The Experts below are selected from a list of 13638 Experts worldwide ranked by ideXlab platform
Reuben P. Siraganian - One of the best experts on this subject based on the ideXlab platform.
-
Protein tyrosine kinase Syk in mast cell signaling.
Molecular immunology, 2002Co-Authors: Reuben P. Siraganian, Juan Zhang, Katsuhiro Suzuki, Kiyonao SadaAbstract: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.
-
phosphorylation of Syk activation loop tyrosines is essential for Syk function an in vivo study using a specific anti Syk activation loop phosphotyrosine antibody
Journal of Biological Chemistry, 2000Co-Authors: Juan Zhang, Melvin L Billingsley, Randall L Kincaid, Reuben P. SiraganianAbstract:Syk is an important protein-tyrosine kinase in immunoreceptor signaling. FceRI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of FceRI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation with its function, an antibody was developed specific for phosphorylated tyrosines in the activation loop of Syk. FceRI aggregation on mast cells induced the phosphorylation of both tyrosine residues of the activation loop. The kinase activity of Syk played the major role in phosphorylating its activation loop tyrosines both in vivoand in vitro. In FceRI-stimulated mast cells, the total Syk tyrosine phosphorylation paralleled the phosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast, the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosine phosphorylation of Syk and minimal histamine release and weak phosphorylation of activation loop tyrosines. These results demonstrate that phosphorylation of the activation loop tyrosines is important for mediating receptor signaling and is a better marker of Syk function than is total Syk tyrosine phosphorylation.
-
cd45 is essential for fceri signaling by zap70 but not Syk in Syk negative mast cells
Journal of Immunology, 1999Co-Authors: Juan Zhang, Reuben P. SiraganianAbstract:The ZAP70/Syk family of protein tyrosine kinases plays an important role in Ag receptor signaling. Structural similarity of Syk and ZAP70 suggests their functional overlap. Previously, it was observed that expression of either ZAP70 or Syk reconstitutes Ag receptor signaling in Syk-negative B cells. However, in CD45-deficient T cells, Syk, but not ZAP70, restores T cell receptor-signaling pathway. To study the function of Syk, ZAP70, and CD45 in mast cells, a Syk/CD45 double-deficient variant of RBL-2H3 cells was characterized. After transfection, stable cell lines were isolated that expressed ZAP70, Syk, CD45, ZAP70 plus CD45, and Syk plus CD45. IgE stimulation did not induce degranulation in parental double-deficient cells, nor in the cells expressing only CD45. ZAP70 expression did not restore FceRI signaling unless CD45 was coexpressed in the cells. However, Syk alone restored the IgE signal transduction pathway. The coexpression of CD45 with Syk had no significant effects on the responses to FceRI-aggregation. There was much better binding of Syk than ZAP70 to the phosphorylated FceRIγ-ITAM. Furthermore, unlike Syk, ZAP70 required CD45 to display receptor-induced increase in kinase activity. Therefore, in mast cells, ZAP70, but not Syk, requires CD45 for Ag receptor-induced signaling.
-
mutations in the activation loop tyrosines of protein tyrosine kinase Syk abrogate intracellular signaling but not kinase activity
Journal of Immunology, 1998Co-Authors: Juan Zhang, Teruaki Kimura, Reuben P. SiraganianAbstract:The protein tyrosine kinase Syk plays a pivotal role in mediating the high-affinity IgE receptor (FceRI)-induced degranulation of mast cells. To examine the mechanism of Syk regulation, the two tyrosine residues at 519 and 520 in the putative activation loop of rat Syk were mutated to phenylalanine either singly or in combination. The various mutants were expressed in a Syk-negative variant of the RBL-2H3 (rat basophilic leukemia 2H3) mast cell line. In these transfected cell lines, mutant Syk did show increased tyrosine phosphorylation in vivo and increased enzymatic activity in vitro after FceRI aggregation. There were conformational changes detected by an Ab when the wild-type and mutant Syk were either tyrosine phosphorylated or bound to tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these mutant Syk were incapable of transducing FceRI signaling. In cells in which the expression level of mutant Syk was similar to that of the wild-type Syk, FceRI cross-linking induced no increase in cellular protein tyrosine phosphorylation, no increase in tyrosine phosphorylation of phospholipase C-γ2 and mitogen-activated protein kinase, and no histamine release. Overexpression of Y519F or Y520F Syk mutants partially reconstituted the signaling pathways. These results indicate that these tyrosines in the putative activation loop are not essential for the enzymatic activity of Syk or for the conformational changes induced by binding of tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these tyrosines are necessary for Syk-mediated propagation of FceRI signaling.
