EphrinB2 induces tyrosine phosphorylation of NR2B via Src-family kinases during inflammatory hyperalgesia

In recent years a role for EphB receptor tyrosine kinases and their ephrinB ligands in activity-dependent synaptic plasticity in the CNS has been identified. The aim of the present study was to test the hypothesis that EphB receptor activation in the adult rat spinal cord is involved in synaptic plasticity and processing of nociceptive inputs, through modulation of the function of the glutamate ionotropic receptor NMDA (N-methyl-D-aspartate). In particular, EphB receptor activation would induce phosphorylation of the NR2B subunit of the NMDA receptor by a Src family non-receptor tyrosine kinase. Intrathecal administration of ephrinB2-Fc in adult rats, which can bind to and activate EphB receptors and induce behavioral thermal hyperalgesia, led to NR2B tyrosine phosphorylation, which could be blocked by the Src family kinase inhibitor PP2. Furthermore animals pre-treated with PP2 did not develop behavioral thermal hyperalgesia following EphrinB2-Fc administration, suggesting that this pathway is functionally significant. Indeed, EphB1-Fc administration, which competes with the endogenous receptor for ephrinB2 binding and prevents behavioral allodynia and hyperalgesia in the carrageenan model of inflammation, also inhibited NR2B phosphorylation in this model. Taken together these findings support the hypothesis that EphB-ephrinB interactions play an important role in NMDA-dependent, activity-dependent synaptic plasticity in the adult spinal cord, inducing the phosphorylation of the NR2B subunit of the receptor via Src family kinases, thus contributing to chronic pain states.     

The Syk family of protein tyrosine kinases in T-cell activation and development

Summary: The processes of T-cell development and activation employ similar immature and mature receptors as well as similar signal transduction pathways to achieve different outcomes. Many signaling molecules are shared between [he receptor signaling pathways, including two families of cytoplasmic protein tyrosine kinases, the Src family and the Syk family. The two Syk family members expressed in T cells, Syk and ZAP-70, are structurally similar but are expressed at different times during rhymic development and during T-cell activation. These two kinases, althongh they share many physical features, differ in terms of biochemical activity and regulation. We discuss the overlapping and distinct characteristics of Syk and ZAP-70 in T-cell signaling and the potential biological importance of their differences.     

Essential role for platelet-activating factor-induced NF-kappaB activation in macrophage-derived angiogenesis

Activated monocyte-macrophages have been implicated in tumor angiogenesis via their capacity to produce many potent angiogenic factors. However, the mechanisms leading to production of these angiogenic factors in macrophages remain to be elucidated. In this study, we demonstrated by use of a mouse Matrigel implantation model that mouse peritoneal macrophages induce angiogenesis. mRNA expression and protein synthesis of macrophage-derived crucial angiogenic factors such as IL-1, TNF-alpha, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF) were blocked by platelet-activating factor (PAF) receptor antagonists. It was also observed that inhibitors of NF-kappaB blocked macrophage production of these angiogenic factors. Gene expression and protein synthesis of the angiogenic factors cited above were also inhibited in IkappaBalpha-mutated macrophages. VEGF is the most potent angiogenic factor in macrophage-induced angiogenesis. PAF antagonists or NF-kappaB inhibitors also inhibit the capacity of conditioned medium from LPS-stimulated human peripheral blood monocytes to induce sprouting of porcine pulmonary arterial endothelial cells. These data indicate that PAF-induced NF-kappaB activation is a common upstream pathway leading to the production of crucial macrophage-derived angiogenic factors. This will provide an important clue for a better understanding of mechanisms involved in tumor angiogenesis.     

The role of G-proteins versus protein tyrosine kinases in the regulation of lymphocyte activation.

The relative roles of G-proteins and protein tyrosine kinases (PTKs) in the regulation of antigen receptor-mediated signalling in B and T cells is controversial. As they, and the biochemical events they control, are potential targets for intervention in various immune dysfunctions, the resolution of the controversy is of great interest. Here, Margaret Harnett and Kevin Rigley provide a timely assessment of current understanding, and propose a model for the interaction of G-proteins and PTKs in antigen receptor-mediated signalling.     

