Two Closely Spaced Tyrosines Regulate NFAT Signaling in B Cells via Syk Association with Vav

Abstract

Syk is a cytoplasmic protein tyrosine kinase that regulates cellular responses mediated by a variety of membrane receptors with intracellular signaling modules known as immunoreceptor tyrosine-based activation motifs (ITAMs) (23, 52). Examples of ITAM/Syk receptors include the B and T cell receptors for antigen, the immunoglobulin receptors FcεRI, Fc␥RI, and Fc␥RIIa, the activating NK cell receptors, and integrins. When these receptors are engaged, a pair of tyrosines within the ITAM become phosphorylated to create a docking site for Syk's N-terminal pair of SH2 domains. Syk binding to the ITAM leads to Syk activation and phosphorylation on several tyrosines to generate sites that participate in protein-protein interactions. Thus, activated Syk at the site of the clustered receptor participates in the formation of a signaling complex. This "signalosome" contains multiple effector proteins that include members of the Vav family of guanine nucleotide exchange factors (GEFs) (13,14).Among the three members of the Vav family, Vav1 is restricted largely to hematopoietic cells while Vav2 and Vav3 are more widely distributed (8,55,67,77). Vav proteins have both unique and redundant functions in the immune system, as a loss of Vav1 produces severe defects in T cell development and signaling, while the loss of more than one Vav family member is needed to produce similar defects in B cells (12,17,21,75,76). Vav proteins enhance many signaling pathways mediated by Syk-associated receptors, including the activation of phospholipase C-␥ (PLC-␥) and the mobilization of intracellular calcium, the activation of phosphoinositide 3-kinase (PI3K) and Akt, the activation of the Ras/Erk pathway, the promotion of cytoskeletal rearrangements, and the inside-out activation of integrins. While Vav proteins serve as GEFs for Rac/Rho family GTPases, they also act as scaffolds, a function that promotes the assembly of signaling complexes but does not require GEF activity (66).Syk associates with Vav family proteins and phosphorylates them on tyrosines that regulate their activity (13,43,50,53,59,69,74,75). This physical association occurs between the Vav SH2 domain and the Syk linker B region, which separates the tandem SH2 domains from the catalytic domain and contains phosphotyrosines 342 and 346 (based on the numbering system for murine Syk). Syk linker B interacts with multiple binding partners depending on which tyrosines in linker B are phosphorylated and the stoichiometry of this phosphorylation (23). As such, Syk linker B harbors the unusual site of two closely space tyrosines, which are recognized by a single SH2 domain of particular Syk binding partners. These interactions influence the ability of the kinase to couple ITAM-bearing receptors to the many different intracellular signaling pathways that are regulated following receptor engagement (23,52).Vav1 SH2 adopts the typical fold of an SH2 domain, including a central ␤-sheet flanked by two ␣-helices (Protein Data Bank [PDB] designation: 2CRH). The nonaromatic hydrophobic Ile r...

Syk Interacts with and Phosphorylates Nucleolin To Stabilize Bcl-xL mRNA and Promote Cell Survival

“…Thus, it is likely that the phosphorylation of both Y342 and Y346 on Syk within an intact cell generates a high-affinity docking site for a protein or proteins that contain an SH2 domain that binds to this region to disrupt the interaction between Syk and nucleolin. This hypothesis is consistent with the fact that several SH2 domains found on Syk-interacting proteins contain two phosphotyrosine-binding pockets and these interact with Syk preferentially when both Y342 and Y346 are phosphorylated (54,55).…”

Syk Is Recruited to Stress Granules and Promotes Their Clearance through Autophagy

“…The recruitment of Syk to SGs requires its phosphorylation on the two tyrosines found within the sequence, YESPYADP, in the linker B region of the protein. Multiple proteins have been described that contain SH2 domains capable of binding within this region, and many of these SH2 domains recognize Syk preferentially when both tyrosines (Tyr-342 and Tyr-346) are phosphorylated (41,44). This was first demonstrated for the C-terminal SH2 domain of phospholipase C-␥ (41).…”

Distinct mechanisms of a phosphotyrosyl peptide binding to two SH2 domains

“…The dissociation constants of pYpY are 7.4 and 0.07 μM, respectively, for Vav1 SH2 and PLC-γ SH2 8, 12 . For Vav1 SH2, the binding affinity is reduced by 10-fold when Y342 is unphosphorylated (abbreviated as YpY) and by 2-fold when Y346 is unphosphorylated (abbreviated as pYY) 12 . The binding affinity of pYY for PLC-γ SH2 is 6-fold lower 8 .…”

“…Phosphorylation of Y317 negatively regulates the function of Syk 10 , whereas phosphorylation of both Y342 and Y346 is required for optimal signaling 11 . The SH2 domains of Vav1 and PLC-γ can both bind to the doubly phosphorylated Syk linker B (referred to as pYpY hereafter), with pY342 in the primary site and pY346 in the secondary site 8, 12 . The dissociation constants of pYpY are 7.4 and 0.07 μM, respectively, for Vav1 SH2 and PLC-γ SH2 8, 12 .…”

“…The SH2 domains of Vav1 and PLC-γ can both bind to the doubly phosphorylated Syk linker B (referred to as pYpY hereafter), with pY342 in the primary site and pY346 in the secondary site 8, 12 . The dissociation constants of pYpY are 7.4 and 0.07 μM, respectively, for Vav1 SH2 and PLC-γ SH2 8, 12 . For Vav1 SH2, the binding affinity is reduced by 10-fold when Y342 is unphosphorylated (abbreviated as YpY) and by 2-fold when Y346 is unphosphorylated (abbreviated as pYY) 12 .…”