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The SYK protein-tyrosine kinase is a well-known mediator of signals elicited by the clustering of BCR complexes and other receptors that bear components that contain one or more ITAM sequences. Additional roles for the kinase in signaling through other receptor classes also have been described. To assist in the identification of SYK-regulated processes, we developed mice lacking endogenous Syk genes but containing instead genes coding for an analogue-sensitive form of SYK (SYK-AQL). SYK-AQL supports the development of B cells, and these can be activated with both anti-IgM F(ab') 2 through the BCR and LPS through TLR4. An orthogonal inhibitor that selectively targets SYK-AQL blocks the activation of B cells by anti-IgM F(ab') 2 in SYK-AQL-expressing but not wild-type cells. The SYK-AQL-specific inhibitor, however, does not block B cell activation in response to LPS in either wild-type or SYK-AQL-expressing cells. Thus, SYK is essential for coupling the BCR but not TLR4 to the activation of B cells., (Copyright © 2019 The Authors.)

The ability of breast cancer cells to transiently transition between epithelial and mesenchymal states contributes to their metastatic potential. Therefore, driving tumor cells into a stable mesenchymal state, as opposed to complete tumor cell eradication, presents an opportunity to pharmacologically limit disease progression by promoting an asymptomatic state of dormancy. Here, we compare a reversible model of epithelial-mesenchymal transition (EMT) induced by TGFβ to a stable mesenchymal phenotype induced by chronic exposure to the ErbB kinase inhibitor lapatinib. Only cells capable of returning to an epithelial phenotype resulted in skeletal metastasis. Gene expression analyses of the two mesenchymal states indicated similar transition expression profiles. A potently downregulated gene in both datasets was spleen tyrosine kinase (SYK). In contrast to this similar diminution in mRNA, kinome analyses using a peptide array and DNA-conjugated peptide substrates showed a robust increase in SYK activity upon TGFβ-induced EMT only. SYK was present in cytoplasmic RNA processing depots known as P-bodies formed during the onset of EMT, and SYK activity was required for autophagy-mediated clearance of P-bodies during mesenchymal-epithelial transition (MET). Genetic knockout of autophagy-related 7 (ATG7) or pharmacologic inhibition of SYK activity with fostamatinib, a clinically approved inhibitor of SYK, prevented P-body clearance and MET, inhibiting metastatic tumor outgrowth. Overall, this study suggests assessment of SYK activity as a biomarker for metastatic disease and the use of fostamatinib as a means to stabilize the latency of disseminated tumor cells. SIGNIFICANCE: These findings present inhibition of spleen tyrosine kinase as a therapeutic option to limit breast cancer metastasis by promoting systemic tumor dormancy. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/8/1831/F1.large.jpg. See related commentary by Farrington and Narla, p. 1756 ., (©2019 American Association for Cancer Research.)

Ableson tyrosine kinase (ABL) plays essential roles in cell differentiation, division, adhesion, and stress response. However, fusion of the breakpoint cluster region (BCR) to ABL produces constitutive kinase activity that causes chronic myelogenous leukemia (CML). Small molecule tyrosine kinase inhibitors (TKIs) such as imatinib revolutionized the treatment of CML and other cancers, but acquired resistance to these inhibitors is rising. Thus, careful dissection of ABL signaling pathways is needed to find novel drug targets. Here we present a refined proteomic approach for elucidation of direct kinase substrates called kinase assay linked phosphoproteomics (KALIP). Our strategy integrates in vitro kinase assays at both the peptide and protein levels with quantitative tyrosine phosphoproteomics in response to treatment by multiple TKIs. Utilizing multiple TKIs permits elimination of off-target effects of these drugs, and overlapping the in vivo and in vitro data sets allows us to define a list of the most probable kinase substrates. Applying our approach produced a list of 60 ABL substrates, including novel and known proteins. We demonstrate that spleen tyrosine kinase (SYK) is a novel direct substrate of ABL, and we predict our proteomic strategy may facilitate identification of substrates in other cancers that have disrupted kinase signaling.

Clustering of the B cell antigen receptor (BCR) by polyvalent antigens is transmitted through the SYK tyrosine kinase to the activation of multiple intracellular pathways that determine the physiological consequences of receptor engagement. To explore factors that modulate the quantity and quality of signals sent by the crosslinked BCR, we developed a novel chemical mediator of dimerization to induce clustering of receptor-associated SYK. To accomplish this, we fused SYK with E. coli dihydrofolate reductase (eDHFR), which binds the small molecule trimethoprim (TMP) with high affinity and selectivity and synthesized a dimer of TMP with a flexible linker. The TMP dimer is able to induce the aggregation of eDHFR-linked SYK in live cells. The induced dimerization of SYK bound to the BCR differentially regulates the activation of downstream transcription factors, promoting the activation of Nuclear Factor of Activated T cells (NFAT) without affecting the activation of NFκB. The dimerization of SYK enhances the duration but not the amplitude of calcium mobilization by enhancing the extent and duration of its interaction with the crosslinked BCR at the plasma membrane., Competing Interests: The authors declare no conflict of interest.

Protein kinases and their substrates comprise extensive signaling networks that regulate many diverse cellular functions. However, methods and techniques to systematically identify kinases directly responsible for specific phosphorylation events have remained elusive. Here we describe a novel proteomic strategy termed fluorescence complementation mass spectrometry (FCMS) to identify kinase-substrate pairs in high throughput. The FCMS strategy employs a specific substrate and a kinase library, both of which are fused with fluorescence complemented protein fragments. Transient and weak kinase-substrate interactions in living cells are stabilized by the association of fluorescence protein fragments. These kinase-substrate pairs are then isolated with high specificity and are identified and quantified by LC-MS. FCMS was applied to the identification of both known and novel kinases of the transcription factor, cAMP response element-binding protein (CREB). Novel CREB kinases were validated by in vitro kinase assays, and the phosphorylation sites were unambiguously located. These results uncovered possible new roles for CREB in multiple important signaling pathways and demonstrated the great potential of this new proteomic strategy.

