Inhibition of tyrosine phosphorylation prevents T-cell receptor-mediated signal transduction.

The binding of antigen to the multicomponent T-cell receptor (TCR) activates several signal transduction pathways via coupling mechanisms that are poorly understood. One event that follows antigen receptor engagement is the activation of inositol phospholipid-specific phospholipase C (PLC). TCR activation by antigen, lectins, or anti-TCR monoclonal antibody has also been shown to cause increases in tyrosine phosphorylation of TCR-zeta and other substrates, suggesting stimulation of protein tyrosine kinase (PTK) activity. A critical question is whether these two pathways, PLC and PTK, are independently activated or whether one initiates and/or regulates the other. In the former case, PLC activation could be coupled to the TCR via a GTP-binding protein (G protein). We have reported, however, that tyrosine phosphorylation of intracellular substrates precedes detection of PLC activation and intracellular calcium elevation, suggesting that inositol phospholipid turnover in T cells is initiated by a PTK pathway. In this study, we test this hypothesis by treating T cells with the drug herbimycin A. We demonstrate that this agent inhibits substrate tyrosine phosphorylation, TCR-mediated inositol phospholipid hydrolysis, and calcium elevation. In contrast, under these conditions G-protein-mediated PLC activity, as tested by addition of aluminum fluoride, remains intact. Furthermore, whereas herbimycin treatment prevents TCR-mediated interleukin 2 production and interleukin 2 receptor expression, phorbol ester-induced effects are substantially resistant to herbimycin. The drug thus appears to abrogate TCR-mediated signaling without affecting distal signaling mechanisms.     

Tyrosine phosphorylation in platelets Potential roles in intracellular signal transduction

Tyrosine phosphorylation has been shown to play a critical role in the induction of cellular responses to extracellular stimuli in a wide variety of cell types. In platelets, many diverse agonists induce multiple waves of tyrosine phosphorylation, suggesting a potential role for these protein modifications throughout the platelet activation process. Tyrosine phosphorylation of several platelet proteins is regulated by fibrinogen binding to its integrin receptor and subsequent platelet aggregation, suggesting specific functions for tyrosine phosphorylation in integrin-regulated intracellular processes. In this article, we review the regulation of tyrosine phosphorylation and the role of tyrosine phosphorylation in platelet activation events.     

Tyrosine kinase signal transduction in rheumatoid synovitis.

Explants of synovial cells in rheumatoid arthritis display a transformed phenotype with focus formation and anchorage-independent growth. Many of the cytokines that activate these fibroblasts mediate their action through tyrosine kinase growth factor receptors. Mechanisms of signal transduction via such tyrosine kinases are therefore relevant to the pathogenesis of rheumatoid lesions. Data are presented using the neu oncogene product p185neu as a model system to explore signal transduction by receptor tyrosine kinases. Evidence is shown that increased tyrosine kinase activity in the oncogenic form of this protein may result from dimerization of the tyrosine kinase receptor. In the normal cellular counterpart of p185neu, dimerization appears to be mediated by the action of an as yet unidentified ligand. Dimerization also appears to be important in signal transduction mediated by epidermal growth factor, platelet-derived growth factor, and colony-stimulating factor 1. These cytokines also alter the phenotype of rheumatoid synovial fibroblasts to resemble transformed fibroblasts. Additionally, preliminary data that suggest increased tyrosine kinase activity in rheumatoid arthritis synovia compared with osteoarthritis synovia are presented. Molecular characterization of tyrosine kinase receptors will be an important direction for future studies of the pathogenesis of rheumatoid disease.     

