Two closely spaced tyrosines regulate NFAT signaling in B cells via Syk association with Vav

Activated Syk, an essential tyrosine kinase in B cell signaling, interacts with Vav guanine nucleotide exchange factors and regulates Vav activity through tyrosine phosphorylation. The Vav SH2 domain binds Syk linker B by an unusual recognition of two closely spaced Syk tyrosines: Y342 and Y346. The binding affinity is highest when both Y342 and Y346 are phosphorylated. An investigation in B cells of the dependence of Vav phosphorylation and NFAT activation on phosphorylation of Y342 and Y346 finds that cellular response levels match the relative binding affinities of the Vav1 SH2 domain for singly and doubly phosphorylated linker B peptides. This key result suggests that the uncommon recognition determinant of these two closely spaced tyrosines is a limiting factor in signaling. Interestingly, differences in affinities for binding singly and doubly phosphorylated peptides are reflected in the on rate, not the off rate. Such a control mechanism would be highly effective for regulating binding among competing Syk binding partners. The nuclear magnetic resonance (NMR) structure of Vav1 SH2 in complex with a doubly phosphorylated linker B peptide reveals diverse conformations associated with the unusual SH2 recognition of two phosphotyrosines. NMR relaxation indicates compensatory changes in loop fluctuations upon binding, with implications for nonphosphotyrosine interactions of Vav1 SH2.     

Phosphorylation of Tyr342 in the linker region of Syk is critical for Fc epsilon RI signaling in mast cells

The linker region of Syk and ZAP70 tyrosine kinases plays an important role in regulating their function. There are three conserved tyrosines in this linker region; Tyr317 of Syk and its equivalent residue in ZAP70 were previously shown to negatively regulate the function of Syk and ZAP70. Here we studied the roles of the other two tyrosines, Tyr342 and Tyr346 of Syk, in Fc epsilon RI-mediated signaling. Antigen stimulation resulted in Tyr342 phosphorylation in mast cells. Syk with Y342F mutation failed to reconstitute Fc epsilon RI-initiated histamine release. In the Syk Y342F-expressing cells there was dramatically impaired receptor-induced phosphorylation of multiple signaling molecules, including LAT, SLP-76, phospholipase C-gamma2, but not Vav. Compared to wild-type Syk, Y342F Syk had decreased binding to phosphorylated immunoreceptor tyrosine-based activation motifs and reduced kinase activity. Surprisingly, mutation of Tyr346 had much less effect on Fc epsilon RI-dependent mast cell degranulation. An anti-Syk-phospho-346 tyrosine antibody indicated that antigen stimulation induced only a very minor increase in the phosphorylation of this tyrosine. Therefore, Tyr342, but not Tyr346, is critical for regulating Syk in mast cells and the function of these tyrosines in immune receptor signaling appears to be different from what has been previously reported for the equivalent residues of ZAP70.     

A novel quantitative proteomics strategy to study phosphorylation-dependent peptide-protein interactions

Phosphorylation-dependent protein-protein interactions provide the mechanism for a large number of intracellular signal transduction pathways. One of the goals of signal transduction research is to understand more precisely the nature of these phosphorylation-dependent interactions. Here, we report a novel strategy based on quantitative proteomics that allows for the rapid analysis of peptide-protein interactions with more than one phosphorylation site involved. The phosphorylation of two tyrosine residues, Y342 and Y346, within the linker B region of the protein-tyrosine kinase Syk is important for optimal signaling from the B cell receptor for antigen. We employed four amino-specific, isobaric reagents to differentially label proteins interacting in vitro with four Syk peptides containing none, one, or two phosphates on tyrosine residues Y342 and Y346, respectively. In total, 76 proteins were identified and quantified, 11 of which were dependent on the phosphorylation of individual tyrosine residues. One of the proteins, peroxiredoxin 1, preferably bound to phosphorylated Y346, which was further verified by Western blotting results. Thus, we demonstrate that the use of 4-fold multiplexing allows for relative protein measurements simultaneously for the identification of interacting proteins dependent on the phosphorylation of specific residues.     

Activation of the high-affinity immunoglobulin E receptor Fc epsilon RI in RBL-2H3 cells is inhibited by Syk SH2 domains.