Processing bodies (P-bodies) are ribonucleoprotein complexes involved in post-transcriptional mRNA metabolism that accumulate in cells exposed to various stress stimuli. The treatment of mammary epithelial cells with transforming growth factor-beta (TGF-β), triggers epithelial-mesenchymal transition (EMT), and induces the formation of P-bodies. Ectopic expression of the transcription factor TWIST, which stimulates EMT downstream of the TGF-β receptor, also promotes P-body formation. Removal of TGF-β from treated cells results in the clearance of P-bodies by a process that is blocked by inhibitors of autophagy. Activators of autophagy enhance P-body clearance and block EMT. Blockage of P-body formation by disruption of the gene for DDX6, a protein essential for P-body assembly, blocks EMT and prevents tumor cell metastasis in vivo . These studies suggest critical roles for P-body formation and autophagy in transitions of cancer cells between epithelial and mesenchymal phenotypes and help explain how autophagy functions to promote or suppress tumor cell growth during different stages of tumorigenesis., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article.

Force-displacement (F-Z) curves are the most commonly used Atomic Force Microscopy (AFM) mode to measure the local, nanoscale elastic properties of soft materials like living cells. Yet a theoretical framework has been lacking that allows the post-processing of F-Z data to extract their viscoelastic constitutive parameters. Here, we propose a new method to extract nanoscale viscoelastic properties of soft samples like living cells and hydrogels directly from conventional AFM F-Z experiments, thereby creating a common platform for the analysis of cell elastic and viscoelastic properties with arbitrary linear constitutive relations. The method based on the elastic-viscoelastic correspondence principle was validated using finite element (FE) simulations and by comparison with the existed AFM techniques on living cells and hydrogels. The method also allows a discrimination of which viscoelastic relaxation model, for example, standard linear solid (SLS) or power-law rheology (PLR), best suits the experimental data. The method was used to extract the viscoelastic properties of benign and cancerous cell lines (NIH 3T3 fibroblasts, NMuMG epithelial, MDA-MB-231 and MCF-7 breast cancer cells). Finally, we studied the changes in viscoelastic properties related to tumorigenesis including TGF-β induced epithelial-to-mesenchymal transition on NMuMG cells and Syk expression induced phenotype changes in MDA-MB-231 cells.

Syk is a cytoplasmic kinase that serves multiple functions within the immune system to couple receptors for antigens and antigen-antibody complexes to adaptive and innate immune responses. Recent studies have identified additional roles for the kinase in cancer cells, where its expression can either promote or suppress tumor cell growth, depending on the context. Proteomic analyses of Syk-binding proteins identified several interacting partners also found to be recruited to stress granules. We show here that the treatment of cells with inducers of stress granule formation leads to the recruitment of Syk to these protein-RNA complexes. This recruitment requires the phosphorylation of Syk on tyrosine and results in the phosphorylation of proteins at or near the stress granule. Grb7 is identified as a Syk-binding protein involved in the recruitment of Syk to the stress granule. This recruitment promotes the formation of autophagosomes and the clearance of stress granules from the cell once the stress is relieved, enhancing the ability of cells to survive the stress stimulus., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Microglial cells in the brains of Alzheimer's patients are known to be recruited to amyloid-beta (Aβ) plaques where they exhibit an activated phenotype, but are defective for plaque removal by phagocytosis. In this study, we show that microglia stressed by exposure to sodium arsenite or Aβ(1-42) peptides or fibrils form extensive stress granules (SGs) to which the tyrosine kinase, SYK, is recruited. SYK enhances the formation of SGs, is active within the resulting SGs and stimulates the production of reactive oxygen and nitrogen species that are toxic to neuronal cells. This sequestration of SYK inhibits the ability of microglial cells to phagocytose Escherichia coli or Aβ fibrils. We find that aged microglial cells are more susceptible to the formation of SGs; and SGs containing SYK and phosphotyrosine are prevalent in the brains of patients with severe Alzheimer's disease. Phagocytic activity can be restored to stressed microglial cells by treatment with IgG, suggesting a mechanism to explain the therapeutic efficacy of intravenous IgG. These studies describe a mechanism by which stress, including exposure to Aβ, compromises the function of microglial cells in Alzheimer's disease and suggest approaches to restore activity to dysfunctional microglial cells.

A longstanding goal in cellular mechanobiology has been to link dynamic biomolecular processes underpinning disease or morphogenesis to spatio-temporal changes in nanoscale mechanical properties such as viscoelasticity, surface tension, and adhesion. This requires the development of quantitative mechanical microscopy methods with high spatio-temporal resolution within a single cell. The Atomic Force Microscope (AFM) can map the heterogeneous mechanical properties of cells with high spatial resolution, however, the image acquisition time is 1-2 orders of magnitude longer than that required to study dynamic cellular processes. We present a technique that allows commercial AFM systems to map quantitatively the dynamically changing viscoelastic properties of live eukaryotic cells at widely separated frequencies over large areas (several 10's of microns) with spatial resolution equal to amplitude-modulation (AM-AFM) and with image acquisition times (tens of seconds) approaching those of speckle fluorescence methods. This represents a ~20 fold improvement in nanomechanical imaging throughput compared to AM-AFM and is fully compatible with emerging high speed AFM systems. This method is used to study the spatio-temporal mechanical response of MDA-MB-231 breast carcinoma cells to the inhibition of Syk protein tyrosine kinase giving insight into the signaling pathways by which Syk negatively regulates motility of highly invasive cancer cells.