Juan Zhang - One of the best experts on this subject based on the ideXlab platform.
-
activation of Syk tyrosine kinase is required for c cbl mediated ubiquitination of fceri and Syk in rbl cells
Journal of Biological Chemistry, 2002Co-Authors: Rossella Paolini, Juan Zhang, Rosa Molfetta, Laurie O. Beitz, Andrew M. Scharenberg, Mario Piccoli, Luigi Frati, Reuben Siraganian, Angela SantoniAbstract:Engagement of the high affinity receptor for IgE (FceRI) on mast cells and basophils results in FceRI β and γ subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FceRI engagement on RBL-2H3 cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FceRI β and γ chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FceRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FceRI and activated Syk to the proteasome for degradation.
-
Protein tyrosine kinase Syk in mast cell signaling.
Molecular immunology, 2002Co-Authors: Reuben P. Siraganian, Juan Zhang, Katsuhiro Suzuki, Kiyonao SadaAbstract: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.
-
phosphorylation of Syk activation loop tyrosines is essential for Syk function an in vivo study using a specific anti Syk activation loop phosphotyrosine antibody
Journal of Biological Chemistry, 2000Co-Authors: Juan Zhang, Melvin L Billingsley, Randall L Kincaid, Reuben P. SiraganianAbstract:Syk is an important protein-tyrosine kinase in immunoreceptor signaling. FceRI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of FceRI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation with its function, an antibody was developed specific for phosphorylated tyrosines in the activation loop of Syk. FceRI aggregation on mast cells induced the phosphorylation of both tyrosine residues of the activation loop. The kinase activity of Syk played the major role in phosphorylating its activation loop tyrosines both in vivoand in vitro. In FceRI-stimulated mast cells, the total Syk tyrosine phosphorylation paralleled the phosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast, the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosine phosphorylation of Syk and minimal histamine release and weak phosphorylation of activation loop tyrosines. These results demonstrate that phosphorylation of the activation loop tyrosines is important for mediating receptor signaling and is a better marker of Syk function than is total Syk tyrosine phosphorylation.
-
cd45 is essential for fceri signaling by zap70 but not Syk in Syk negative mast cells
Journal of Immunology, 1999Co-Authors: Juan Zhang, Reuben P. SiraganianAbstract:The ZAP70/Syk family of protein tyrosine kinases plays an important role in Ag receptor signaling. Structural similarity of Syk and ZAP70 suggests their functional overlap. Previously, it was observed that expression of either ZAP70 or Syk reconstitutes Ag receptor signaling in Syk-negative B cells. However, in CD45-deficient T cells, Syk, but not ZAP70, restores T cell receptor-signaling pathway. To study the function of Syk, ZAP70, and CD45 in mast cells, a Syk/CD45 double-deficient variant of RBL-2H3 cells was characterized. After transfection, stable cell lines were isolated that expressed ZAP70, Syk, CD45, ZAP70 plus CD45, and Syk plus CD45. IgE stimulation did not induce degranulation in parental double-deficient cells, nor in the cells expressing only CD45. ZAP70 expression did not restore FceRI signaling unless CD45 was coexpressed in the cells. However, Syk alone restored the IgE signal transduction pathway. The coexpression of CD45 with Syk had no significant effects on the responses to FceRI-aggregation. There was much better binding of Syk than ZAP70 to the phosphorylated FceRIγ-ITAM. Furthermore, unlike Syk, ZAP70 required CD45 to display receptor-induced increase in kinase activity. Therefore, in mast cells, ZAP70, but not Syk, requires CD45 for Ag receptor-induced signaling.