Tyrosine phosphorylation of the erythropoietin receptor: role for differentiation and mitogenic signal transduction

The erythropoietin (Epo) receptor belongs to the cytokine receptor superfamily. Although the cytokine receptors do not possess a tyrosine kinase consensus sequence in the intracellular domain, rapid stimulation of a tyrosine kinase activity occurs after activation by the ligand. We and others have shown that Epo induces the tyrosine phosphorylation of its cognate receptor as well as phosphorylation of other proteins. In this report, we examined the role of the receptor tyrosine residues in signal transduction. Eight tyrosine residues are located within the intracellular domain of the murine Epo receptor. A single tyrosine residue is present in the region previously shown to be sufficient for proliferative signal transduction. This tyrosine (Tyr 343) was mutated to phenylalanine. Moreover, mutant receptors were also generated with either a tyrosine residue or a phenylalanine residue at position 343 and with a COOH terminal truncation that removed the 7 other tyrosine residues. Expression vectors carrying these mutated receptors were transfected into the interleukin-3-dependent murine cell line Ba/F3. Epo-induced growth was sustained efficiently by all these receptors, although receptors without any tyrosine residues conferred a significantly reduced mitogenic activity. Moreover, all receptors were able to mediate Epo-dependant accumulation of beta-globin mRNA. The mutated receptors all induced the tyrosine phosphorylation of several cellular proteins after Epo stimulation. However, the truncated receptors induced the phosphorylation of a reduced number of proteins, suggesting that phosphorylated tyrosines of the receptor could have a role in the recruitment either of a tyrosine kinase or of tyrosine kinase substrate proteins. The receptors were all able to mediate Epo- induced activation of phosphatidylinositol 3-kinase, although truncated receptors no longer bound phosphatidylinositol 3-kinase.     

Distinct regions of c-Mpl cytoplasmic domain are coupled to the JAK-STAT signal transduction pathway and Shc phosphorylation.

c-Mpl, a member of the hematopoietic cytokine receptor family, is the receptor for thrombopoietin. To investigate signal transduction by c-Mpl, a chimeric receptor, composed of the extracellular domain of human growth hormone receptor and the intracellular domain of c-Mpl, was introduced into the interleukin 3-dependent cell line Ba/F3. In response to growth hormone, this chimeric receptor induced growth in the absence of interleukin 3. Deletion analysis of the 123-amino acid intracellular domain indicated that the elements responsible for this effect are present within the 63 amino acids proximal to the transmembrane domain. Mutation of the recently described box 1 motif abrogated the proliferative response. Tyrosine phosphorylation of the tyrosine kinase JAK-2 and activation of STAT proteins were dependent on box 1 and sequences within 63 amino acids of the plasma membrane. STAT proteins activated by thrombopoietin in a megakaryocytic cell line were purified and shown to be STAT1 and STAT3. A separate region located at the C terminus of the c-Mpl intracellular domain was found to be required for induction of Shc phosphorylation and c-fos mRNA accumulation, suggesting involvement of the Ras signal transduction pathway. Thus, at least two distinct regions are involved in signal transduction by the c-Mpl.     

Insulin signal transduction: the role of protein phosphorylation.

Recent evidence suggests that the mechanism of insulin action depends in part on protein phosphorylation on tyrosine residues. A cascade of phosphorylation/dephosphorylation reactions is proposed to modulate multiple enzymes involved in metabolism, protein synthesis, and cell growth. Direct evidence is presented for the phosphorylation of myelin basic protein and microtubule-associated protein 2 on tyrosine residues by the insulin receptor.     

Redox regulation of signal transduction: tyrosine phosphorylation and calcium influx.

Studies presented here show that altering the intracellular redox balance by decreasing glutathione levels profoundly affects early signal transduction events in human T cells. In a T-cell receptor (TCR) signaling model, short-term pretreatment with buthionine sulfoximine, which specifically decreases intracellular glutathione, essentially abrogates the stimulation of calcium influx by anti-CD3 antibodies without significantly impairing other aspects of TCR-initiated signal transduction, such as overall levels of TCR-stimulated tyrosine phosphorylation. In an inflammatory-cytokine signaling model, the failure of tumor necrosis factor alpha to stimulate more than minimal tyrosine phosphorylation in lymphocytes is overcome by buthionine sulfoximine pretreatment--i.e., tumor necrosis factor alpha stimulates extensive tyrosine phosphorylation in glutathione-depleted lymphocytes. These redox-dependent changes in T-cell responsiveness suggest that the glutathione deficiency that we and others have demonstrated in human immunodeficiency virus-infected individuals may contribute significantly to the immunodeficiency and the increased inflammatory reactions in these individuals.     