The kinase Syk is intricately involved in early signaling events in B cells and is required for proper response when antigens bind to B cell receptors (BCRs). Experiments using an analog-sensitive version of Syk (Syk-AQL) have better elucidated its role, but have not completely characterized its behavior. We present a computational model for BCR signaling, using dynamical systems, which incorporates both wild-type Syk and Syk-AQL. Following the use of sensitivity analysis to identify significant reaction parameters, we screen for parameter vectors that produced graded responses to BCR stimulation as is observed experimentally. We demonstrate qualitative agreement between the model and dose response data for both mutant and wild-type kinases. Analysis of our model suggests that the level of NF- κ B activation, which is reduced in Syk-AQL cells relative to wild-type, is more sensitive to small reductions in kinase activity than Erkp activation, which is essentially unchanged. Since this profile of high Erkp and reduced NF- κ B is consistent with anergy, this implies that anergy is particularly sensitive to small changes in catalytic activity. Also, under a range of forward and reverse ligand binding rates, our model of Erkp and NF- κ B activation displays a dependence on a power law affinity: the ratio of the forward rate to a non-unit power of the reverse rate. This dependence implies that B cells may respond to certain details of binding and unbinding rates for ligands rather than simple affinity alone.

Tandem mass spectrometry (MS/MS) has enabled researchers to analyze complex biological samples since the original concept inception. It facilitates the identification and quantification of modifications within tens of thousands of proteins in a single large-scale proteomic experiment. Phosphorylation analysis, as one of the most common and important post-translational modifications, has particularly benefited from such progress in the field. Here we showcase the technique through in-depth analyses of B cell signaling based on quantitative phosphoproteomics. As a complement to the previously described PolyMAC-Ti (polymer-based metal ion affinity capture using titanium) reagent, we introduce here PolyMAC-Fe, which utilizes a different metal ion, Fe(III). An extensive comparison using the different available MS/MS fragmentations techniques was made between PolyMAC-Fe, PolyMAC-Ti and IMAC (immobilized metal ion affinity chromatography) reagents in terms of specificity, reproducibility and type of phosphopeptides being enriched. PolyMAC-Fe based chelation demonstrated good selectivity and unique specificity toward phosphopeptides, making it useful in specialized applications. We have combined PolyMAC-Ti and PolyMAC-Fe, along with SILAC-based quantitation and large-scale fractionation, for quantitative B cell phosphoproteomic analyses. The complementary approach allowed us to identify a larger percentage of multiply phosphorylated peptides than with PolyMAC-Ti alone. Overall, out of 13,794 unique phosphorylation sites identified, close to 20% were dependent on BCR signaling. These sites were further mapped to a variety of major signaling networks, offering more detailed information about the biochemistry of B cell receptor engagement.

The Syk protein-tyrosine kinase, a well-characterized modulator of immune recognition receptor signaling, also plays important, but poorly characterized, roles in tumor progression, acting as an inhibitor of cellular motility and metastasis in highly invasive cancer cells. Multiharmonic atomic force microscopy (AFM) was used to map nanomechanical properties of live MDA-MB-231 breast cancer cells either lacking or expressing Syk. The expression of Syk dramatically altered the cellular topography, reduced cell height, increased elasticity, increased viscosity, and allowed visualization of a more substantial microtubule network. The microtubules of Syk-expressing cells were more stable to nocodazole-induced depolymerization and were more highly acetylated than those of Syk-deficient cells. Silencing of MAP1B, a major substrate for Syk in MDA-MB-231 cells, attenuated Syk-dependent microtubule stability and reversed much of the effect of Syk on cellular topography, stiffness, and viscosity. This study illustrates the use of multiharmonic AFM both to quantitatively map the local nanomechanical properties of living cells and to identify the underlying mechanisms by which these properties are modulated by signal transduction machinery.

SYK (spleen tyrosine kinase) is well-characterized in the immune system as an essential enzyme required for signaling through multiple classes of immune recognition receptors. As a modulator of tumorigenesis, SYK has a bit of a schizophrenic reputation, acting in some cells as a tumor promoter and in others as a tumor suppressor. In many hematopoietic malignancies, SYK provides an important survival function and its inhibition or silencing frequently leads to apoptosis. In cancers of non-immune cells, SYK provides a pro-survival signal, but can also suppress tumorigenesis by restricting epithelial-mesenchymal transition, enhancing cell-cell interactions and inhibiting migration., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The Syk protein tyrosine kinase, a well-characterized regulator of immune cell function, plays an increasingly recognized role in tumorigenesis as a promoter of cell survival in both hematological and nonhematological malignancies. We show here that the expression of Syk in MCF7 or MDA-MB-231 breast cancer cells or in DG75 B-lymphoma cells protects cells from apoptosis induced by oxidative or genotoxic stress by stabilizing the mRNA for Bcl-x(L), an antiapoptotic protein. Syk binds robustly to nucleolin and phosphorylates it on tyrosine, enhancing its ability to bind the Bcl-x(L) mRNA. Consequently, reducing the level of nucleolin by RNA interference attenuates the ability of Syk to protect cells from stress-induced cell death., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Spleen tyrosine kinase (Syk) is a cytoplasmic protein tyrosine kinase well known for its ability to couple immune cell receptors to intracellular signaling pathways that regulate cellular responses to extracellular antigens and antigen-immunoglobulin (Ig) complexes of particular importance to the initiation of inflammatory responses. Thus, Syk is an attractive target for therapeutic kinase inhibitors designed to ameliorate the symptoms and consequences of acute and chronic inflammation. Its more recently recognized role as a promoter of cell survival in numerous cancer cell types ranging from leukemia to retinoblastoma has attracted considerable interest as a target for a new generation of anticancer drugs. This review discusses the biological processes in which Syk participates that have made this kinase such a compelling drug target., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events. Most studies to date have focused on the BCR and protein tyrosine phosphorylation. Because spleen tyrosine kinase, Syk, is an upstream kinase in multiple BCR-regulated signaling pathways, it also affects many downstream events that are modulated through the phosphorylation of proteins on serine and threonine residues. Here, we report a novel phosphopeptide enrichment strategy and its application to a comprehensive quantitative phosphoproteomics analysis of Syk-dependent downstream signaling events in B cells, focusing on serine and threonine phosphorylation. Using a combination of the Syk inhibitor piceatannol, SILAC quantification, peptide fractionation, and complementary PolyMAC-Ti and PolyMAC-Zr enrichment techniques, we analyzed changes in BCR-stimulated protein phosphorylation that were dependent on the activity of Syk. We identified and quantified over 13,000 unique phosphopeptides, with a large percentage dependent on Syk activity in BCR-stimulated B cells. Our results not only confirmed many known functions of Syk, but more importantly, suggested many novel roles, including in the ubiquitin proteasome pathway, that warrant further exploration.

