Regulation of Syk

In cells, it has been shown that Syk becomes active after binding to the phosphorylated ITAMs located on the BCR. Syk binds the two ITAM phosphotyrosines through its tandem SH2 domains. Hydrogen/deuterium exchange and mass spectrometric analyses show that the binding of Syk to a diphosphorylated ITAM peptide induces a structural change that stabilizes the linker A helix. The binding of the SH2 domains of Syk to the phosphotyrosine residues of the ITAM most likely occurs in a two step process. There is an initial binding of one of the SH2 domains to one phosphotyrosine in the ITAM. A conformational change occurs that is followed by the binding of the second SH2 domain to the second ITAM phosphotyrosine. Linker A is crucial for Syk’s binding to the receptor and for the subsequent relay of signals. The crystal structure of Syk’s tandem SH2 domains bound to an ITAM and the solution structure of the C-terminal SH2 domain indicate that the SH2 domains of Syk also can function independently from each other. Studies using a gain of function S2 insect cell system that reconstitutes BCR signaling show that the binding of the tandem SH2 domains of Syk to the BCR is absolutely crucial for Syk’s activation in cells. In fact in vitro experiments using a diphosphorylated ITAM demonstrate that binding to the ITAM peptide alone is sufficient for the full activation of Syk.

Syk undergoes autophosphorylation in vitro. The tyrosine residues that are phosphorylated have been identified through in vitro kinase assays, phosphopeptide mapping, and mass spectrometric analyses as Y130, Y290, Y317, Y342, Y346, Y519, Y520, Y624, and Y625. Syk’s autophosphorylation leads to its activation. Syk can autophosphorylate in vitro in the presence of ATP and presumably this also is one mechanism of its activation in cells. In fact it has been found that phosphorylation alone also can fully activate Syk.

The phosphorylated tyrosine residues on Syk have different roles including changes in conformation and changes in protein binding. Tyrosine 130 of murine Syk is phosphorylated in cells following BCR ligation. Phosphorylation of Y130 within linker A leads to a conformational change within the protein. This change increases the activity of Syk in cells, but decreases the affinity of Syk for the ITAMs. Tyrosine 290 on Syk is phosphorylated in vitro during an autophosphorylation reaction, but has not been found to be phosphorylated in intact cells of the immune system. Using mutants of Syk with Y290 mutated to a phenylalanine, no significant contribution of this residue to the ability of Syk to rescue FcεRI-mediated degranulation in Syk-deficient mast cells or antigen-induced tyrosine phosphorylation in T cells can be found. Tyrosine 317 on murine Syk is important for the regulation of Syk in B cells by acting as a site for the binding of the E3 ubiquitin ligase, cellular homolog of Casitas B lineage lymphoma (c-Cbl). When Y317 of Syk is mutated to a phenylalanine, Syk-dependent, anti-IgM-induced B cell signaling events such as NFAT- and ELK-1-mediated gene transcription are enhanced, suggesting a negative regulatory role for this site. Phosphorylated Y317 on Syk also is important for the binding of p85, the regulatory subunit of phosphoinositide-3-kinase (PI3K). Using gene knockout studies and dominant negative mutants, a Syk-dependent increase in phosphatidylinositol-3,4,5-trisphosphate following BCR-ligation is observed. These studies indicate that Syk is important for the regulation of the PI3K pathway. However the exact role that the phosphorylation of Y317 plays in the activation of PI3K in B cells has not been examined fully.

Tyrosine residues 342 and 346 of Syk are phosphorylated both in vitro by autophosphorylation as demonstrated using kinase assays in conjunction with phosphopeptide mapping and in intact, anti-IgM stimulated cells as shown using mass spectrometry, site specific antibodies, and phosphopeptide mapping. The phosphorylation of these sites in cells occurs by autophosphorylation or through phosphorylation by Lyn kinase following either BCR-ligation or treatment with H2O2 or pervanadate. Phosphorylation of Y342 and Y346 are crucial for the efficient activation of B cells. DT40 B cells expressing a mutant version of Syk in which tyrosines 342 and/or 346 are mutated to phenylalanines have defects in calcium mobilization and NFAT mediated transcription following BCR ligation. Tyrosines 342 and 346 of Syk can bind to different proteins depending on the site that is phosphorylated and the stoichiometry of phosphorylation. The phosphorylation of tyrosines 342 and 346 of Syk also enhances the catalytic activity of Syk. The zeta chain associated protein kinase 70, Zap-70, which is a second member of the Syk family of tyrosine kinases, contains corresponding tyrosine residues that contribute structurally to the stability of the autoinhibited state of the kinase. If these tyrosines are phosphorylated, structural studies suggest that they would
no longer stabilize this autoinhibited state resulting in the activation of the kinase. It is likely that Syk is regulated in an analogous manner.