Src-family kinases: rheostats of immune cell signaling

In immune cells, Src-family kinases have been implicated as critical regulators of a large number of intracellular signaling pathways. Studies of gene knockout mice lacking various family members have both confirmed the requirement for these kinases in some pathways and provided surprising insight into their roles in other pathways. In many cases, loss of a single kinase produces only a modest/moderate defect in the pathway being studied, while combinatorial mutants have more profound defects. In several cases, loss of Src-family kinase activity results in activation of signaling, illuminating the fact that these molecules play important roles in inhibitory signaling. This review will focus on both the positive and negative roles these kinases play in immunoreceptor and integrin signaling pathways. The overall observation that these molecules play such diverse signaling functions leads one to the conclusion that this family of proteins serves as general modulators of immune cell signaling.     

Sustained activation of Src-family tyrosine kinases by ischemia: a potential mechanism mediating extracellular signal-regulated kinase cascades in hippocampal dentate gyrus

In the present report, we investigated the association between the sustained activation of Src family tyrosine kinases (primarily Src kinase) with the biphasic phosphorylation of extracellular signal-regulated kinase (ERK) induced by ischemia in the rat hippocampal CA3/dentate gyrus subfield. Post-ischemia reperfusion resulted in the phosphorylation of ERK in a Ras-dependent manner; down-regulation of NMDA receptors or Src family protein kinases by ketamine or 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) potently antagonized the activation of ERK, indicating that NMDA receptors and Src family tyrosine kinases are essential for the up-regulation of ERK activity following ischemic stimuli. Additionally, an ischemia-induced association between RKIP and Raf-1 resulted in the inhibition of the ERK signaling cascade through an inhibition of Src-mediated Raf-1 phosphorylation at Tyr340/341 residues. This ischemia-induced inhibition of ERK was not associated with other downstream pathways involving Raf-1 phosphorylation at Ser 259 elicited by protein kinase B (Akt). Dissociation of Raf-1 from RKIP by 24 h reperfusion or (4S)-3-[(E)-but-2-enoyl]-4-benzyl-2-oxazolidinone (locostatin) influenced the second phase of ERK activation elicited by the Src-Raf cassette. We propose that, following ischemia, the Src family tyrosine kinases are critical for modulation of the Ras/Raf/MEK/ERK cascade, in which RKIP is involved in biphasic phosphorylation of ERK via a blockade of Src-Raf cascades.     

The role of the protein tyrosine phosphatase CD45 in regulation of B lymphocyte activation

The transmembrane protein tyrosine phosphatase CD45 is expressed throughout B cell development and differentiation, with the exception of terminally differentiated plasma cells on which its expression is down regulated. Numerous studies using CD45-deficient B cell lines and CD45-deficient mice have clearly demonstrated that CD45 plays an important role in modulating the signal that is transduced via the B cell antigen receptor by regulating the phosphorylation state of Src family kinases. Spatial and temporal controls enable CD45 to promote B cell antigen receptor signal transduction by constitutively maintaining Src family kinases in a partially active state, such that the B cell is able to effectively respond to an antigenic challenge. Moreover, CD45 is required for optimal activation of Ca2+-dependent and MAP kinase-dependent signal transduction pathways in the B cell. The net result is that CD45 affects the B cell response by controlling the relative threshold of sensitivity to a given antigenic stimulus. Thus, CD45 expression and function is required for normal B cell development, tolerance induction, and responsiveness to antigen.     

Involvement of protein tyrosine kinases in activation of human eosinophils by platelet-activating factor

Activation of human eosinophils by platelet-activating factor (PAF) involves multiple signal transduction pathways. Among these, protein kinase C has been demonstrated both to mediate respiratory burst and to suppress an alternative pathway of activation of respiratory burst and arachidonic acid metabolism in eosinophils. We utilized inhibitors of protein tyrosine kinases (PTK) to elucidate the role of PTK in PAF-induced activation of eosinophils. Eosinophils were isolated from peripheral blood of atopic donors and stimulated with PAF in the absence or presence of broad-spectrum PTK inhibitors-genistein or lavendustin A; an inhibitor of mitogen-activated protein (MAP) kinase activation-tyrphostin AG126; or an inhibitor of Janus kinase 2 (Jak2)-tyrphostin B42 (AG490). PAF induced superoxide anion (O2-*) generation, leukotriene C4 (LTC4) release, intracellular calcium ion mobilization and tyrosine phosphorylation of multiple eosinophil proteins in a concentration-dependent manner. All of these responses were concentration-dependently inhibited by genistein; lavendustin A also exhibited potent inhibition of PAF-induced LTC4 release. AG126 had no effect on either O2-* generation or LTC4 release, while AG490 inhibited both responses, albeit less effectively than genistein. We conclude that PAF activates PTK in human eosinophils and that this signalling pathway is involved in eliciting respiratory burst and leukotriene production. The specific PTK(s) involved are unknown but may include Jak2.     