The association of spleen tyrosine kinase (Syk), a central tyrosine kinase in B cell signaling, with Vav SH2 domain is controlled by phosphorylation of two closely spaced tyrosines in Syk linker B: Y342 and Y346. Previous studies established both singly phosphorylated and doubly phosphorylated forms play a role in signaling. The structure of the doubly phosphorylated form identified a new recognition of phosphotyrosine whereby two phosphotyrosines bind simultaneously to the Vav SH2 domain, one in the canonical pTyr pocket and one in the specificity pocket on the opposite side of the central β-sheet. It is unknown if the specificity pocket can bind phosphotyrosine independent of phosphotyrosine binding the pTyr pocket. To address this gap in knowledge, we determined the structure of the complex between Vav1 SH2 and a peptide (SykLB-YpY) modeling the singly phosphorylated-Y346 form of Syk with unphosphorylated Y342. The nuclear magnetic resonance (NMR) data conclusively establish that recognition of phosphotyrosine is swapped between the two pockets; phosphorylated pY346 binds the specificity pocket of Vav1 SH2, and unphosphorylated Y342 occupies what is normally the pTyr binding pocket. Nearly identical changes in chemical shifts occurred upon binding all three forms of singly and doubly phosphorylated peptides; however, somewhat smaller shift perturbations for SykLB-YpY from residues in regions of high internal mobility suggest that internal motions are coupled to binding affinity. The differential recognition that includes this swapped binding of phosphotyrosine to the specificity pocket of Vav SH2 increases the repertoire of possible phosphotyrosine binding by SH2 domains in regulating protein-protein interactions in cellular signaling., (Copyright © 2013 Wiley Periodicals, Inc.)

Syk is a 72kDa non-receptor tyrosine kinase that is best characterized in hematopoietic cells. While Syk is pro-tumorigenic in some cancer cell types, it also has been reported as a negative regulator of metastatic cell growth in others. An examination of the RelA (p65) subunit of NF-κB expressed in MCF7 breast cancer cells indicated that either treatment with pervanadate or stable expression of Syk protected RelA from calpain-mediated proteolysis. Similar results were observed with the tyrosine phosphatase, PTP1B, another sensitive calpain substrate. The activity of calpain in MCF7 cell lysates was inhibited by both treatment with hydrogen peroxide and expression of Syk, the former due to oxidative inactivation of calpain and the latter to enhanced expression of calpastatin (CAST), the endogenous calpain inhibitor. The level of CAST was elevated in the cytosolic fraction of Syk-positive breast cancer cells resulting in more CAST present in complex with calpain in cell lysates. The high levels of CAST coincided with elevated basal levels of calcium-and of intracellular calpain activity-in Syk-expressing cells resulting from decreased levels of Bcl-2, an inhibitor of IP3-receptor-mediated calcium release. The inhibition of cellular calpain stimulated the Syk-mediated enhancement of NF-κB induced by TNF-α, enhanced tyrosine phosphorylation resulting from integrin crosslinking, and increased the localization of Syk to the plasma membrane., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Protein kinases are implicated in multiple diseases such as cancer, diabetes, cardiovascular diseases, and central nervous system disorders. Identification of kinase substrates is critical to dissecting signaling pathways and to understanding disease pathologies. However, methods and techniques used to identify bona fide kinase substrates have remained elusive. Here we describe a proteomic strategy suitable for identifying kinase specificity and direct substrates in high throughput. This approach includes an in vitro kinase assay-based substrate screening and an endogenous kinase dependent phosphorylation profiling. In the in vitro kinase reaction route, a pool of formerly phosphorylated proteins is directly extracted from whole cell extracts, dephosphorylated by phosphatase treatment, after which the kinase of interest is added. Quantitative proteomics identifies the rephosphorylated proteins as direct substrates in vitro. In parallel, the in vivo quantitative phosphoproteomics is performed in which cells are treated with or without the kinase inhibitor. Together, proteins phosphorylated in vitro overlapping with the kinase-dependent phosphoproteome in vivo represents the physiological direct substrates in high confidence. The protein kinase assay-linked phosphoproteomics was applied to identify 25 candidate substrates of the protein-tyrosine kinase SYK, including a number of known substrates and many novel substrates in human B cells. These shed light on possible new roles for SYK in multiple important signaling pathways. The results demonstrate that this integrated proteomic approach can provide an efficient strategy to screen direct substrates for protein tyrosine kinases.