Antigen-mediated aggregation of the high-affinity receptor for immunoglobulin E, Fc epsilon RI, results in the activation of multiple signaling pathways, leading to the release of mediators of the allergic response. One of the earliest responses to receptor stimulation is the tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI and the association of the tyrosine kinase Syk with the phosphorylated receptor. This association is mediated by the SH2 domains of Syk and is believed to be critical for activating signaling pathways resulting in mediator release. To examine the importance of the interaction of Syk with Fc epsilon RI in signaling events following receptor activation, we introduced a protein containing the SH2 domains of Syk into streptolysin O-permeabilized RBL-2H3 cells. The Syk SH2 domains completely inhibited degranulation and leukotriene production following receptor aggregation, and they blocked the increase in protein tyrosine phosphorylation observed after receptor activation. Inhibition was specific for Fc epsilon RI-mediated signaling, since degranulation of cells activated by alternative stimuli was not blocked by the Syk SH2 domains. A protein containing a point mutation in the carboxy-terminal SH2 domain which abolishes phosphotyrosine binding was not inhibitory. In addition, inhibition of degranulation was reversed by pretreatment of the SH2 domains with a tyrosine phosphorylated peptide corresponding to the tyrosine-based activation motif found in the gamma subunit of Fc epsilon RI, the nonphosphorylated peptide had no effect. The association of Syk with the tyrosine-phosphorylated gamma subunit of the activated receptor was blocked by the Syk SH2 domains, and deregulation in cells activated by clustering of Syk directly without Fc epsilon RI aggregation was not affected by the Syk SH2 domains. These results demonstrate that the association of Syk with the activated Fc epsilon RI is critical for both early and late events following receptor activation and confirm the key role Syk plays in signaling through the high-affinity IgE receptor.     

Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase Cgamma Src homology 2-Src homology 3 linker

Tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCgamma in vitro, the specific kinase(s) controlling BCR-dependent PLCgamma activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCgamma2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCgamma2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCgamma2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr(753) and Tyr(759). Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCgamma2 carboxyl-terminal sites, Tyr(1197) and Tyr(1217), was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCgamma2 SH2-SH3 linker.     

Molecular basis for a direct interaction between the Syk protein-tyrosine kinase and phosphoinositide 3-kinase

After engagement of the B cell receptor for antigen, the Syk protein-tyrosine kinase becomes phosphorylated on multiple tyrosines, some of which serve as docking sites for downstream effectors with SH2 or other phosphotyrosine binding domains. The most frequently identified binding partner for catalytically active Syk identified in a yeast two-hybrid screen was the p85 regulatory subunit of phosphoinositide 3-kinase. The C-terminal SH2 domain of p85 was sufficient for mediating an interaction with tyrosine-phosphorylated Syk. Interestingly, this domain interacted with Syk at phosphotyrosine 317, a site phosphorylated in trans by the Src family kinase, Lyn, and identified previously as a binding site for c-Cbl. This site interacted preferentially with the p85 C-terminal SH2 domain compared with the c-Cbl tyrosine kinase binding domain. Molecular modeling studies showed a good fit between the p85 SH2 domain and a peptide containing phosphotyrosine 317. Tyr-317 was found to be essential for Syk to support phagocytosis mediated by FcgammaRIIA receptors expressed in a heterologous system. These studies establish a new type of p85 binding site that can exist on proteins that serve as substrates for Src family kinases and provide a molecular explanation for observations on direct interactions between Syk and phosphoinositide 3-kinase.     

Activation of Syk protein tyrosine kinase through interaction with integrin beta cytoplasmic domains.

Syk protein tyrosine kinase is essential for immune system development and function [1]and for the maintenance of vascular integrity [2,3]. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs)[1]. Syk can also be activated by integrin adhesion receptors [4,5], but the mechanism of its activation is unknown. Here we report a novel mechanism for Syk's recruitment and activation, which requires that Syk bind to the integrin beta3 cytoplasmic tail. We found that both Syk and the related kinase ZAP-70 bound the beta3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction was independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the beta3 cytoplasmic tail [beta3(759X)] decreased Syk binding and disrupted its physical association with integrin alphaIIbbeta3. Furthermore, cells expressing alphaIIbbeta3(759X) failed to exhibit Syk activation or lamellipodia formation upon cell adhesion to the alphaIIbbeta3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation were unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin beta3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase.     