Conserved aspartate residues and phosphorylation in signal transduction by the chemotaxis protein CheY.

The CheY protein is phosphorylated by CheA and dephosphorylated by CheZ as part of the chemotactic signal transduction pathway in Escherichia coli. Phosphorylation of CheY has been proposed to occur on an aspartate residue. Each of the eight aspartate residues of CheY was replaced by using site-directed mutagenesis. Substitutions at Asp-12, Asp-13, or Asp-57 resulted in loss of chemotaxis. Most of the mutant CheY proteins were still phosphorylated by CheA but exhibited modified biochemical properties, including reduced ability to accept phosphate from CheA, altered phosphate group stability, and/or resistance to CheZ-mediated dephosphorylation. The properties of CheY proteins bearing a substitution at position 57 were most aberrant, consistent with the hypothesis that Asp-57 is the normal site of acyl phosphate formation. Evidence for an alternate site of phosphorylation in the Asp-57 mutants is presented. Phosphorylated CheY is believed to cause tumbling behavior. However, a dominant mutant CheY protein that was not phosphorylated in vitro caused tumbling in vivo in the absence of CheA. This phenotype suggests that the role of phosphorylation in the wild-type CheY protein is to stabilize a transient conformational change that can generate tumbling behavior.     

Tyrosine phosphorylation of GluK2 up-regulates kainate receptor-mediated responses and downstream signaling after brain ischemia

Although kainate receptors play important roles in ischemic stroke, the molecular mechanisms underlying postischemic regulation of kainate receptors remain unclear. In this study we demonstrate that Src family kinases contribute to the potentiation of kainate receptor function. Brain ischemia and reperfusion induce rapid and sustained phosphorylation of the kainate receptor subunit GluK2 by Src in the rat hippocampus, implicating a critical role for Src-mediated GluK2 phosphorylation in ischemic brain injury. The NMDA and kainate receptors are involved in the tyrosine phosphorylation of GluK2. GluK2 binds to Src, and the tyrosine residue at position 590 (Y590) on GluK2 is a major site of phosphorylation by Src kinases. GluK2 phosphorylation at Y590 is responsible for increases in whole-cell currents and calcium influx in response to transient kainate stimulation. In addition, GluK2 phosphorylation at Y590 facilitates the endocytosis of GluK2 subunits, and the activation of JNK3 and its substrate c-Jun after long-term kainate treatment. Thus, Src phosphorylation of GluK2 plays an important role in the opening of kainate receptor channels and downstream proapoptosis signaling after brain ischemia. The present study reveals an additional mechanism for the regulation of GluK2-containing kainate receptors by Src family kinases, which may be of pathological significance in ischemic stroke.Kainate receptors are widely expressed in the mammalian central nervous system, particularly in the hippocampus, where they are involved in synaptic transmission (1), neuronal plasticity (2), and excitotoxic lesions (3). Overactivation of postsynaptic kainate receptor-mediated responses is associated with neurological disorders resulting from ischemic stroke (4). However, the intracellular processes responsible for the postischemic up-regulation of kainate receptors, and its molecular consequences, have not yet been elucidated.Reversible phosphorylation is one of the most common mechanisms regulating the function of receptor proteins. In particular, serine/threonine phosphorylation is important in the functional regulation of NMDA (5), AMPA (6), and kainate receptors (6–9), with tyrosine phosphorylation of NMDA receptors the most extensively studied (10, 11). There is accumulating evidence to show that tyrosine phosphorylation of NMDA receptors modulates their assembly at synapses after brain ischemia (12–15). However, less is known about the regulation of kainate receptor activity by tyrosine phosphorylation. Src is an important member of Src family kinases, the largest family of nonreceptor protein tyrosine kinases, and is highly expressed in the brain. Brain ischemia increases Src kinase activity in vulnerable brain regions, including the hippocampus (15–18), but it is not known whether Src phosphorylates kainate receptors.Kainate receptors are tetrameric glutamate-gated ion channels consisting of GluK1–GluK5 subunits, formerly known as GluR5–GluR7, KA1, and KA2, respectively. Functional kainate receptors can be expressed as homomers and heteromers of GluK1–3 subunits, whereas GluK4 and GluK5 subunits combine with GluK1–3 to form functional channels. It has been reported that GluK2-deficient mice are resistant to kainic acid-induced neuronal degeneration and seizures (19), and GluK2 knockdown protects against postischemic neuronal loss in the rat hippocampal CA1 region (20). In addition to sodium and potassium ions, GluK2-containing kainate receptors are permeable to Ca²⁺ (21, 22). Glutamate-induced intracellular Ca²⁺ ([Ca²⁺]_i) overload is a major mechanism underlying excitotoxicity and ischemic cell death. Furthermore, excessive activation of GluK2-containing kainate receptors triggers the proapoptotic JNK signal cascade, which contributes to ischemic brain damage (23, 24). These findings suggest that GluK2-containing kainate receptor-mediated responses are critical events in the induction of neuronal cell death after stroke.In this study we found that Src family kinases are involved in kainate-evoked whole-cell currents. In the vulnerable hippocampal CA1 region, GluK2 is phosphorylated on tyrosine 590 (Y590) by Src family kinases after brain ischemia. Conversely, the mutation of the Y590 residue on GluK2 decreases whole-cell peak currents and [Ca²⁺]_i increases elicited by kainate, and deficiency of GluK2 phosphorylation at Y590 attenuates the endocytosis of GluK2 subunits and JNK3–c-Jun activation in response to kainate. These data indicate that Src-mediated phosphorylation promotes the opening of GluK2-containing kainate receptor channels and facilitates GluK2–JNK3 signaling. Our results contribute to the elucidation of molecular mechanisms underlying brain ischemic excitotoxicity.     

