Inhibition of signaling through the B cell antigen receptor by the protooncogene product, c-Cbl, requires Syk tyrosine 317 and the c-Cbl phosphotyrosine-binding domain.

The Syk protein-tyrosine kinase couples the B cell Ag receptor (BCR) to intracellular biochemical pathways. Syk becomes phosphorylated on multiple tyrosine residues upon receptor cross-linking. Tyrosine 317 is a site of phosphorylation located within the linker region of Syk that separates the amino-terminal, tandem pair of SH2 domains from the carboxyl-terminal catalytic domain. The amino acid sequence surrounding phosphotyrosine 317 matches the consensus sequence for recognition by the phosphotyrosine-binding (PTB) domain of the protooncogene product, c-Cbl. The overexpression of c-Cbl in DT40 B cells inhibits Ag receptor-mediated activation of the NF-AT transcription factor. The ability of overexpressed c-Cbl to inhibit signaling requires both Syk tyrosine 317 and a functional c-Cbl PTB domain. Mutant forms of Syk lacking tyrosine 317 exhibit an enhanced ability to couple the BCR to pathways leading to the activation of both NF-AT and Elk-1. Coimmunoprecipitation experiments indicate that Syk phosphotyrosine 317 and the c-Cbl PTB domain enhance, but are not required for, all interactions between these two proteins. In unstimulated cells, c-Cbl and Syk can be isolated in a complex that also contains tubulin. A mutant form of Syk lacking tyrosine at position 317 exhibits an enhanced ability to interact with a diphosphopeptide modeled on the immunoreceptor tyrosine-based activation motif of the CD79a component of the Ag receptor. These studies indicate that c-Cbl may contribute to the regulation of BCR signaling by modulating the ability of Syk to associate with the BCR and couple the receptor to intracellular signaling pathways.

Phosphorylation- and activation-independent association of the tyrosine kinase Syk and the tyrosine kinase substrates Cbl and Vav with tubulin in B-cells.

Aggregation of the B-cell antigen receptor leads to the activation of the 72-kDa Syk protein-tyrosine kinase and the phosphorylation of tubulin on tyrosine. To explore the requirement of Syk catalytic activity for tubulin phosphorylation, tubulin was isolated from cytosolic fractions from anti-IgM-activated B-cells (DT40) that lacked endogenous Syk and immunoblotted with anti-phosphotyrosine antibodies. Tubulin was not tyrosine-phosphorylated in Syk- B-cells. Phosphorylation could be restored by the expression of wild-type, but not catalytically inactive, Syk. However, both catalytically inactive and wild-type Syk were capable of constitutive association with tubulin, indicating that tubulin phosphorylation is not required for this interaction. Anti-phosphotyrosine antibody immunoblotting of proteins adsorbed to colchicine-agarose revealed the presence of three major tubulin-associated phosphoproteins of 110, 90, and 74 kDa, the phosphorylation of which was dependent on Syk expression. The proteins of 110 and 90 kDa were identified as Cbl and Vav, two proto-oncogene products known to become prominently phosphorylated following receptor engagement. Both proteins were shown to be constitutively associated with tubulin.

Syk- and Lyn-dependent phosphorylation of Syk on multiple tyrosines following B cell activation includes a site that negatively regulates signaling.

The Syk protein tyrosine kinase is an essential component of the B cell Ag receptor signaling pathway. Syk is phosphorylated on tyrosine following B cell activation. However, the sites that are modified and the kinases responsible for these modifications have yet to be determined. To approach this problem, we used a mapping strategy based on the electrophoretic separation of peptides on alkaline polyacrylamide gels to identify the tryptic phosphopeptides derived from metabolically labeled Syk. In this work, we report that Syk from activated B cells is phosphorylated principally on six tyrosines: one located between the tandem SH2 domains (Tyr130); three in the linker region (Tyr317, Tyr342, and Tyr346); and two in the catalytic domain (Tyr519 and Tyr520). The linker region sites are the primary targets of the Src family protein tyrosine kinase, Lyn, and include a site that negatively (Tyr317) regulates receptor signaling. Efficient phosphorylation of the catalytic domain and inter-SH2 domain tyrosines is catalyzed primarily by Syk itself, but only occurs to an appreciable extent in cells that express Lyn. We propose that these sites are phosphorylated following the binding of Syk to immunoreceptor tyrosine-based activation motif.

Syk activation and dissociation from the B-cell antigen receptor is mediated by phosphorylation of tyrosine 130.

Syk (p72(syk)) is a 72-kDa cytoplasmic protein-tyrosine kinase that serves as an essential component of the signal transduction machinery coupled to the B-cell antigen receptor. Syk is recruited to the receptor when it is cross-linked and, in response, becomes tyrosine-phosphorylated and activated before it dissociates from the receptor and appears in the cytoplasm. To begin to explore how tyrosine phosphorylation affects Syk activation and receptor binding, Tyr-130, which is localized within the Syk inter-Src homology 2 domain region, was substituted with Phe or Glu. Substitution of Tyr-130 with Phe enhanced the binding of Syk to the receptor and increased receptor-mediated protein tyrosine phosphorylation, while substitution with Glu greatly reduced this interaction. Replacement of Tyr-130 with Glu also increased the basal activity of the kinase, while replacement with Phe decreased its activity and uncoupled kinase activation from receptor engagement. These data suggest that the phosphorylation of Tyr-130 normally plays an important role in mediating both the activation of Syk and its release from the antigen receptor.

Enzyme-linked immunosorbent assays for the measurement of blood group A and B glycosyltransferase activities.

ELISA assays have been developed for alpha(1-3)N-acetylgalactosaminyltransferase (blood group A transferase) and alpha(1-3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc alpha 1-2Gal beta-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuc alpha 1-2(GalNAc alpha 1-3)Gal beta-R-BSA or Fuc alpha 1-2(Gal alpha 1-3)Gal beta-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min.