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Results for “Syk Kinase”

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PubMed2005

Clinical significance of nuclear expression of spleen tyrosine kinase (Syk) in gastric cancer.

Spleen tyrosine kinase (Syk) expression was immunohistochemically examined in 250 gastric cancer patients. The rate of positive Syk expression was 42.4%. Syk expression was significantly associated with T1 tumors, lymphatic invasion, venous invasion and lymph node metastasis (P<0.0001). The 5-year survival rate was significantly higher among patients with nuclear Syk expression than among those who were negative for Syk expression (P=0.0003). However, Syk expression was not an independent prognostic factor. Loss of Syk expression was closely related to the malignant property of gastric cancer in the context of tumor depth and lymph node metastasis, especially in early gastric cancer.

Adult↗

PubMed1997

Identification of the major sites of autophosphorylation of the murine protein-tyrosine kinase Syk.

The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.

Amino Acid Sequence↗

PubMed1999

The tyrosine kinases Syk and Lyn exert opposing effects on the activation of protein kinase Akt/PKB in B lymphocytes.

The protein kinase Akt/PKB is a crucial regulator of cell survival in response to mitogenic signals. The increased kinase activity of v-akt, an oncogenic form of Akt/PKB, causes mouse T cell lymphoma, and overexpression of Akt/PKB is associated with progression of several tumor types in human. In this study, we demonstrate that ligation of B cell antigen receptor (BCR) leads to activation of Akt/PKB in B lymphocytes. BCR-induced activation of Akt/PKB required the tyrosine kinase Syk, which was not previously known to regulate Akt/PKB. In contrast, BCR crosslinking of Lyn-deficient B cells resulted in markedly enhanced hyperphosphorylation and activation of Akt/PKB compared with wild-type B cells, indicating that this Src-family kinase acts as an endogenous antagonist of BCR-induced Akt/PKB activation. Lyn inhibited Akt/PKB additively with an okadaic acid-sensitive endogenous phosphatase(s). Expression of exogenous Lyn in mutant cells restored normal BCR-induced phosphorylation of Akt/PKB. Negative regulation of Akt/PKB by Lyn was not dependent on the protein phosphatases SHP-1, SHP-2, or SHIP. Our results show that Lyn provides a mechanism for negative regulation and opposes the effect of Syk on BCR-mediated activation of Akt/PKB. Deregulation of Akt/PKB correlates with the hyperresponsiveness of B cells from Lyn-deficient mice stimulated by BCR crosslinking and may contribute to the autoimmune syndrome that develops in Lyn-deficient animals.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2002

Protein tyrosine kinase Syk in mast cell signaling.

The tyrosine kinase Syk is essential for signaling from FcrepsilonRI in mast cells. The Src homology domain mediated binding of Syk to the phosphorylated immunoreceptor tyrosine-based motif (ITAM) of the receptor subunits results in a conformational change and activation. Studies in Syk deficient mast cells have defined the pathways that are activated upstream and downstream of Syk and have demonstrated the functional importance of the linker region of Syk in signaling in mast cells.

Animals↗

PubMed2004

Alpha-melanocyte stimulating hormone protects against H2O2-induced inhibition of wound restitution in IEC-6 cells via a Syk kinase- and NF-kappabeta-dependent mechanism.

Epithelial injury and repair are central consequences of ischemia and reperfusion of the gut. Intestinal mucosal wounds are repaired in part by epithelial restitution. However, the signaling mechanisms regulating restitution remain poorly understood, and few therapies to enhance restitution have been described. Previously we demonstrated that alpha-melanocyte-stimulating hormone (alpha-MSH) protected against postischemic gut injury in the rat. In this report, we tested the effects and mechanisms of alpha-MSH on wound restitution of rat small intestine (IEC-6) cells subjected to H2O2 stress with or without scrape wounding. H2O2 treatment resulted in tyrosine phosphorylation of Syk kinase and its downstream target IkappaBalpha, with subsequent NF-kappaB activation. Alpha-MSH and the Syk kinase inhibitor piceatannol blocked these processes. In scrape-wounded cells, H2O2 inhibited wound restitution, and this was partially restored by cotreatment with alpha-MSH or piceatannol. In contrast, overexpression of NF-kappaB p65 or Syk kinase, but not a dominant-negative mutant of Syk kinase, aggravated H2O2 inhibition of wound restitution, and inhibitors of c-Src tyrosine kinase or phosphatidylinositol-3 kinase were without effect. The results indicate an important role for Syk tyrosine kinase and the NF-kappaB pathway in the response to oxidant stress and the impairment of epithelial restitution in IEC-6 cells. The data also disclose that the beneficial effects of alpha-MSH on gut ischemia/reperfusion injury may relate to its acceleration of epithelial restitution.