Anandamide, an endogenous cannabimimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor-mediated signal transduction.

Arachidonylethanolamide (anandamide), a candidate endogenous cannabinoid ligand, has recently been isolated from porcine brain and displayed cannabinoid-like binding activity to synaptosomal membrane preparations and mimicked cannabinoid-induced inhibition of the twitch response in isolated murine vas deferens. In this study, anandamide and several congeners were evaluated as cannabinoid agonists by examining their ability to bind to the cloned cannabinoid receptor, inhibit forskolin-stimulated cAMP accumulation, inhibit N-type calcium channels, and stimulate one or more functional second messenger responses. Synthetic anandamide, and all but one congener, competed for [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the rat cannabinoid receptor. The ability of anandamide to activate receptor-mediated signal transduction was evaluated in Chinese hamster ovary (CHO) cells expressing the human cannabinoid receptor (HCR, termed CHO-HCR cells) and compared to control CHO cells expressing the muscarinic m5 receptor (CHOm5 cells). Anandamide inhibited forskolin-stimulated cAMP accumulation in CHO-HCR cells, but not in CHOm5 cells, and this response was blocked with pertussis toxin. N-type calcium channels were inhibited by anandamide and several active congeners in N18 neuroblastoma cells. Anandamide stimulated arachidonic acid and intracellular calcium release in both CHOm5 and CHO-HCR cells and had no effect on the release of inositol phosphates or phosphatidylethanol, generated after activation of phospholipase C and D, respectively. Anandamide appears to exhibit the essential criteria required to be classified as a cannabinoid/anandamide receptor agonist and shares similar nonreceptor effects on arachidonic acid and intracellular calcium release as other cannabinoid agonists.     