SH2 domains are attractive targets for chemotherapeutic agents due to their involvement in the formation of protein-protein interactions critical to many signal transduction cascades. Little is known, however, about how synthetic SH2 domain ligands would influence the growth properties of tumor cells or with which intracellular proteins they would interact due to their highly charged nature and enzymatic lability. In this study, a prodrug delivery strategy was used to introduce an enzymatically stable, phosphotyrosine peptidomimetic into tumor cells. When tested in a human tumor cell panel, the prodrug exhibited a preference for inhibiting the growth of leukemia and lymphoma cells. In these cells, it was largely cytostatic and induced endoreduplication and the appearance of midbodies. Proteomic analyses identified multiple targets that included mitotic centromere-associated kinesin (MCAK). Molecular modeling studies suggested the ATP-binding site on MCAK as the likely site of drug interaction. Consistent with this, ATP inhibited the drug-MCAK interaction and the drug inhibited MCAK ATPase activity. Accordingly, the effects of the prodrug on the assembly of the mitotic spindle and alignment of chromosomes were consistent with the identification of MCAK as an important intracellular target., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Characterization of ligand-protein binding is of crucial importance in drug discovery. Classical competition binding assays measure the binding of a labeled ligand in the presence of various concentrations of unlabeled ligand and typically use single purified proteins. Here, we introduce a high-throughput approach to study ligand-protein interactions by coupling competition binding assays with mass spectrometry-based quantitative proteomics. With the use of a phosphorylated immunoreceptor tyrosine-based activation motif (pITAM) peptide as a model, we characterized pITAM-interacting partners in human lymphocytes. The shapes of competition binding curves of various interacting partners constructed in a single set of quantitative proteomics experiments reflect relative affinities for the pITAM peptide. This strategy can provide an efficient approach to distinguish specific interacting partners, including two signaling kinases possessing tandem SH2 domains, SYK and ZAP-70, as well as other SH2 domain-containing proteins such as CSK and PI3K, from contaminants and to measure relative binding affinities of multiple proteins in a single experiment.

The Syk protein-tyrosine kinase can have multiple effects on cancer cells, acting in some as a tumor suppressor by inhibiting motility and in others as a tumor promoter by enhancing survival. Phosphoproteomic analyses identified PKA as a Syk-specific substrate. Syk catalyzes the phosphorylation of the catalytic subunit of PKA (PKAc) both in vitro and in cells on Tyr-330. Tyr-330 lies within the adenosine-binding motif in the C-terminal tail of PKAc within a cluster of acidic amino acids (DDYEEEE), which is a characteristic of Syk substrates. The phosphorylation of PKAc on Tyr-330 by Syk strongly inhibits its catalytic activity. Molecular dynamics simulations suggest that this additional negative charge prevents the C-terminal tail from interacting with the substrate and the nucleotide-binding site to stabilize the closed conformation of PKAc, thus preventing catalysis from occurring. Phosphoproteomic analyses and Western blotting studies indicate that Tyr-330 can be phosphorylated in a Syk-dependent manner in MCF7 breast cancer cells and DT40 B cells. The phosphorylation of a downstream substrate of PKAc, cAMP-responsive element-binding protein (CREB), is inhibited in cells expressing Syk but can be rescued by a selective inhibitor of Syk. Modulation of CREB activity alters the expression of the CREB-regulated gene BCL2 and modulates cellular responses to genotoxic agents. Thus, PKA is a novel substrate of Syk, and its phosphorylation on Tyr-330 inhibits its participation in downstream signaling pathways.

We report here for the first time the multiplexed quantitation of phosphorylation and protein expression based on a functionalized soluble nanopolymer. The soluble nanopolymer, pIMAGO, is functionalized with Ti (IV) ions for chelating phosphoproteins in high specificity and with infrared fluorescent tags for direct, multiplexed assays. The nanopolymer allows for direct competition for epitopes on proteins of interest, thus facilitating simultaneous detection of phosphorylation by pIMAGO and total protein amount by protein antibody in the same well of microplates. The new strategy has a great potential to measure cell signaling events by clearly distinguishing actual phosphorylation signals from protein expression changes, thus providing a powerful tool to accurately profile cellular signal transduction in healthy and disease cells. We anticipate broad applications of this new strategy in monitoring cellular signaling pathways and discovering new signaling events.

Spleen tyrosine kinase (Syk) has been implicated in a number of pathologies including cancer and rheumatoid arthritis and thus has been pursued as a novel therapeutic target. Because of the complex relationship between Syk's auto- and other internal phosphorylation sites, scaffolding proteins, enzymatic activation state and sites of phosphorylation on its known substrates, the role of Syk's activity in these diseases has not been completely clear. To approach such analyses, we developed a Syk-specific artificial peptide biosensor (SAStide) to use in a cell-based assay for direct detection of intracellular Syk activity and inhibition in response to physiologically relevant stimuli in both laboratory cell lines and primary splenic B cells. This peptide contains a sequence derived from known Syk substrate preference motifs linked to a cell permeable peptide, resulting in a biosensor that is phosphorylated in live cells in a Syk-dependent manner, thus serving as a reporter of Syk catalytic activity in intact cells. Because the assay is compatible with live, primary cells and can report pharmacodynamics for drug action on an intended target, this methodology could be used to facilitate a better understanding of Syk's function and the effect of its inhibition in disease.

Many protein-protein interactions in cells are mediated by functional domains that recognize and bind to motifs containing phosphorylated serine and threonine residues. To create small molecules that inhibit such interactions, we developed methodology for the synthesis of a prodrug that generates a phosphoserine peptidomimetic in cells. For this study, we synthesized a small molecule inhibitor of 14-3-3 proteins that incorporates a nonhydrolyzable difluoromethylenephosphoserine prodrug moiety. The prodrug is cytotoxic at low micromolar concentrations when applied to cancer cells and induces caspase activation resulting in apoptosis. The prodrug reverses the 14-3-3-mediated inhibition of FOXO3a resulting from its phosphorylation by Akt1 in a concentration-dependent manner that correlates well with its ability to inhibit cell growth. This methodology can be applied to target a variety of proteins containing phosphoserine and other phosphoamino acid binding domains., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The engagement of antigen receptors on lymphocytes leads to the activation of phospholipase C-γ, the mobilization of intracellular calcium and the activation of the NFAT transcription factor. The coupling of antigen receptors to the activation of NFAT is modulated by numerous cellular effectors including phospho-inositide 3-kinase (PI3K), which is activated following receptor cross-linking. The activation of PI3K has both positive and negative effects on the receptor-mediated activation of NFAT. An increase in the level and activity of Akt2, a target of activated PI3K, potently inhibits the subsequent activation of NFAT. In contrast, an elevation in Akt1 has no effect on signaling. Signaling pathways operating both upstream and downstream of inositol 1,4,5-trisphosphate (IP3)-stimulated calcium release from intracellular stores are unaffected by Akt2. An increase in the level of Akt2 has no significant effect on the initial amplitude, but substantially reduces the duration of calcium mobilization. The ability of Akt2 to inhibit prolonged calcium mobilization is abrogated by the administration of a cell permeable peptide that blocks the interaction between Bcl-2 and the IP3 receptor. Thus, Akt2 is a negative regulator of NFAT activation through its ability to inhibit calcium mobilization from the ER., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity.