Animals↗

PubMed2006

The Vav binding site of the non-receptor tyrosine kinase Syk at Tyr 348 is critical for beta2 integrin (CD11/CD18)-mediated neutrophil migration.

Leukocyte adhesion via beta(2) integrins (CD11/CD18) activates the tyrosine kinase Syk. We found that Syk was enriched at the lamellipodium during N-formyl-Met-Leu-Phe-induced migration of neutrophil-like differentiated HL-60 cells. Here, Syk colocalized with Vav, a guanine nucleotide exchange factor for Rac and Cdc42. The enrichment of Syk at the lamellipodium and its colocalization with Vav were absent upon expression of a Syk kinase-dead mutant (Syk K402R) or a Syk mutant lacking the binding site of Vav (Syk Y348F). Live cell imaging revealed that both mutations resulted in excessive lamellipodium formation and severely compromised migration compared with control cells. Similar results were obtained upon down-regulation of Syk by RNA interference (RNAi) technique as well as in Syk(-/-) neutrophils from wild-type mice reconstituted with Syk(-/-) bone marrow. A pivotal role of Syk in vivo was demonstrated in the Arthus reaction, where neutrophil extravasation, edema formation, and hemorrhage were profoundly diminished in Syk(-/-) bone marrow chimeras compared with those in control animals. In the inflamed cremaster muscle, Syk(-/-) neutrophils revealed a defect in adhesion and migration. These findings indicate that Syk is critical for beta(2) integrin-mediated neutrophil migration in vitro and plays a fundamental role in neutrophil recruitment during the inflammatory response in vivo.

Animals↗

PubMed2000

Tyrosine kinase SYK: essential functions for immunoreceptor signalling.

The tyrosine kinase SYK plays critical roles in signalling through immune receptors. Gene-targeting studies have identified the cell types that require SYK for development and function, and the receptors that use SYK as well as their downstream signalling effectors. There is also evidence of a role for SYK in non-immune cells and in the maintenance of vascular integrity.

Animals↗

PubMed2003

A role for Syk-kinase in the control of the binding cycle of the beta2 integrins (CD11/CD18) in human polymorphonuclear neutrophils.

A fine control of beta(2) integrin (CD11/CD18)-mediated firm adhesion of human neutrophils to the endothelial cell monolayer is required to allow ordered emigration. To elucidate the molecular mechanisms that control this process, intracellular protein tyrosine signaling subsequent to beta(2) integrin-mediated ligand binding was studied by immunoprecipitation and Western blotting techniques. The 72-kDa Syk-kinase, which was tyrosine-phosphorylated upon adhesion, was found to coprecipitate with CD18, the beta-subunit of the beta(2) integrins. Moreover, inhibition of Syk-kinase by piceatannol enhanced adhesion and spreading but diminished N-formyl-Met-Leu-Phe-induced chemotactic migration. The enhancement of adhesiveness was associated with integrin clustering, which results in increased integrin avidity. In contrast, piceatannol had no effect on the surface expression or on the affinity of beta(2) integrins. Altogether, this suggests that Syk-kinase controls alternation of beta(2) integrin-mediated ligand binding with integrin detachment.

Blotting, Western↗

PubMed2006

Protein-tyrosine kinase Syk is required for pathogen engulfment in complement-mediated phagocytosis.

The protein tyrosine kinase Syk plays a central role in Fcgamma receptor-mediated phagocytosis in the adaptive immune system. We show here that Syk also plays an essential role in complement-mediated phagocytosis in innate immunity. Macrophage-like differentiated HL60 cells and C3bi-opsonized zymosan comprised the pathogen-phagocyte system. C3bi-opsonized zymosan particles promptly attached to the cells and were subsequently engulfed via complement receptor 3. During this process, Syk became tyrosine phosphorylated and accumulated around the nascent phagosomes. The transfer of Syk-siRNA or dominant-negative Syk (DN-Syk) into HL60 cells resulted in impaired phagocytosis. Quenching assays using fluorescent zymosan revealed that most of the attached zymosan particles were located inside parental HL60 cells, whereas few were ingested by the mutant cells. These data indicated that Syk is required for the engulfment of C3bi-opsonized zymosan. During C3bi-zymosan-induced phagocytosis, actin accumulation occurred around phagosomes and was followed by depolymerization, and further RhoA was activated together with tyrosine phosphorylation of Vav. These responses including the actin remodeling were suppressed in Syk-siRNA- or DN-Syk-expressing cells. Our results demonstrated that Syk plays an indispensable role in complement-mediated phagocytosis by regulating both actin dynamics and the RhoA activation pathway and that these functions of Syk lead to phagosome formation and pathogen engulfment.