Unique allosteric regulation of 5-hydroxytryptamine receptor-mediated signal transduction by oleamide

The effects of oleamide, an amidated lipid isolated from the cerebrospinal fluid of sleep-deprived cats, on serotonin receptor-mediated responses were investigated in cultured mammalian cells. In rat P11 cells, which endogenously express the 5-hydroxytryptamine_2A (5HT_2A) receptor, oleamide significantly potentiated 5HT-induced phosphoinositide hydrolysis. In HeLa cells expressing the 5HT₇ receptor subtype, oleamide caused a concentration-dependent increase in cAMP accumulation but with lower efficacy than that observed by 5HT. This effect was not observed in untransfected HeLa cells. Clozapine did not prevent the increase in cAMP elicited by oleamide, and ketanserin caused an ≈65% decrease. In the presence of 5HT, oleamide had the opposite effect on cAMP, causing insurmountable antagonism of the concentration-effect curve to 5HT, but had no effect on cAMP levels elicited by isoproterenol or forskolin. These results indicate that oleamide can modulate 5HT-mediated signal transduction at different subtypes of mammalian 5HT receptors. Additionally, our data indicate that oleamide acts at an apparent allosteric site on the 5HT₇ receptor and elicits functional responses via activation of this site. This represents a unique mechanism of activation for 5HT G protein-coupled receptors and suggests that G protein-coupled neurotransmitter receptors may act like their iontropic counterparts (i.e., γ-aminobutyric acid type A receptors) in that there may be several binding sites on the receptor that regulate functional activity with varying efficacies.     

Prolactin receptor signal transduction.

Within the immune system, multiple isoforms of the human prolactin receptor (PRLr) serve to mediate the effects of its ligand (PRL). Now numbering four, these isoforms are structurally and functionally distinct, demonstrating significant differences in ligand affinities, kinetics of transduction and the transduction proteins activated. The proximal transduction pathways activated during PRLr-associated signaling include the tyrosine kinases Jak2, Fyn and Tec, the phosphatase SHP-2, the guanine nucleotide exchange factor Vav, and the signaling suppressor SOCS. Differential activation of these pathways may contribute to the pleiotropism of PRL action in tissues of the immune system.     

Insulin signal transduction pathways.

Insulin initiates its pleiotropic effects by activating the insulin receptor tyrosine kinase to phosphorylate several intracellular proteins. Recent studies have demonstrated that phosphotyrosine residues bind specifically to proteins that contain src homology 2 (SH2) domains, and that this interaction mediates the regulation of multiple intracellular signaling pathways. This article reviews recent progress in elucidating the detailed pathways that lead from the insulin receptor to the ultimate biologic actions of insulin.     

Activin signal transduction pathways.

Many of the signal transduction pathways required for mammalian endocrine cell function are conserved from flies and worms. These model organisms permitted the illumination of the biological properties of ligands and provided systems in which cellular coactivating molecules could be identified rapidly. Our knowledge about the activin signaling components has been advanced tremendously by the work carried out in these systems. Subsequent research is beginning to reveal the complex interactions that serve to regulate this signaling pathway.     

Tyrosine phosphorylation in the human duodenum.

Many growth factor receptors including the epidermal growth factor receptor function through tyrosine kinase activity. The aim of this study was to examine the constitutive level of tyrosine phosphorylation in the normal duodenum and in the hyperproliferative coeliac duodenum. A flow cytometric assay was devised using monoclonal antibody to phosphorylated (but not native) tyrosine residues to determine the levels of tyrosine phosphorylation in both CD3 positive intraepithelial lymphocytes and CD3 negative epithelial cells obtained by EDTA treatment of endoscopically obtained duodenal biopsy specimens. In addition, immunohistochemistry was performed on 18 formalin fixed coeliac duodenal biopsy specimens and eight control specimens. Tyrosine phosphorylation could be detected by flow cytometry on duodenal enterocytes and this expression was up regulated by pretreatment with epidermal growth factor. Tyrosine phosphorylation decreased with progression from the villus to the crypt, however, and was virtually undetectable on crypt enterocytes. Immunohistochemistry of the coeliac duodenum showed virtually absent tyrosine phosphorylation in the crypt. Increased tyrosine phosphorylation was detected in the infiltrating T cells. In conclusion, tyrosine phosphorylation in the duodenum is confined to the non-proliferative villous epithelium and is virtually undetectable in the proliferative crypt compartment. These findings suggest that tyrosine kinase activity is not a significant factor in the regulation of crypt cell proliferation in the human duodenum either in normal subjects or in coeliac disease patients.     