Syk is a 72-kDa protein-tyrosine kinase that regulates signaling through multiple cell surface receptors including those for antigens, immunoglobulins and proteins of the extracellular matrix. As part of its function, Syk binds a variety of downstream effectors through interactions that are often mediated by motifs that recognize phosphotyrosines. In a search for novel Syk-interacting proteins by yeast two-hybrid analysis, we identified tensin2 as a Syk-binding protein. Syk interacts with a fragment of tensin2 located near the C-terminus that contains SH2 and PTB domains. In epithelial cells, tensin2 localizes both to focal adhesions and to large cytoplasmic puncta. It is within these punctuate structures that Syk and tensin2 are co-localized. The clustering of Syk within these structures leads to its phosphorylation on tyrosine., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Green fluorescent protein (GFP) and variants have become powerful tools to study protein localization, interactions, and dynamics. We present here a mass spectrometry-based proteomics strategy to examine protein-protein interactions using anti-GFP single-chain antibody V(H)H in a combination with a novel stable isotopic labeling reagent, isotope tag on amino groups (iTAG). We demonstrate that the single-chain V(H)H (GFP nanotrap) allows us to identify interacting partners of the Syk protein-tyrosine kinase bearing a GFP epitope tag with high efficiency and high specificity. Interacting proteins identified include CrkL, BLNK, α- and β-tubulin, Csk, RanBP5 and DJ-1. The iTAG reagents were prepared with simple procedures and characterized with high accuracy in the determination of peptides in model peptide mixtures and as well as in complex mixture. Applications of the iTAG method and GFP nanotrap to an analysis of the nucleocytoplasmic trafficking of Syk led to the identification of location-specific associations between Syk and multiple proteins. While the results reveal that the new quantitative proteomic strategy is generally applicable to integrate protein interaction data with subcellular localization, extra caution should be taken in evaluating the results obtained by such affinity purification strategies as many interactions appear to occur following cell lysis., (© American Society for Mass Spectrometry, 2011)

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.

Transport of protein and RNA cargoes between the nucleus and cytoplasm (nucleocytoplasmic transport) is vital for a variety of cellular functions. The studies of kinetics involved in such processes have been hindered by the lack of quantitative tools for measurement of the nuclear and cytosolic fractions of an intracellular protein at the single cell level for a cell population. In this report, we describe using a novel method, microfluidic electroporative flow cytometry, to study kinetics of nucleocytoplasmic transport of an important transcription factor NF-κB. With data collected from single cells, we quantitatively characterize the population-averaged kinetic parameters such as the rate constants and apparent activation barrier for NF-κB transport. Our data demonstrate that NF-κB nucleocytoplasmic transport fits first-order kinetics very well and is a fairly reversible process governed by equilibrium thermodynamics.

Protein translocation, or the change in a protein's location between different subcellular compartments, is a critical process by which intracellular proteins carry out their cellular functions. Aberrant translocation events contribute to various diseases ranging from metabolic disorders to cancer. In this study, we demonstrate the use of a newly developed single-cell tool, microfluidic total internal reflection fluorescence flow cytometry (TIRF-FC), for detecting both cytosol to plasma membrane and cytosol to nucleus translocations using the tyrosine kinase Syk and the transcription factor NF-κB as models. This technique detects fluorescent molecules at the plasma membrane and in the membrane-proximal cytosol in single cells. We were able to record quantitatively changes in the fluorescence density in the evanescent field associated with these translocation processes for large cell populations with single cell resolution. We envision that TIRF-FC will provide a new approach to explore the molecular biology and clinical relevance of protein translocations.

The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro- and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling.

Conventional biochemical analysis mainly focuses on the expression level of cellular proteins from entire cells. However, it has been increasingly acknowledged that the subcellular location of proteins often carries important information. Analysis of subcellular proteins conventionally requires subcellular fractionation which involves two steps: cell lysis to release proteins and high-speed centrifugation to separate the homogenate. Such approach requires bulky and expensive equipment and is not compatible with processing scarce cell samples of limited volume. In this study, we apply microfluidic flow-through electroporation to breach cell membranes and extract cytosolic proteins selectively in a single step. We demonstrate that this approach allows monitoring the translocation of the transcription factor NF-kappaB from the cytosol to the nucleus without the need of subcellular fractionation. Our technique is compatible with the processing of samples of various sizes and provides a simple and universal tool for bioanalytical analysis and spatial proteomics.

The B cell receptor (BCR) transduces antigen binding into alterations in the activity of intracellular signaling pathways through its ability to recruit and activate the cytoplasmic protein-tyrosine kinase Syk. The recruitment of Syk to the receptor, its activation and its subsequent interactions with downstream effectors are all regulated by its phosphorylation on tyrosine. This review discusses our current understanding of how this phosphorylation regulates the activity of Syk and its participation in signaling through the BCR.