Tyrosine residues in phospholipase Cgamma 2 essential for the enzyme function in B-cell signaling

Phospholipase Cgamma (PLCgamma) isoforms are regulated through activation of tyrosine kinase-linked receptors. The importance of growth factor-stimulated phosphorylation of specific tyrosine residues has been documented for PLCgamma1; however, despite the critical importance of PLCgamma2 in B-cell signal transduction, neither the tyrosine kinase(s) that directly phosphorylate PLCgamma2 nor the sites in PLCgamma2 that become phosphorylated after stimulation are known. By measuring the ability of human PLCgamma2 to restore calcium responses to the B-cell receptor stimulation or oxidative stress in a B-cell line (DT40) deficient in PLCgamma2, we have demonstrated that two tyrosine residues, Tyr(753) and Tyr(759), were important for the PLCgamma2 signaling function. Furthermore, the double mutation Y753F/Y759F in PLCgamma2 resulted in a loss of tyrosine phosphorylation in stimulated DT40 cells. Of the two kinases that previously have been proposed to phosphorylate PLCgamma2, Btk, and Syk, purified Btk had much greater ability to phosphorylate recombinant PLCgamma2 in vitro, whereas Syk efficiently phosphorylated adapter protein BLNK. Using purified proteins to analyze the formation of complexes, we suggest that function of Syk is to phosphorylate BLNK, providing binding sites for PLCgamma2. Further analysis of PLCgamma2 tyrosine residues phosphorylated by Btk and several kinases from the Src family has suggested multiple sites of phosphorylation and, in the context of a peptide incorporating residues Tyr(753) and Tyr(759), shown preferential phosphorylation of Tyr(753).     

Distinct roles for the linker region tyrosines of Syk in FcepsilonRI signaling in primary mast cells

Stimulation of FcepsilonRI, the high affinity IgE receptor of mast cells results in the rapid binding of the Syk tyrosine kinase to cytoplasmic domains of FcepsilonRI and to its subsequent activation. Syk plays an essential role in signal transduction from FcepsilonRI as shown by Syk-deficient mast cells, which are defective in receptor-induced degranulation, cytokine synthesis, and intracellular pathways. However the mechanism by which Syk activates these pathways remains unclear. Activation of Syk is associated with its phosphorylation on several tyrosine residues, including the linker tyrosines Tyr317, Tyr342, and Tyr346. These residues have been proposed to play important roles in the transduction of signals by binding to other signaling proteins. To test these hypotheses in primary murine mast cells, we used retroviral infection of Syk-deficient mast cells to generate cells expressing Syk proteins bearing mutations in the linker tyrosines. We show that Tyr342 and Tyr346 contribute positively to the function of Syk and have both overlapping as well as distinct functions. Mutations in either Tyr342 or Tyr346 alone had no effect on FcepsilonRI-induced degranulation or calcium flux, whereas mutation of both residues caused a significant reduction in both pathways. In contrast, phosphorylation of PLCgamma1, PLCgamma2, and Vav1 was strongly decreased by a mutation in Tyr342 alone, whereas phosphorylation of ERK and Akt was more dependent on Tyr346. Finally we show that Tyr317 functions as a negative regulatory site and that its mutation can partially compensate for the loss of both Tyr342 and Tyr346.     