The effects of dasatinib on IgE receptor-dependent activation and histamine release in human basophils

Dasatinib is a multitargeted drug that blocks several tyrosine kinases. Apart from its well-known antileukemic activity, the drug has attracted attention because of potential immunosuppressive and anti-inflammatory effects. We report that dasatinib at 1 microM completely blocks anti-IgE-induced histamine release in blood basophils in healthy donors, and allergen-induced release of histamine in sensitized individuals. In addition, dasatinib inhibited FcepsilonRI-mediated release of IL-4 and IgE-mediated up-regulation of CD13, CD63, CD164, and CD203c in basophils. The effects of dasatinib were dose-dependent (IC(50): 50-500 nM) and specific for FcepsilonRI activation in that the drug failed to inhibit C5a-induced or Ca-ionophore-induced histamine release. Interestingly, at lower concentrations, dasatinib even promoted FcepsilonRI-dependent histamine release in basophils in allergic subjects. In consecutive studies, dasatinib was found to interact with and block several FcepsilonRI downstream targets in basophils, including Btk. Correspondingly, FcepsilonRI-mediated histamine secretion in basophils was markedly reduced in Btk knockout mice and in a patient with Btk deficiency. However, the remaining "low-level" mediator secretion in Btk-deficient cells was fully blocked down again by 1 muM dasatinib. Together, these data suggest that dasatinib inhibits FcepsilonRI-mediated activation of basophils through multiple signaling molecules including Btk. Dasatinib may be an interesting agent for immunologic disorders involving Btk-dependent responses or/and FcepsilonRI activation of basophils.     

Tyrosine phosphorylation provides an early and requisite signal for the activation of natural killer cell cytotoxic function.

Natural killer (NK) cells are a unique subpopulation of lymphocytes with the capability to kill malignant cells via one of two alternative mechanisms: (i) Fc receptor-dependent cytotoxicity against antibody-coated targets or (ii) direct cell-mediated cytotoxicity. However, the molecular mechanisms that trigger and subsequently regulate NK cell cytotoxicity are incompletely understood. We have therefore investigated the role of protein tyrosine phosphorylation in the transmembrane signaling initiated after Fc receptor stimulation or direct tumor cell contact in clonal CD16+/CD3- human NK cells. We report that stimulation of the Fc receptor rapidly induced the tyrosine phosphorylation of a number of NK cell proteins. These effects occurred within 2 min, were maximal at 10 min, and declined toward baseline after 60 min. In addition, Fc receptor ligation increased the in vitro protein kinase activity of NK cell phosphotyrosyl proteins. We have also demonstrated that direct contact of NK cells with K562 tumor cells induced the rapid phosphorylation of distinct NK cell phosphotyrosyl proteins. Furthermore, the protein-tyrosine kinase inhibitor herbimycin A blocked NK cell cytotoxic function in a concentration-dependent manner. Our results suggest that protein tyrosine phosphorylation is an obligatory early proximal signal in activating the cytotoxic function of NK cells.     

The chemistry of signal transduction.

Several disciplines, including chemical ecology, seek to understand the molecular basis of information transfer in biological systems, and general molecular strategies are beginning to emerge. Often these strategies are discovered by a careful analysis of natural products and their biological effects. Cyclosporin A, FK506, and rapamycin are produced by soil microorganisms and are being used or considered as clinical immunosuppressive agents. They interrupt the cytoplasmic portion of T-cell signaling by forming a complex with a binding protein--FKBP12 in the case of FK506 and rapamycin and cyclophilin A (CyPA) in the case of cyclosporin A (CsA). This complex in turn inhibits a protein target, and the best understood target is calcineurin, which is inhibited by FK506-FKBP12 and CyPA-CsA. Mutational and structural studies help define how FK506-FKBP12 interacts with calcineurin, and the results of these studies are summarized. The existence of strong FK506-FKBP12 binding suggests that FK506 is mimicking some natural ligand for FKBP12. Synthetic and structural studies to probe this mimicry are also described.