-
mutations in the activation loop tyrosines of protein tyrosine kinase Syk abrogate intracellular signaling but not kinase activity
Journal of Immunology, 1998Co-Authors: Juan Zhang, Teruaki Kimura, Reuben P. SiraganianAbstract:The protein tyrosine kinase Syk plays a pivotal role in mediating the high-affinity IgE receptor (FceRI)-induced degranulation of mast cells. To examine the mechanism of Syk regulation, the two tyrosine residues at 519 and 520 in the putative activation loop of rat Syk were mutated to phenylalanine either singly or in combination. The various mutants were expressed in a Syk-negative variant of the RBL-2H3 (rat basophilic leukemia 2H3) mast cell line. In these transfected cell lines, mutant Syk did show increased tyrosine phosphorylation in vivo and increased enzymatic activity in vitro after FceRI aggregation. There were conformational changes detected by an Ab when the wild-type and mutant Syk were either tyrosine phosphorylated or bound to tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these mutant Syk were incapable of transducing FceRI signaling. In cells in which the expression level of mutant Syk was similar to that of the wild-type Syk, FceRI cross-linking induced no increase in cellular protein tyrosine phosphorylation, no increase in tyrosine phosphorylation of phospholipase C-γ2 and mitogen-activated protein kinase, and no histamine release. Overexpression of Y519F or Y520F Syk mutants partially reconstituted the signaling pathways. These results indicate that these tyrosines in the putative activation loop are not essential for the enzymatic activity of Syk or for the conformational changes induced by binding of tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these tyrosines are necessary for Syk-mediated propagation of FceRI signaling.
Hirohei Yamamura - One of the best experts on this subject based on the ideXlab platform.
-
Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk.
The Journal of general virology, 2008Co-Authors: Sachiko Inubushi, Hirohei Yamamura, Motoko Nagano-fujii, Kikumi Kitayama, Motofumi Tanaka, Hiroshi Yokozaki, Hideko Nuriya, Michinori Kohara, Kiyonao SadaAbstract:Hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma. However, the precise mechanism underlying the carcinogenesis is yet to be elucidated. It has recently been reported that Syk, a non-receptor protein tyrosine kinase, functions as a potent tumour suppressor in human breast carcinoma. This study first examined the possible effect of HCV infection on expression of Syk in vivo. Immunohistochemical analysis revealed that endogenous Syk, which otherwise was expressed diffusely in the cytoplasm of normal hepatocytes, was localized near the cell membrane with a patchy pattern in HCV-infected hepatocytes. The possible interaction between HCV proteins and Syk in human hepatoma-derived Huh-7 cells was then examined. Immunoprecipitation analysis revealed that NS5A interacted strongly with Syk. Deletion-mutation analysis revealed that an N-terminal portion of NS5A (aa 1-175) was involved in the physical interaction with Syk. An in vitro kinase assay demonstrated that NS5A inhibited the enzymic activity of Syk and that, in addition to the N-terminal 175 residues, a central portion of NS5A (aa 237-302) was required for inhibition of Syk. Moreover, Syk-mediated phosphorylation of phospholipase C-gamma1 was downregulated by NS5A. An interaction of NS5A with Syk was also detected in Huh-7.5 cells harbouring an HCV RNA replicon or infected with HCV. In conclusion, these results demonstrated that NS5A interacts with Syk resulting in negative regulation of its kinase activity. The results indicate that NS5A may be involved in the carcinogenesis of hepatocytes through the suppression of Syk kinase activities.
-
protein tyrosine kinase Syk is required for cxcl12 induced polarization of b cells
Biochemical and Biophysical Research Communications, 2005Co-Authors: Satoshi Matsusaka, Yumi Tohyama, Yuhong Shi, Tomomi Kadono, Ryoichi Hazama, Rina Kurihara, Kaoru Tohyama, Hirohei YamamuraAbstract:Cell polarization and migration in response to CXCL12 is essential for hematopoiesis. To investigate the role of Syk in CXCL12/CXCR4-induced signaling, wild-type Syk or its dominant-negative form (DN-Syk) was introduced in mouse pro-B cells, BAF3. With CXCL12 stimulation, BAF3 cells became polarized with the formation of a leading edge and contractile uropod at the rear end with increased motility. Overexpression of wild-type Syk caused enhanced polarization, whereas DN-Syk inhibited cell polarity due to the loss of contractile structure at the rear end, and the altered phenotype was enhanced after CXCL12 stimulation. Motility of mutant BAF3 containing DN-Syk increased independent of CXCL12 stimulation. As β1 integrin-mediated cell adhesion was inhibited, decreased adhesion might promote motility. CXCL12 stimulation led to prompt activation of RhoA, but expression of DN-Syk suppressed RhoA activation. These results demonstrate that Syk participates in CXCL12-induced cell polarization, which occurs in concert with cell adhesion mediated by β1 integrin.