Redundant role for Zap70 in B cell development and activation

Expression of the Syk family tyrosine kinase Zap70 is strongly correlated with poor clinical outcome in chronic lymphocytic leukemia, the most common human leukemia characterized by B cell accumulation. The expression of Zap70 may reflect the specific cell of origin of the tumor or may contribute to pathology. Thus, the normal role of Zap70 in B cell physiology is of great interest. While initial studies reported that Zap70 expression in the mouse was limited to T and NK cells, more recent work has shown expression in early B cell progenitors and in splenic B cells, suggesting that the kinase may play a role in the development or activation of B cells. In this study, we show that Zap70 is expressed in all developing subsets of B cells as well as in recirculating B cells, marginal zone B cells and peritoneal B1 cells. Analysis of Zap70-deficient mice shows no unique role for Zap70 in either the development of B cells or in their in vitro and in vivo activation. However, we show that Zap70 can rescue the defective positive selection of immature B cells into the recirculating pool in Syk-deficient mice, demonstrating functional redundancy between these two kinases.     

酪氨酸家族激酶在大鼠脊髓背角LTP中的作用及其机制

目的: 探讨酪氨酸家族激酶(SFKs)在大鼠脊髓背角C纤维诱发电位的长时程增强(LTP)中的作用及其机制。 方法: 用在体电生理方法检测SFKs抑制剂对高频刺激坐骨神经诱导的脊髓背角LTP的影响;用Western blotting方法检测高频刺激诱导脊髓LTP的不同时点大鼠脊髓背角磷酸化SFKs的表达情况;用免疫荧光双染方法检测高频刺激诱导脊髓LTP后大鼠脊髓背角磷酸化SFKs表达的细胞学定位。 结果: SFKs的抑制剂(PP2或SU6656)可以完全阻断LTP的诱导,甚至将LTP逆转为长时程抑制(LTD);高频电刺激大鼠坐骨神经诱导脊髓LTP后15 min开始,刺激同侧的脊髓背角中磷酸化SFKs表达明显增加,并且这些高表达的磷酸化SFKs仅仅存在于脊髓小胶质细胞中。 结论: 在脊髓背角,小胶质细胞中的SFKs 是诱导LTP的必要条件,抑制SFKs及其下游分子可能有助于治疗病理性疼痛。     

Role of protein tyrosine phosphatases in T cell activation

The last decade has seen an exponentially increasing interest in the molecular mechanisms of signal transduction. In T cells, much of the focus has been on protein tyrosine kinase (PTK)‐mediated signaling from the T cell receptor (TCR) and cytokine receptors, while the study of protein tyrosine phosphatases (PTPases) has lagged behind. However, recent discoveries have revealed that several PTPases play important roles in many different aspects of T cell physiology. We predict that the phosphatases will become a ‘hot topic’ in the field within the next few years. This review summarizes the current understanding of the regulation and biology of PTPases in T lymphocyte activation.     

Essential role of LAT in T cell development

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement. Phosphorylated LAT binds many critical signaling molecules. The central role of this molecule in TCR-mediated signaling has been demonstrated by experiments in a LAT-deficient cell line. To probe the role of LAT in T cell development, the LAT gene was disrupted by targeting. LAT-deficient mice appeared healthy. Flow cytometric analysis revealed normal B cell populations but the absence of any mature peripheral T cells. Intrathymic development was blocked within the CD4- CD8- stage. No gross abnormality of NK or platelet function was observed. LAT is thus critical to both T cell activation and development.     