The expression of the Syk protein tyrosine kinase in breast cancer cells is inversely correlated with invasive growth and metastasis. The expression of Syk inhibits cell motility while supporting the formation of cell clusters by enhancing cell-cell contacts and promoting the redistribution of the adhesion proteins cortactin and vinculin to these contacts. Syk associates physically with cortactin and catalyzes its phosphorylation on tyrosine. The clustering of integrins leads to the phosphorylation of Syk and of numerous cellular proteins in a manner dependent on the activity of the kinase and on the presence of tyrosine 342 located in the linker B region. The ability of Syk to participate in integrin-mediated protein tyrosine phosphorylation correlates well with its ability to inhibit cell motility.

The nonreceptor, protein-tyrosine kinase Syk is a suppressor of breast cancer progression whose expression is inversely correlated with the invasive behavior of cancer cells. In contrast, Syk has a positive function in murine mammary tumor virus-mediated tumorigenesis. A yeast two-hybrid screen using a library from human mammary gland identified tumor necrosis factor (TNF) receptor-associated factor-interacting protein (TRIP) as an Syk-binding partner. This interaction is mediated by the C-terminal region of TRIP and is enhanced by the treatment of cells with TNF and the tyrosine phosphorylation of Syk. Syk and TRIP have opposing functions in TNF-signaling pathways. Syk enhances the activation of nuclear factor-kappaB by TNF and this is antagonized by TRIP. The overexpression of TRIP sensitizes cells to TNF-induced apoptosis, an effect that can be reversed by the coexpression of Syk.

Total internal reflection fluorescence microscopy (TIRFM) has been widely used to explore biological events that are close to the cell membrane by illuminating fluorescent molecules using the evanescent wave. However, TIRFM is typically limited to the examination of a low number of cells, and the results do not reveal potential heterogeneity in the cell population. In this report, we develop an analytical tool referred to as total internal reflection fluorescence flow cytometry (TIRF-FC) to examine the region of the cell membrane with a throughput of approximately 100-150 cells/s and single cell resolution. We use an elastomeric valve that is partially closed to force flowing cells in contact with the glass surface where the evanescent field resides. We demonstrate that TIRF-FC is able to detect the differences in the subcellular location of an intracellular fluorescent protein. Proper data processing and analysis allows TIRF-FC to be quantitative. With the high throughput, TIRF-FC will be a very useful tool for generating information on cell populations with events and dynamics close to the cell surface.

The Syk protein-tyrosine kinase plays a major role in signaling through the B cell receptor for antigen (BCR). Syk binds the receptor via its tandem pair of SH2 domains interacting with a doubly phosphorylated immunoreceptor tyrosine-based activation motif (dp-ITAM) of the BCR complex. Upon phosphorylation of Tyr-130, which lies between the two SH2 domains distant to the phosphotyrosine binding sites, Syk dissociates from the receptor. To understand the structural basis for this dissociation, we investigated the structural and dynamic characteristics of the wild type tandem SH2 region (tSH2) and a variant tandem SH2 region (tSH2(pm)) with Tyr-130 substituted by Glu to permanently introduce a negative charge at this position. NMR heteronuclear relaxation experiments, residual dipolar coupling measurements and analytical ultracentrifugation revealed substantial differences in the hydrodynamic behavior of tSH2 and tSH2(pm). Although the two SH2 domains in tSH2 are tightly associated, the two domains in tSH2(pm) are partly uncoupled and tumble in solution with a faster correlation time. In addition, the equilibrium dissociation constant for the binding of tSH2(pm) to dp-ITAM (1.8 microM) is significantly higher than that for the interaction between dp-ITAM and tSH2 but is close to that for a singly tyrosine-phosphorylated peptide binding to a single SH2 domain. Experimental data and hydrodynamic calculations both suggest a loss of domain-domain contacts and change in relative orientation upon the introduction of a negative charge on residue 130. A long-distance structural mechanism by which the phosphorylation of Y130 negatively regulates the interaction of Syk with immune receptors is proposed.

Directed localization of kinases within cells is important for their activation and involvement in signal transduction. Detection of these events has been largely carried out based on imaging of a low number of cells and subcellular fractionation/Western blotting. These conventional techniques either lack the high throughput desired for probing an entire cell population or provide only the average behaviors of cell populations without information from single cells. Here we demonstrate a new tool, referred to as microfluidic electroporative flow cytometry, to detect the translocation of an EGFP-tagged tyrosine kinase, Syk, to the plasma membrane in B cells at the level of the cell population. We combine electroporation with flow cytometry and observe the release of intracellular kinase out of the cells during electroporation. We found that the release of the kinase was strongly influenced by its subcellular localization. Cells stimulated through the antigen receptor have a fraction of the kinase at the plasma membrane and retain more kinase after electroporation than do cells without stimulation and translocation. We are able to differentiate a cell population with translocation from one without it with the information collected from individual cells of the entire population. This technique potentially allows detection of protein translocation at the single-cell level. Due to the frequent involvement of kinase translocations in disease processes such as oncogenesis, our approach will have utility for kinase-related drug discovery and tumor diagnosis and staging.

Aberrant phosphorylation of tau protein on serine and threonine residues has been shown to be critical in neurodegenerative disorders called tauopathies. An increasing amount of data suggest that tyrosine phosphorylation of tau might play an equally important role in pathology, with at least three putative tyrosine kinases of tau identified to date. It was recently shown that the tyrosine kinase Syk could efficiently phosphorylate alpha-synuclein, the aggregated protein found in Parkinson's disease and other synucleinopathies. We report herein that Syk is also a tau kinase, phosphorylating tau in vitro and in CHO cells when both proteins are expressed exogenously. In CHO cells, we have also demonstrated by co-immunoprecipitation that Syk binds to tau. Finally, by site-directed mutagenesis substituting the tyrosine residues of tau with phenylalanine, we established that tyrosine 18 was the primary residue in tau phosphorylated by Syk. The identification of Syk as a common tyrosine kinase of both tau and alpha-synuclein may be of potential significance in neurodegenerative disorders and also in neuronal physiology. These results bring another clue to the intriguing overlaps between tauopathies and synucleinopathies and provide new insights into the role of Syk in neuronal physiology.