-
Activation of Syk protein tyrosine kinase in response to osmotic stress requires interaction with p21-activated protein kinase Pak2/γ-PAK
Molecular and cellular biology, 2004Co-Authors: S. M. Shahjahan Miah, Kiyonao Sada, Polygena T. Tuazon, Jun Ling, Koichiro Maeno, Shinkou Kyo, Yumi Tohyama, Jolinda A. Traugh, Hirohei YamamuraAbstract:The p21-activated serine/threonine protein kinase Pak2/γ-PAK and the nonreceptor type of protein tyrosine kinase Syk are known to be activated when the cells are exposed to osmotic stress. The purpose of the present study was to examine whether Pak2 and Syk functionally cooperate in cellular signaling. Cotransfection studies revealed that Pak2 associates with Syk in COS cells. The constitutively active form of Cdc42 increases the association of Pak2 with Syk. Pak2 coexpressed with an inactive form of Cdc42 or kinase-inactive Pak2 interacts to a lesser extent with Syk, suggesting that Pak2-Syk association is enhanced by Pak2 activation. Interaction with Pak2 enhances the intrinsic kinase activity of Syk. This is supported by in vitro studies showing that Pak2 phosphorylates and activates Syk. Treatment of cells with sorbitol to induce hyperosmolarity results in the translocation of Pak2 and Syk to the region surrounding the nucleus and in dramatic enhancement of their association. Furthermore, cotransfection of Pak2 and Syk leads to the activation of c-Jun N-terminal kinase (JNK) under hyperosmotic conditions. Pak2 short interfering RNA suppresses sorbitol-mediated activation of endogenous Syk and JNK, thus identifying a novel pathway for JNK activation by Cdc42. These results demonstrate that Pak2 and Syk positively cooperate to regulate cellular responses to stress.
-
protein tyrosine kinase Syk expressed in human nasal fibroblasts and its effect on rantes production
Journal of Immunology, 2001Co-Authors: Takechiyo Yamada, Hirohei Yamamura, Shigeru Yanagi, Ryoko Inatome, Shigeharu Fujieda, Hiroshi Sunaga, Hitoshi SaitoAbstract:Fibroblasts, a rich source of chemokines, interact with eosinophils and play a key role in the pathogenesis of airway disease. RANTES is produced by fibroblasts to attract and activate eosinophils. LPS is known to induce RANTES and cause protein tyrosine phosphorylation. Nonreceptor protein tyrosine kinase Syk is widely expressed and an important role in intracellular signal transduction in hemopoietic cells. In the present study, we examined whether Syk was expressed in a number of primary human nasal polyp tissue-derived fibroblast lines and whether it played some role in cellular function. Syk proteins were expressed in human nasal fibroblasts, but the expression level varied. There were positive correlations between the level of Syk expression and RANTES production induced by LPS. Overexpression of wild-type Syk by gene transfer enhanced RANTES production from nasal fibroblasts stimulated with LPS. The decrease of Syk expression by the administration of Syk antisense inhibited RANTES production. These results suggest that Syk expression affects RANTES production in fibroblasts of nasal polyps.
-
purification of a 72 kda protein tyrosine kinase from rat liver and its identification as Syk involvement of Syk in signaling events of hepatocytes
Journal of Biochemistry, 2000Co-Authors: Shinobu Tsuchida, Shigeru Yanagi, Ryoko Inatome, Junyi Ding, Patrice Hermann, Toshiaki Tsujimura, Nobuzo Matsui, Hirohei YamamuraAbstract:Syk protein-tyrosine kinase (PTK) has been implicated in a variety of hematopoietic cell responses including immunoreceptor signaling. However, so far, there has been no evidence of the expression of Syk or Syk-related PTK in non-hematopoietic tissues. In this study, we have purified from blood cell-depleted rat liver a 72-kDa cytoplasmic PTK which shows cross-reactivity with anti-Syk antibody. Partial amino acid sequence analysis revealed that this 72-kDa PTK is identical to Syk. Immunohistochemical and RT-PCR analyses demonstrated that Syk is expressed in human hepatocytes and two rat liver-derived cell lines, JTC-27 and RLC-16. Furthermore, Syk is significantly tyrosine-phosphorylated in response to angiotensin II in JTC-27 cells, and angiotensin II-induced MAP kinase activation is blocked by the treatment of cells with a Syk-selective inhibitor, piceatannol. These results suggest that Syk plays an important role in signaling events of hepatocytes, such as signaling steps leading to MAP kinase activation by G-protein-coupled receptors. This is the first report of the expression of Syk in non-hematopoietic tissue.