Phosphorylation on Syk Y342 is important for both ITAM and hemITAM signaling in platelets

Immune cells express receptors bearing an immune tyrosine activation motif (ITAM) containing two YXXL motifs or hemITAMs containing only one YXXL motif. Phosphorylation of the ITAM/hemITAM is mediated by Src family kinases allowing for the binding and activation of spleen tyrosine kinase (Syk). It is believed that Syk must be phosphorylated on tyrosine residues for activation, and Tyr342, а conserved tyrosine in the interdomain B region, has been shown to be critical for regulating Syk in FcεR1-activated mast cells. Syk is a key mediator of signaling pathways downstream of several platelet pathways including the ITAM bearing glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor and the hemITAM containing C-type lectin-like receptor-2 (CLEC-2). Since platelet activation is a crucial step in both hemostasis and thrombosis, we evaluated the importance of Syk Y342 in these processes by producing an Syk Y342F knock-in mouse. When using a CLEC-2 antibody as an agonist, reduced aggregation and secretion were observed in Syk Y342F mouse platelets when compared with control mouse platelets. Platelet reactivity was also reduced in response to the GPVI agonist collagen-related peptide. Signaling initiated by either GPVI or CLEC-2 was also greatly inhibited, including Syk Y519/520 phosphorylation. Hemostasis, as measured by tail bleeding time, was not altered in Syk Y342F mice, but thrombus formation in response to FeCl₃ injury was prolonged in Syk Y342F mice. These data demonstrate that phosphorylation of Y342 on Syk following stimulation of either GPVI or CLEC-2 receptors is important for the ability of Syk to transduce a signal.     

Structural basis for recruitment of the adaptor protein APS to the activated insulin receptor

The adaptor protein APS is a substrate of the insulin receptor and couples receptor activation with phosphorylation of Cbl to facilitate glucose uptake. The interaction with the activated insulin receptor is mediated by the Src homology 2 (SH2) domain of APS. Here, we present the crystal structure of the APS SH2 domain in complex with the phosphorylated tyrosine kinase domain of the insulin receptor. The structure reveals a novel dimeric configuration of the APS SH2 domain, wherein the C-terminal half of each protomer is structurally divergent from conventional, monomeric SH2 domains. The APS SH2 dimer engages two kinase molecules, with pTyr-1158 of the kinase activation loop bound in the canonical phosphotyrosine binding pocket of the SH2 domain and a second phosphotyrosine, pTyr-1162, coordinated by two lysine residues in beta strand D. This structure provides a molecular visualization of one of the initial downstream recruitment events following insulin activation of its dimeric receptor.     

Domain-dependent function of the rasGAP-binding protein p62Dok in cell signaling

p62Dok, the rasGAP-binding protein, is a common target of protein-tyrosine kinases. It is one of the major tyrosine-phosphorylated molecules in v-Src-transformed cells. Dok consists of an amino-terminal Pleckstrin homology domain, a putative phosphotyrosine binding domain, and a carboxyl-terminal tail containing multiple tyrosine phosphorylation sites. The importance and function of these sequences in Dok signaling remain largely unknown. We have demonstrated here that the expression of Dok can inhibit cellular transformation by the Src tyrosine kinase. Both the phosphotyrosine binding domain and the carboxyl-terminal tail of Dok (in particular residues 336-363) are necessary for such activity. Using a combinatorial peptide library approach, we have shown that the Dok phosphotyrosine binding domain binds phosphopeptides with the consensus motif of Y/MXXNXL-phosphotyrosine. Furthermore, Dok can homodimerize through its phosphotyrosine binding domain and Tyr(146) at the amino-terminal region. Mutations of this domain or Tyr(146) that block homodimerization significantly reduce the ability of Dok to inhibit Src transformation. Our results suggest that Dok oligomerization through its multiple domains plays a critical role in Dok signaling in response to tyrosine kinase activation.     

The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains

Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N termini. Engagement of these SH2 domains by tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs leads to kinase activation and downstream signaling. These kinases are also regulated by beta3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the beta3 integrin cytoplasmic domain. Here, we report that the interaction of integrins with Syk and ZAP-70 depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta cytoplasmic domains with varying affinities (beta3 (Kd = 24 nm) > beta2 (Kd = 38 nm) > beta1 (Kd = 71 nm)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains.     

Molecular mechanism of the Syk activation switch

Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340-356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression.     