Myocardial nuclear protein kinases during postnatal development

Chromatin template activity of isolated rat myocardial cells declines progressively during the first few weeks of postnatal life, a time period also characterized by restriction in myocardial proliferative capacity. Multiple protein kinases have been isolated from the nuclei of myocardial cells. Protein kinase activity decreased progressively during the first 3 wk of postnatal development, mostly in two fractions (III and V). Cyclic AMP dependency, substrate specificity, and phosphorylation patterns of endogenous substrates did not change appreciably with age. Higher phosphorylating activity of protein kinases from younger rats correlated with increased capacity to enhance myocardial RNA synthesis in vitro. This suggests the possibility that the decrease in chromatin template activity with age may be, in part, related to the decline in nuclear phosphokinase activities.     

Expression of protein tyrosine kinases in head and neck squamous cell carcinomas

Protein tyrosine kinases (TKs) are overexpressed in many carcinomas and sarcomas. We studied the expression of the following TKs in head and neck squamous cell carcinoma (HNSCC): platelet-derived growth factor receptor (PDGFR), c-kit, epidermal growth factor receptor (EGFR), and a serine-threonine kinase, Akt. Formalin-fixed, paraffin-embedded tumor blocks from 44 consecutive patients with primary HNSCC and 5 specimens of benign pharyngeal and laryngeal mucosa were retrieved for immunohistochemical analysis. Of the specimens, 38 had enough material to stain for all 4 antibodies. The study included 21 pharyngeal (base of tongue, 14; tonsil, 6; soft palate, 1), 16 laryngeal, and 1 floor of the mouth carcinoma. All 4 kinases in the tumor samples were expressed highly (PDGFR, 95%-100%; EGFR, 38%-43%; c-kit, 50%-86%; p-Akt, 57%-81%), with EGFR, c-kit, and p-Akt significantly higher than in benign samples. None of the kinase expressions correlated with disease-free survival. The expression of the kinases raises the possibility of treatment of HNSCC by tyrosine and serine-threonine kinase inhibitors.     

Rethinking the role of Src family protein tyrosine kinases in the allergic response

Antigen-induced cross-linking of immunoglobulin E (IgE) antibodies bound to the high-affinity IgE receptor (FcepsilonRI), on mast cells results in the release of mediators that initiate an inflammatory response. This normal immune response has been abducted by immunological adaptation, through the production of IgE antibodies to normally innocuous substances, to cause allergic disease. Therefore, understanding the molecular requirements in IgE-dependent mast-cell activation holds promise for therapeutic intervention in disease. Recent investigation on the functional coupling of FcepsilonRI to the intracellular signaling apparatus has provided paradigm-altering insights on the importance and function of Src family protein tyrosine kinases (Src PTK) in mast-cell activation. In this synopsis, we review the current knowledge on the role of the Src PTKs, Fyn and Lyn, in mast-cell activation and discuss the implications of our findings on allergic disease.     

Essential role of the adaptor protein Nck1 in Jurkat T cell activation and function

The non-catalytic region of tyrosine kinase (Nck) is proposed to play an essential role in T cell activation. However, evidence based on functional and biochemical studies has brought into question the critical function of Nck. Therefore, the aim of the present work was to investigate the role of Nck in T cell activation. To study this, the human Jurkat T cell line was used as a model for human T lymphocytes. The short interfering (si) RNA targeting Nck1 gene was used with electroporation to knock-down Nck1 protein expression in Jurkat T cells. Primary human CD4 T cells were also transfected with the siRNA of Nck1. The results showed that decreased Nck1 protein expression did not affect the apoptosis of the transfected Jurkat T cells compared with control siRNA-transfected cells and non-transfected cells. Upon CD3ε/CD28 stimulation, knock-down of Nck1 in Jurkat T cells caused a decrease in CD69 expression and in interleukin (IL)-2 secretion. Similarly, knock-down of Nck1 in primary CD4 T cells also caused decreased CD69 expression. However, no significant alterations of CD69 and IL-2 expression were found upon phytohaemagglutinin (PHA)/phorbol myristate acetate (PMA) stimulation. Knock-down of Nck1 had no effect on the proliferation of Jurkat T cells stimulated with either PHA or anti-T cell receptor (TCR) monoclonal antibody (C305). The reduced Nck1 expression in Jurkat cells was also associated with a reduced phosphorylation of extracellular regulated kinase (Erk)1 and Erk2 proteins upon CD3ε/CD28 stimulation. In conclusion, the decreased Nck1 protein in Jurkat T cells resulted in an impairment of TCR-CD3-mediated activation involving a defective Erk phosphorylation pathway.