Dimitar G Efremov - One of the best experts on this subject based on the ideXlab platform.
-
phosphorylation of the activation loop tyrosines is required for sustained Syk signaling and growth factor independent b cell proliferation
Cellular Signalling, 2009Co-Authors: Laura Carsetti, Stefania Gobessi, Luca Laurenti, Pablo G Longo, Giuseppe Leone, Dimitar G EfremovAbstract:The Syk kinase is regarded as a promising target for the treatment of antigen-driven B-cell malignancies, considering its essential role in propagating antigenic stimuli through the B-cell receptor (BCR). In certain common B-cell malignancies Syk is activated even in the absence of BCR engagement, suggesting a wider role for this kinase in lymphomagenesis. In this paper, we have profiled molecular differences between BCR-induced and constitutive Syk activation in terms of phosphorylation of regulatory tyrosine residues, downstream signaling properties and capacity to sustain B-cell proliferation. Analysis of primary chronic lymphocytic leukemia B-cells and diffuse large B-cell lymphoma cell lines revealed that constitutive and BCR-induced Syk activation differ with respect to the phosphorylation status of the regulatory tyrosines at positions 352 and 525/526, with only the first site being phosphorylated in the case of constitutive and both sites in the case of BCR-induced Syk activation. Syk phosphorylated only on Y352 is capable of downstream signaling, as evidenced by experiments with a phosphomimetic mutant in which the activation loop tyrosines (YY525/526) were replaced with phenylalanines. However, phosphorylation at YY525/526 was shown to significantly increase the enzymatic activity of Syk and to be required for sustained PLCγ2, Akt and ERK signaling as well as B-cell transformation. These data demonstrate that constitutively active Syk and Syk activated by BCR crosslinking represent separate stages of Syk activation with distinct signaling properties and transforming capacities.
-
inhibition of constitutive and bcr induced Syk activation downregulates mcl 1 and induces apoptosis in chronic lymphocytic leukemia b cells
Leukemia, 2009Co-Authors: Stefania Gobessi, Luca Laurenti, Pablo G Longo, Laura Carsetti, V Berno, Simona Sica, Giuseppe Leone, Dimitar G EfremovAbstract:The protein kinase Syk is a key mediator of proximal B-cell receptor (BCR) signaling. Following antigen stimulation, Syk is recruited to the BCR and becomes activated by phosphorylation at Y352. Recently, Syk was found to be constitutively phosphorylated in several common B-cell lymphoma subtypes, indicating a role for antigen-independent Syk activation in the pathogenesis of these diseases. We now report that Syk is constitutively phosphorylated on the activating Y352 residue in chronic lymphocytic leukemia (CLL) B cells. To examine the effects of constitutive Syk activity on intracellular signaling and leukemic cell survival, we performed in vitro studies with the Syk inhibitor R406. Treatment with R406 induced leukemic cell apoptosis in the majority of investigated cases and affected the basal activity or expression of several pro-survival molecules regulated by Syk, including the Akt and extracellular signal-regulated (ERK) kinases, and the anti-apoptotic protein Mcl-1. In addition, R406 prevented the increase in leukemic cell viability induced by sustained BCR engagement and inhibited BCR-induced Akt activation and Mcl-1 upregulation. Collectively, these data identify Syk as a potential target for CLL treatment and suggest that inhibition of this kinase could provide a double therapeutic benefit by disrupting both antigen-dependent and antigen-independent signaling pathways that regulate leukemic cell survival.