Actins↗

PubMed2004

Protein tyrosine kinase Syk modulates EGFR signalling in human mammary epithelial cells.

Signalling through protein tyrosine kinases (PTKs) is critical in the regulation of important cellular processes and its deregulation is associated with pathophysiological disorders such as cancer. We investigated the function of the PTK spleen tyrosine kinase (Syk) in the regulation of growth factor signalling pathways in human mammary epithelial cells. Our results show that downregulation of endogenous Syk expression enhances the ligand-induced activity of the epidermal growth factor receptor (EGFR) but not that of the closely related human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3) receptors. Moreover, Syk function interfered with EGFR-mediated cell responses such as proliferation and survival of mammary epithelial cells. A mechanistic link between Syk and EGFR is further supported by the colocalisation of the two PTKs in membrane fractions as well as the regulatory feedback effects of the EGFR kinase on Syk activity. Our findings demonstrate that Syk acts a negative control element of EGFR signalling.

Cell Line↗

PubMed2000

Characterisation and specificity of two single-chain Fv antibodies directed to the protein tyrosine kinase Syk.

In order to obtain single chain Fv fragments (scFv) specific for the protein tyrosine kinase Syk, we screened a human synthetic phage-display library. Two glutathione S-transferase (GST):Syk fusion proteins containing both SH2 domains of Syk were used to perform three rounds of selection of the library. Among the scFv fragments resulting from the third round of selection, the ones specific for the GST portion of the fusion proteins were eliminated by performing enzyme-linked immunosorbent assay tests on GST:Syk versus GST coated plates, and the monoclonal scFv fragments binding only to the GST:Syk coated plates with high affinities were further analysed. We report here the in vitro characterisation of G4G11 and G6G2 anti-Syk scFvs. G4G11 shows the best performance in immunoprecipitation and immunofluorescence experiments, and G6G2 is able to detect Syk in immunoprecipitation, immunofluorescence and on Western blots. Both scFvs are also able to detect the phosphorylated form of Syk, and neither of them binds to Zap-70, the other member of the Syk family of protein tyrosine kinases.

Animals↗

PubMed2001

Cross-linking of beta2 integrins caused diminished responses of neutrophils to priming agents like lipopolysaccharide or tumor necrosis factor-alpha: possible involvement of tyrosine kinase Syk.

Neutrophils up-regulate beta2 integrins like CD11b/CD18 in response to lipopolysaccharide (LPS). Up-regulation of beta2 integrins causes neutrophils to adhere to surfaces, and to release superoxide anion (O2-). When neutrophils are exposed to LPS plus plasma under conditions not favorable for adherence (absence of Mg2+), the cells do not spontaneously release O2-, but instead they are primed for enhanced release of O2- after subsequent triggering by fMLP. In the presence of Mg2+, neutrophils adhere in response to LPS but fMLP-triggered O2- release by LPS-primed neutrophils is diminished. To understand why adherence interferes with the response of neutrophils to N-formyl-methionyl-leucyl-phenylalanine (fMLP), beta2 integrins were cross-linked by mouse monoclonal antibodies that had been immobilized by surface-bound anti-mouse antibody. When unprimed neutrophils were trapped on the surface by these cross-linked monoclonal antibodies, O2- release was triggered, and priming by LPS for fMLP-triggered O2- release was diminished, indicating that this cross-linking of beta2 integrins mimicked adherence. Alkaline phosphatase is up-regulated by LPS or tumor necrosis factor-alpha, and this response was also diminished by the cross-linking antibodies. The diminished alkaline phosphatase up-regulation was reversed by genistein, a general inhibitor of tyrosine kinases, and by piceatannol, an inhibitor for Syk kinase. Piceatannol also inhibited the phosphorylation of Syk caused by cross-linking of beta2 integrins. These results suggested that adherence-induced triggering and Syk kinase activation might be responsible for the diminished response of LPS-primed neutrophils to fMLP when neutrophils were adherent.

Alkaline Phosphatase↗

PubMed2007

Tyrosine kinase Syk associates with toll-like receptor 4 and regulates signaling in human monocytic cells.