Mutational investigation of the specificity determining region of the Src SH2 domain

SH2 domains are protein modules which bind tyrosine phosphorylated sequences in many signaling pathways. These domains contain two regions with specialized functions: residues in one region form a deep pocket into which the phosphotyrosine of the target inserts, while the other region contains the so-called "specificity determining residues" which interact with the three residues C-terminal to the phosphotyrosine in the target. Here, titration calorimetry and site-directed mutagenesis have been used to probe the importance of eight specificity determining residues of the SH2 domain of the Src kinase involved in contacts with its tyrosine phosphorylated consensus peptide target (sequence pYEEI where pY indicates a phosphotyrosine). Mutating six of these eight residues to Ala individually, resulted in a threefold or less loss in binding affinity; hence the majority of the residues in the specificity determining region are by themselves of minimal importance for binding. Two residues were found to have significant effects on binding: Tyr betaD5 and Lys betaD3. Tyr betaD5 was the most crucial residue as evidenced by the 30-fold loss in affinity when Tyr betaD5 is mutated to Ile. However, while this mutation eliminated the specificity of the Src SH2 domain for the pYEEI peptide sequence, it was not sufficient to switch the specificity of the Src SH2 domain to that of a related SH2 domain which has an Ile at the betaD5 position. Mutation of Lys betaD3 to an Ala residue resulted in a modest reduction in binding affinity (sevenfold). It is interesting that this mutation resulted in a change of specificity affecting the selection of the +1 position residue C-terminal to the phosphotyrosine. Except for the Lys betaD3-+1 Glu interaction which is significantly coupled, only weak energetic coupling was observed across the binding interface, as assessed using double mutant cycles. The results of this study suggest that interactions involving the specificity determining region of SH2 domains may be insufficient by themselves to target single SH2 domains to particular phosphorylated sites.     

Structural basis for a novel intrapeptidyl H-bond and reverse binding of c-Cbl-TKB domain substrates

The c-Cbl tyrosine kinase binding domain (Cbl-TKB), essentially an 'embedded' SH2 domain, has a critical role in targeting proteins for ubiquitination. To address how this domain can bind to disparate recognition mofits and to determine whether this results in variations in substrate-binding affinity, we compared crystal structures of the Cbl-TKB domain complexed with phosphorylated peptides of Sprouty2, Sprouty4, epidermal growth factor receptor, Syk, and c-Met receptors and validated the binding with point-mutational analyses using full-length proteins. An obligatory, intrapeptidyl H-bond between the phosphotyrosine and the conserved asparagine or adjacent arginine is essential for binding and orients the peptide into a positively charged pocket on c-Cbl. Surprisingly, c-Met bound to Cbl in the reverse direction, which is unprecedented for SH2 domain binding. The necessity of this intrapeptidyl H-bond was confirmed with isothermal titration calorimetry experiments that also showed Sprouty2 to have the highest binding affinity to c-Cbl; this may enable the selective sequestration of c-Cbl from other target proteins.     

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.     

