (Syk(Y290F))
The ability of each to restore BCR-dependent signaling to Sykdeficient cells was measured by monitoring the activation of NFAT, a transcription factor regulated through changes in intracellular calcium
Summary
To its phosphorylation on tyrosines that regulate its activity and interactions with downstream effectors that bear Src homology 2 or other phosphotyrosine binding domains. Protein Interaction Assays—DT40 cells stably expressing Syk-EGFP or Syk-EGFP(S291A) and treated with anti-IgM (5 g/ml), PMA (100 ng/ml), or DMSO carrier as indicated were lysed in 1% Nonidet P-40, 25 mM HEPES, pH 7.2, 150 mM NaCl, 5 mM EDTA, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM sodium vanadate, and 2 mM NaF for 15 min on ice. Lysates were adsorbed to GST or GST-14-3-3 or GST-14-3-3 expressed in bacteria and bound to glutathione-Sepharose.
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