Toll-like receptor 4 (TLR4) induces an innate immune response in mammals by recognizing lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria. In this study, we show that tyrosine kinase Syk constitutively associates with TLR4 in THP-1 cells. As previously reported in peripheral blood mononuclear cells, TLR4 gets inducibly tyrosine phosphorylated upon LPS engagement in THP-1 cells. Piceatannol, a pharmacological inhibitor of the tyrosine kinase Syk, abrogates TLR4 tyrosine phosphorylation at low doses. The kinetics of TLR4 tyrosine phosphorylation in THP-1 cells coincides with an early wave of Syk tyrosine phosphorylation. Additionally, serine threonine kinase interleukin-1 (IL1) receptor-associated kinase 1 (IRAK-1) is transiently recruited to the complex containing adaptor molecule MyD88, TLR4 and Syk within 1 min of LPS engagement and dissociates by 30 min. Finally, the inhibition of Syk with piceatannol has no effect on LPS-mediated release of cytokines IL6, IL1beta, tumor necrosis factor-alpha, neither on chemokines macrophage inhibitory protein (MIP)1alpha, MIP1beta, monocyte chemoattractant protein -1, IL8, Groalpha and RANTES. However, IL10 and IL12p40 releases are significantly inhibited. Our findings implicate Syk as a novel modulator of LPS-mediated TLR4 responses in human monocytic cells and shed insight into the kinetics of early complex formation upon LPS engagement.

Cell Line, Tumor↗

PubMed2006

Differential expression of spleen tyrosine kinase Syk isoforms in tissues: Effects of the microbial flora.

Spleen tyrosine kinase (Syk) is expressed widely in hematopoietic and non-hematopoietic cells. The widespread distribution of Syk and its involvement in host defense and allergic reactions, prompted us analyze the influence of microbial exposure on Syk expression. We compared the distribution of Syk in various tissues of germ-free and conventional mice using immunohistochemistry, Western blot analysis and real time RT-PCR. Total Syk expression was similar between germ-free and conventional mice. Since it has been claimed that Syk isoforms are differentially expressed, we studied the distribution and abundance of Syk (L) and Syk (S) isoforms in tissues from these mice. In contrast to previous reports, we found broad tissue expression of Syk (S). Interestingly, in germ-free mice the amount of Syk (S) but not Syk L protein was selectively increased in lung and spleen. In summary, our study reveals new and broad tissue expression of both Syk isoforms and demonstrates that lack of microbial flora results in selectively increased expression of Syk (S) isoform in lung and spleen.

Animals↗

PubMed1997

Protein tyrosine kinases Syk and ZAP-70 display distinct requirements for Src family kinases in immune response receptor signal transduction.

Engagement of immunoreceptors in hemopoietic cells leads to activation of Src family tyrosine kinases as well as Syk or ZAP-70. Current models propose that Src family kinases are critical in immune response signal transduction through their role in phosphorylation of tyrosine residues within immunoreceptor tyrosine activation motifs (ITAMs; which recruit the SH2 domains of Syk or ZAP-70) and by direct phosphorylation of Syk and ZAP-70. Several lines of evidence suggest that Syk may not show the same dependence on activation by Src family kinases as ZAP-70. In this report, we used COS cells transiently transfected with components of the Fc epsilon RI complex (Lyn, Syk, and a chimeric CD8 receptor containing the cytoplasmic domain of the gamma subunit of Fc epsilon RI (CD8-gamma)) to examine the regulation of Syk activity. Syk was activated and phosphorylated in COS cells cotransfected with Lyn; however, in cells expressing CD8-gamma, activation of Syk and phosphorylation of CD8-gamma did not require coexpression of Lyn. Additional experiments indicate that gamma phosphorylation is dependent on Syk kinase activity and is independent of endogenous COS cell kinases. In parallel experiments, ZAP-70 was not activated by cotransfection with CD8-gamma, nor was CD8-gamma phosphorylated when coexpressed with ZAP-70 alone. Taken together, these studies indicate that Syk can be distinguished from ZAP-70 in its ability to be activated by coexpression with an ITAM-containing receptor without coexpression of a Src family kinase, and that Syk is capable of phosphorylating ITAM tyrosines under certain experimental conditions.

Animals↗

PubMed2005

The non-receptor tyrosine kinase Syk regulates lamellipodium formation and site-directed migration of human leukocytes.