-
constitutive activation of the protein tyrosine kinase Syk in chronic lymphocytic leukemia b cells
Blood, 2007Co-Authors: Stefania Gobessi, Luca Laurenti, Pablo G Longo, Laura Carsetti, Simona Sica, Giuseppe Leone, Dimitar G EfremovAbstract:The protein tyrosine kinase Syk is a key mediator of proximal B-cell receptor (BCR) signaling. Following antigen stimulation Syk is recruited to the BCR and becomes activated by sequential phosphorylation at conserved tyrosine (Tyr) residues. The first event involves phosphorylation at Tyr352 by Lyn or other Src family kinases, followed by autophosphorylation of Tyr525/Tyr526 in the activation loop. Once activated, Syk further propagates the BCR signal by associating with adaptor proteins and phosphorylating downstream signaling molecules. Recently, translocations involving Syk have been identified in MDS and T-cell lymphoma, indicating that Syk may also function as a protooncogene. In line with this possibility, expression of a constitutively active TEL-Syk fusion protein was shown to result in growth factor-independent proliferation and transformation of mouse B-cells. These findings prompted us to investigated the activation status of Syk in primary unstimulated CLL B-cells. Western blot analysis with a phospho-specific antibody revealed substantial levels of Syk phosphorylated at Tyr352 in 29 of 54 freshly isolated CLL B-cell samples. Constitutive phosphorylation of Syk at Tyr352 was confirmed by immunofluorescence and confocal microscopy, which showed punctuate staining distributed across the plasma membrane and cytoplasm of unstimulated CLL B-cells. In contrast, control experiments with BJAB lymphoma B-cells showed phosphorylation of Syk at Tyr352 only following BCR crosslinking. To investigate which downstream signaling pathways are affected by Syk activation, we produced a constitutively active Syk mutant in which Tyr352 was substituted with aspartic acid. Transfection of Syk Tyr352Asp in HEK293, Jurkat and BJAB cells resulted in tyrosine phosphorylation of cellular proteins and autophosphorylation of Syk at Tyr525/Tyr526, whereas no changes were observed following transfection with wild type Syk. In addition, transfection of the Syk Tyr352Asp mutant in the human B-CLL cell line MEC1 resulted in increased phosphorylation of ERK, Akt and GSK3, indicating that these important cellular regulatory pathways are targeted by constitutively active Syk in CLL B-cells. To determine the effect of Syk activation on CLL cell survival, we cultured leukemic B-cells in the presence of R406, a recently developed and specific Syk kinase inhibitor (kindly provided by Rigel Pharmaceutics, Inc.). Assessment of CLL cell viability after 48 hours in culture showed moderate induction of apoptosis at concentrations above 600 nM in 12 of 18 investigated cases, with a maximal cytotoxic effect at 2.5 μM (20–50% apoptotic cells after normalization for spontaneous apoptosis). Interestingly, R406 at even lower concentrations (0.16 to 0.62 μM) inhibited the proliferation of CLL B-cells that was induced by stimulation with unmethylated CpG oligonucleotides, indicating that Syk kinase activity is required for leukemic cell proliferation in this setting. In summary, these data show that Syk is frequently activated in CLL B-cells, even in the absence of BCR engagement. Expression of constitutively active Syk results in activation of pathways that regulate cellular proliferation and survival, whereas inhibition of Syk kinase activity with R406 induces apoptosis and blocks CLL cell proliferation in an in vitro model. Together, these findings suggest that Syk may be a potential candidate for targeted therapy of CLL.
Kiyonao Sada - One of the best experts on this subject based on the ideXlab platform.
-
Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk.