Engagement of the T-cell antigen receptor (TCR) results in the proximal activation of the Src family tyrosine kinase Lck. The activation of Lck leads to the downstream activation of the Ras/Raf/MEK/ERK signaling pathway (where ERK is extracellular signal-related kinase). Under conditions of weak, but not strong, stimulation through the TCR, a version of Lck that contains a single point mutation in the SH3 (Src homology 3) domain (W97ALck) fails to support the activation of ERK, despite initiating signaling through the TCR, as demonstrated by the robust activation of ZAP-70, PLC-gamma, and Ras. We determined that the signaling lesion in W97ALck-expressing cells lies at the level of Raf-1 activation and is dependent on the presence of tyrosines 340/341 in the Raf-1 sequence. These data demonstrate a second function for Lck in TCR-mediated signaling to ERK. Additionally, we found that a significant fraction of Lck is localized to the Golgi apparatus and that, compared with wild-type Lck, W97ALck displays aberrant Golgi membrane localization. Our results support a model where under conditions of weak stimulation through the TCR, in addition to activated Ras, Golgi apparatus-localized Lck is needed for the full activation of Raf-1.

The Syk protein-tyrosine kinase is an essential component of the signaling machinery that couples the B cell receptor for antigen to multiple downstream signal transduction pathways. Syk is phosphorylated and activated rapidly and transiently following receptor engagement, but many signaling events, such as the activation of transcription factors occur over the course of several minutes or hours. To investigate a role for the continued activation of Syk in these processes, we generated an analog-sensitive mutant with an engineered ATP-binding pocket to render the kinase uniquely sensitive to an orthogonal inhibitor. Mutation of the gatekeeper residue in Syk yielded an enzyme with very low activity. Second-site mutations, selected based on structural comparisons between Syk and Src, were introduced that restored catalytic activity to the mutant Syk. Syk-deficient DT40 B cells were prepared expressing the analog-sensitive Syk (Syk-AQL). Inhibition of the activity of Syk prior to, concomitant with or shortly following receptor engagement led to the rapid inhibition of receptor-mediated tyrosine phosphorylation and blocked the activation of extracellular signal-regulated kinase, NF-kappaB, and NFAT. The receptor-mediated activation of NF-kappaB required active Syk for a relatively short period of time, whereas the activation of NFAT required active kinase for a prolonged (>1 h) period. Receptor cross-linking led to the recruitment of Syk to the clustered receptor. Retention of these receptor-kinase complexes on the cell surface was dependent on the continued activity of Syk. Thus, despite the apparent transient nature of the activation of Syk, the catalytic activity of the Syk was required for sustained signaling from ligated receptors.

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.

A novel approach to the intracellular delivery of aryl phosphates has been developed that utilizes a phosphoramidate-based prodrug approach. The prodrugs contain an ester group that undergoes reductive activation intracellularly with concomitant expulsion of a phosphoramidate anion. This anion undergoes intramolecular cyclization and hydrolysis to generate aryl phosphate exclusively with a t(1/2) = approximately 20 min. Phosphoramidate prodrugs (8-10) of phosphate-containing peptidomimetics that target the SH2 domain were synthesized. Evaluation of these peptidomimetic prodrugs in a growth inhibition assay and in a cell-based transcriptional assay demonstrated that the prodrugs had IC50 values in the low micromolar range. Synthesis of phosphorodiamidate analogues containing a P-NH-Ar linker (16-18) was also carried out in the hope that the phosphoramidates released might be phosphatase-resistant. Comparable activation rates and cell-based activities were observed for these prodrugs, but the intermediate phosphoramidate dianion underwent spontaneous hydrolysis with a t(1/2) = approximately 30 min.

The protein tyrosine kinase Syk couples the B-cell receptor (BCR) for antigen to multiple intracellular signaling pathways and also modulates cellular responses to inducers of oxidative stress in a receptor-independent fashion. In B cells, Syk is found in both the nuclear and cytoplasmic compartments but contains no recognizable nuclear localization or export signals. Through the analysis of a series of deletion mutants, we identified the presence of an unconventional shuttling sequence near the junction of the catalytic domain and the linker B region that accounts for Syk's subcellular localization. This localization is altered following prolonged engagement of the BCR, which causes Syk to be excluded from the nucleus. Nuclear exclusion requires the receptor-mediated activation of protein kinase C and new protein synthesis. Both of these processes also potentiate the activation of caspase 3 in cells in response to oxidative stress in a manner that is dependent on the localization of Syk outside of the nucleus. In contrast, restriction of Syk to the nucleus greatly diminishes the stress-induced activation of caspase 3.

The protein-tyrosine kinase Syk couples immune recognition receptors to multiple signal transduction pathways, including the mobilization of calcium and the activation of NFAT. The ability of Syk to regulate signaling is influenced by its phosphorylation on tyrosine residues within the linker B region. The phosphorylation of both Y342 and Y346 is necessary for optimal signaling from the B cell receptor for antigen. The SH2 domains of multiple signaling proteins share the ability to bind this doubly phosphorylated site. The NMR structure of the C-terminal SH2 domain of PLCgamma (PLCC) bound to a doubly phosphorylated Syk peptide reveals a novel mode of phosphotyrosine recognition. PLCC undergoes extensive conformational changes upon binding to form a second phosphotyrosine-binding pocket in which pY346 is largely desolvated and stabilized through electrostatic interactions. The formation of the second binding pocket is distinct from other modes of phosphotyrosine recognition in SH2-protein association. The dependence of signaling on simultaneous phosphorylation of these two tyrosine residues offers a new mechanism to fine-tune the cellular response to external stimulation.

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.