The tyrosine kinase Syk is associated with CD18, the beta-subunit of the leukocyte adhesion molecules of the beta(2) integrin family (CD11/CD18), and becomes activated upon beta(2) integrin-mediated adhesion. In this study, we elucidated the role of Syk in polarization and site-directed migration of neutrophil-like differentiated HL-60 cells and monocytic THP-1 cells. By means of confocal microscopy, we detected a homogenous distribution of Syk in unstimulated cells in suspension. The stimulation of HL-60 cells by formyl-methionyl-leucyl-phenylalanine (fMLP, 100 nM) or the activation of THP-1 cells by monocyte chemoattractant protein-1 (10 ng/ml) induced beta(2) integrin-mediated cell adhesion and polarization on immobilized fibrinogen which was associated with an enrichment of Syk at the lamellipodium forming site. This effect was abolished by function blocking anti-CD18 antibody or by treatment of the cells with the Syk inhibitor piceatannol (30 microM) suggesting that the redistribution of Syk required both, beta(2) integrin-mediated adhesion and Syk activation. Moreover, the inhibition of Syk by piceatannol or the downregulation of Syk by antisense technique resulted in an excessive formation of lamellipodia indicating that Syk may act as a negative regulator that limits lamellipodium formation. The analysis of chemotaxis revealed that the inhibition of Syk impaired the ability of the cells to follow a chemotactic gradient whereas random migration was intact. Taken together, our data suggest a novel role for Syk in the maintenance of a bipolar phenotype by regulating lamellipodium formation, which is a critical prerequisite for site-directed migration of leukocytes.

Cell Adhesion↗

PubMed2006

Genomic and expression profiling identifies the B-cell associated tyrosine kinase Syk as a possible therapeutic target in mantle cell lymphoma.

Among B-cell lymphomas mantle cell lymphoma (MCL) has the worst prognosis. By using a combination of genomic and expression profiling (Affymetrix GeneChip Mapping 10k Xba131 and U133 set), we analysed 26 MCL samples to identify genes relevant to MCL pathogenesis and that could represent new therapeutic targets. Recurrent genomic deletions and gains were detected. Genes were identified as overexpressed in regions of DNA gain on 3q, 6p, 8q, 9q, 16p and 18q, including the cancer genes BCL2 and MYC. Among the transcripts with high correlation between DNA and RNA, we identified SYK, a tyrosine kinase involved in B-cell receptor signalling. SYK was amplified at DNA level, as validated by fluorescence in situ hybridisation (FISH) analysis, and overexpressed at both RNA and protein levels in the JeKo-1 cell line. Low-level amplification, with protein overexpression of Syk was demonstrated by FISH in a small subset of clinical samples. After treatment with low doses of the Syk inhibitor piceatannol, cell proliferation arrest and apoptosis were induced in the cell line overexpressing Syk, while cells expressing low levels of Syk were much less sensitive. A combination of genomic and expression profiling suggested Syk inhibition as a new therapeutic strategy to be explored in lymphomas.

Apoptosis↗

PubMed1995

Solution structure of the C-terminal SH2 domain of the human tyrosine kinase Syk complexed with a phosphotyrosine pentapeptide.

BACKGROUND: Recruitment of the intracellular tyrosine kinase Syk to activated immune-response receptors is a critical early step in intracellular signaling. In mast cells, Syk specifically associates with doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) that are found within the IgE receptor. The mechanism by which Syk recognizes these motifs is not fully understood. Both Syk SH2 (Src homology 2) domains are required for high-affinity binding to these motifs, but the C-terminal SH2 domain (Syk-C) can function independently and can bind, in isolation, to the tyrosine-phosphorylated IgE receptor in vitro. In order to improve understanding of the cellular function of Syk, we have determined the solution structure of Syk-C complexed with a phosphotyrosine peptide derived from the gamma subunit of the IgE receptor. RESULTS: The Syk-C:peptide structure is compared with liganded structures of both the SH2 domain of Src and the C-terminal SH2 domain of ZAP-70 (the 70 kDa zeta-associated protein). The topologies of these domains are similar, although significant differences occur in the loop regions. In the Syk-C structure, the phosphotyrosine and leucine residues of the peptide ligand interact with pockets on the protein, and the intervening residues are extended. CONCLUSIONS: Syk-C resembles other SH2 domains in its peptide-binding interactions and overall topology, a result that is consistent with its ability to function as an independent SH2 domain in vitro. This result suggests that Syk-C plays a unique role in the intact Syk protein. The determinants of the binding affinity and selectivity of Syk-C may reside in the least-conserved structural elements that comprise the phosphotyrosine- and leucine-binding sites. These structural features can be exploited for the design of Syk-selective SH2 antagonists for the treatment of allergic disorders and asthma.

Amino Acid Sequence↗