The Journal of general virology, 2008Co-Authors: Sachiko Inubushi, Hirohei Yamamura, Motoko Nagano-fujii, Kikumi Kitayama, Motofumi Tanaka, Hiroshi Yokozaki, Hideko Nuriya, Michinori Kohara, Kiyonao SadaAbstract:Hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma. However, the precise mechanism underlying the carcinogenesis is yet to be elucidated. It has recently been reported that Syk, a non-receptor protein tyrosine kinase, functions as a potent tumour suppressor in human breast carcinoma. This study first examined the possible effect of HCV infection on expression of Syk in vivo. Immunohistochemical analysis revealed that endogenous Syk, which otherwise was expressed diffusely in the cytoplasm of normal hepatocytes, was localized near the cell membrane with a patchy pattern in HCV-infected hepatocytes. The possible interaction between HCV proteins and Syk in human hepatoma-derived Huh-7 cells was then examined. Immunoprecipitation analysis revealed that NS5A interacted strongly with Syk. Deletion-mutation analysis revealed that an N-terminal portion of NS5A (aa 1-175) was involved in the physical interaction with Syk. An in vitro kinase assay demonstrated that NS5A inhibited the enzymic activity of Syk and that, in addition to the N-terminal 175 residues, a central portion of NS5A (aa 237-302) was required for inhibition of Syk. Moreover, Syk-mediated phosphorylation of phospholipase C-gamma1 was downregulated by NS5A. An interaction of NS5A with Syk was also detected in Huh-7.5 cells harbouring an HCV RNA replicon or infected with HCV. In conclusion, these results demonstrated that NS5A interacts with Syk resulting in negative regulation of its kinase activity. The results indicate that NS5A may be involved in the carcinogenesis of hepatocytes through the suppression of Syk kinase activities.
-
Activation of Syk protein tyrosine kinase in response to osmotic stress requires interaction with p21-activated protein kinase Pak2/γ-PAK
Molecular and cellular biology, 2004Co-Authors: S. M. Shahjahan Miah, Kiyonao Sada, Polygena T. Tuazon, Jun Ling, Koichiro Maeno, Shinkou Kyo, Yumi Tohyama, Jolinda A. Traugh, Hirohei YamamuraAbstract:The p21-activated serine/threonine protein kinase Pak2/γ-PAK and the nonreceptor type of protein tyrosine kinase Syk are known to be activated when the cells are exposed to osmotic stress. The purpose of the present study was to examine whether Pak2 and Syk functionally cooperate in cellular signaling. Cotransfection studies revealed that Pak2 associates with Syk in COS cells. The constitutively active form of Cdc42 increases the association of Pak2 with Syk. Pak2 coexpressed with an inactive form of Cdc42 or kinase-inactive Pak2 interacts to a lesser extent with Syk, suggesting that Pak2-Syk association is enhanced by Pak2 activation. Interaction with Pak2 enhances the intrinsic kinase activity of Syk. This is supported by in vitro studies showing that Pak2 phosphorylates and activates Syk. Treatment of cells with sorbitol to induce hyperosmolarity results in the translocation of Pak2 and Syk to the region surrounding the nucleus and in dramatic enhancement of their association. Furthermore, cotransfection of Pak2 and Syk leads to the activation of c-Jun N-terminal kinase (JNK) under hyperosmotic conditions. Pak2 short interfering RNA suppresses sorbitol-mediated activation of endogenous Syk and JNK, thus identifying a novel pathway for JNK activation by Cdc42. These results demonstrate that Pak2 and Syk positively cooperate to regulate cellular responses to stress.
-
Protein tyrosine kinase Syk in mast cell signaling.
Molecular immunology, 2002Co-Authors: Reuben P. Siraganian, Juan Zhang, Katsuhiro Suzuki, Kiyonao SadaAbstract: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.
-
relocation of Syk protein tyrosine kinase to the actin filament network and subsequent association with fak
FEBS Journal, 1997Co-Authors: Kiyonao Sada, Yasuhiro Minami, Hirohei YamamuraAbstract:Previous studies demonstrated that Syk protein-tyrosine kinase (Syk) is activated by thrombin in platelets. To elucidate the function of Syk in platelets, we have biochemically examined the intracellular location of Syk and the molecules associated with Syk, following platelet activation. In human platelets, thrombin induces the relocation of Syk to the cytoskeletal fraction presumably via Syk tyrosine phosphorylation. Relocated Syk is associated with the actin filament network, and the early phase (10–90 s) of this association can be partially inhibited by the pretreatment of platelets with cytochalasin D, an inhibitor of actin polymerization. Upon thrombin stimulation, Syk becomes associated with Fak as demonstrated by co-immunoprecipitation. The association of both kinases can be inhibited by pretreatment of platelets with cytochalasin D. Interestingly, reconstitution experiments, using COS cells transfected with various porcine Syk mutants, revealed that the kinase domain, but not the kinase activity, of Syk is required for the association of Syk with the actin filament network. These findings suggest that thrombin-induced association of Syk with Fak correlates with the state of actin polymerization, and may play an important role in platelet activation.