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y¹⁹⁴ impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y¹⁹⁴ on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases.Src homology 2 (SH2) domains are modular protein structures that are important for signal transduction due to their ability to bind phosphotyrosine (pY)-containing polypeptides within defined amino acid sequence motifs (1). SH2 domains are found in various signaling enzymes and adaptor proteins. Given the reversibility of protein tyrosine phosphorylation and the affinity of SH2-pY binding, the interactions of SH2 domains are inherently dynamic and diverse. Indeed, selective, transient binding to pY motifs is a key mechanism through which intracellular signaling networks are dynamically assembled, localized, and regulated. In addition to mediating protein interactions in trans, SH2 domains bind intramolecularly (2). For example, in Src family kinases (SFKs), the SH2 domain binds in cis to the phosphorylated C-terminal tail as a mechanism to constrain and thereby auto-inhibit the intervening tyrosine kinase domain (3, 4). As well, SH2 domains of cytoplasmic tyrosine kinases have been shown to affect the kinase activity of adjacent kinase domains through allosteric interactions (5). The SFKs are therefore highly regulated as a function of their SH2 domains, which exist in dynamic equilibrium between intra- and intermolecular interactions (6). Hence, as discussed by Pawson (7), the transient and diverse interactions of an SH2 domain can regulate signaling enzymes and constitutes a major mechanism of signal transduction in response to extracellular signals.The structure of the SH2 domain has been extensively characterized. At its core is a conserved antiparallel β-sheet sandwiched between two α-helices (8). SH2 domains bind phosphotyrosine-containing peptides in an extended conformation across the central β-sheet, with the pY residue inserted in a deep recognition pocket formed by conserved residues from strands βB, βC, and βD, helix αA, and the phosphate binding loop. Peptide binding specificity is determined by more variable binding surfaces on the SH2 domain, which recognize residues C-terminal to the pY residue. For the SFK SH2 domains, the three residues C-terminal to the pY residue (pY+1,+2,+3) are dominant determinants of specificity (9, 10), with the domain binding most tightly to sequences containing the motif pYEEI (11, 12). The hydrophobic pY+3 residue inserts in a deep hydrophobic specificity pocket defined by residues of the EF and BG loops (8, 13, 14). Indeed, structural analysis of the SH2 domain revealed that the configuration of the EF and BG loops is critical in dictating SH2 domain specificity by shaping the ligand-binding surface and controlling accessibility of the pY+3 binding pocket (15). Mutation of a single residue of the EF loop can drastically impact peptide binding specificity by altering the pY+3 pocket (15–17), indicating the importance of the pY+3 pocket in substrate selectivity for the SFK SH2 domains.In addition to binding pY-containing polypeptides, SH2 domains themselves may be modulated by phosphorylation. For example, phosphorylation of the Src SH2 domain at conserved Y²¹³ resulted in activation of the cognate kinase domain, possibly by impairing SH2 binding to the phosphorylated C-terminal tail (18). Similarly, phosphorylation of Lck at the equivalent SH2 residue (Y¹⁹²) generally reduced binding to pY-peptides and proteins (19). Phosphorylation at S⁶⁹⁰ in the SH2 domain of the p85α subunit of PI 3-kinase decreased its affinity for pY-containing proteins and promoted feedback inhibition of PI 3-kinase and Akt in response to cellular starvation (20). Conversely, tyrosine phosphorylation of the tensin-3 SH2 domain stimulated substrate binding and biological activity (21). Therefore, phosphorylation of SH2 domains appears to be a general mechanism for modulating their binding properties.Here, we report that Y¹⁹⁴ in the SH2 domain of the SFK Lyn, a residue conserved in SFK SH2 domains, is frequently phosphorylated in hematopoietic and other cancers. In vitro protein and peptide interactions with the Lyn SH2 domain were affected by this phosphorylation. Our results suggest that tyrosine phosphorylation of the SFK SH2 domain modulates both its binding affinity and specificity and may constitute another layer of regulation in signaling networks.     

Phospholipase C-gamma1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk.

Antigen receptor ligation on lymphocytes activates protein tyrosine kinases and phospholipase C-gamma (PLC-gamma) isoforms. Glutathione S-transferase fusion proteins containing the C-terminal Src-homology 2 [SH2(C)] domain of PLC-gamma1 bound to tyrosyl phosphorylated Syk. Syk isolated from antigen receptor-activated B cells phosphorylated PLC-gamma1 on Tyr-771 and the key regulatory residue Tyr-783 in vitro, whereas Lyn from the same B cells phosphorylated PLC-gamma1 only on Tyr-771. The ability of Syk to phosphorylate PLC-gamma1 required antigen receptor ligation, while Lyn was constitutively active. An mCD8-Syk cDNA construct could be expressed as a tyrosyl-phosphorylated chimeric protein tyrosine kinase in COS cells, was recognized by PLC-gamma1 SH2(C) in vitro, and induced tyrosyl phosphorylation of endogenous PLC-gamma1 in vivo. Substitution of Tyr-525 and Tyr-526 at the autophosphorylation site of Syk in mCD8-Syk substantially reduced the kinase activity and the binding of this variant chimera to PLC-gamma1 SH2(C) in vitro; it also failed to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. In contrast, substitution of Tyr-348 and Tyr-352 in the linker region of Syk in mCD8-Syk did not affect the kinase activity of this variant chimera but almost completely eliminated its binding to PLC-gamma1 SH(C) and completely eliminated its ability to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. Thus, an optimal kinase activity of Syk and an interaction between the linker region of Syk with PLC-gamma1 are required for the tyrosyl phosphorylation of PLC-gamma1.