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PubMed2001

Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel.

Tyrosine side chains participate in several distinct signaling pathways, including phosphorylation and membrane trafficking. A nonsense suppression procedure was used to incorporate a caged tyrosine residue in place of the natural tyrosine at position 242 of the inward rectifier channel Kir2.1 expressed in Xenopus oocytes. When tyrosine kinases were active, flash decaging led both to decreased K(+) currents and also to substantial (15-26%) decreases in capacitance, implying net membrane endocytosis. A dominant negative dynamin mutant completely blocked the decaging-induced endocytosis and partially blocked the decaging-induced K(+) channel inhibition. Thus, decaging of a single tyrosine residue in a single species of membrane protein leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited. Oocyte membrane proteins were also labeled with the thiol-reactive fluorophore tetramethylrhodamine-5-maleimide, and manipulations that decreased capacitance also decreased surface membrane fluorescence, confirming the net endocytosis. In single-channel studies, tyrosine kinase activation decreased the membrane density of active Kir2.1 channels per patch but did not change channel conductance or open probability, in agreement with the hypothesis that tyrosine phosphorylation results in endocytosis of Kir2.1 channels. Despite the Kir2.1 inhibition and endocytosis stimulated by tyrosine kinase activation, neither Western blotting nor (32)P labeling produced evidence for direct tyrosine phosphorylation of Kir2.1. Therefore, it is likely that tyrosine phosphorylation affects Kir2.1 function indirectly, via interactions between clathrin adaptor proteins and a tyrosine-based sorting motif on Kir2.1 that is revealed by decaging the tyrosine side chain. These interactions inhibit a fraction of the Kir2.1 channels, possibly via direct occlusion of the conduction pathway, and also lead to endocytosis, which further decreases Kir2.1 currents. These data establish that side chain decaging can provide valuable time-resolved data about intracellular signaling systems.

Animals↗
PubMed2004

The inhibitory effects of isoflurane on protein tyrosine phosphorylation-modulated contraction of rat aortic smooth muscle.

BACKGROUND: Tyrosine kinase-catalyzed protein tyrosine phosphorylation plays an important role in initiating and modulating vascular smooth muscle contraction. The aim of the current study was to examine the effects of isoflurane on sodium orthovanadate (Na3VO4), a potent protein tyrosine phosphatase inhibitor-induced, tyrosine phosphorylation-mediated contraction of rat aortic smooth muscle. METHODS: The Na3VO4-induced contraction of rat aortic smooth muscle and tyrosine phosphorylation of proteins including phospholipase Cgamma-1 (PLCgamma-1) and p44/p42 mitogen-activated protein kinase (MAPK) were assessed in the presence of different concentrations of isoflurane, using isometric force measurement and Western blotting methods, respectively. RESULTS: Na3VO4 (10(-4) m) induced a gradually sustained contraction and significant increase in protein tyrosine phosphorylation of a set of substrates including PLCgamma-1 and p42MAPK, all of which were markedly inhibited by genistein (5 x 10(-5) m), a tyrosine kinase inhibitor. Isoflurane (1.2-3.5%) dose-dependently depressed the Na3VO4-induced contraction (P < 0.05-0.005; n = 8). Isoflurane also attenuated the total density of the Na3VO4-induced, tyrosine-phosphorylated substrate bands and the density of tyrosine-phosphorylated PLCgamma-1 band and p42MAPK band (P < 0.05-0.005; n = 4) in a concentration-dependent manner. CONCLUSION: The findings of the current study, that isoflurane dose-dependently inhibits both the Na3VO4-stimulated contraction and tyrosine phosphorylation of a set of proteins including PLCgamma-1 and p42MAPK in rat aortic smooth muscle, suggest that isoflurane depresses protein tyrosine phosphorylation-modulated contraction of vascular smooth muscle, especially that mediated by the tyrosine-phosphorylated PLCgamma-1 and MAPK signaling pathways.

Anesthetics, Inhalation↗
PubMed1995

TrkA tyrosine residues involved in NGF-induced neurite outgrowth of PC12 cells.

The proto-oncogene product gp140prototrk (TrkA) is the receptor tyrosine kinase that mediates nerve growth factor-induced neuronal survival and differentiation. In receptor tyrosine kinases, specific intracellular tyrosine residues become phosphorylated after ligand binding and the phosphorylated tyrosines induce the cascade of signal transduction. Here we have identified intracellular tyrosine residues of TrkA involved in nerve growth factor-induced neurite outgrowth of PC12 cells, using site-directed mutagenesis and a PC12 cell line expressing very low levels of endogenous TrkA (PC12nnr5 cells). We analysed eight conserved intracellular tyrosine residues of TrkA while the three putative autophosphorylation sites conferring tyrosine kinase activity were left intact. Five tyrosine residues, Y499, Y643, Y704, Y760 and Y794, in rat TrkA were involved in nerve growth factor-induced neurite outgrowth. None of these tyrosines mediated the full activity of wild-type TrkA, and a pair of these tyrosines, Y760 and Y794, promoted neurite outgrowth in an additive manner. These data indicate that no single tyrosine is sufficient to induce complete neurite outgrowth but the five tyrosine residues Y499, Y643, Y704, Y760 and Y794 cooperate to exhibit the full activity of wild-type TrkA.

Animals↗
PubMed2006

Platelet nitric oxide synthase is activated by tyrosine dephosphorylation: possible role for SHP-1 phosphatase.

BACKGROUND: Endothelial nitric oxide synthase (eNOS) activity in endothelial cells is regulated by post-translational phosphorylation of critical serine, threonine and tyrosine residues in response to a variety of stimuli. However, the post-translational regulation of eNOS in platelets is poorly defined. OBJECTIVES: We investigated the role of tyrosine phosphorylation in the regulation of platelet eNOS activity. METHODS: Tyrosine phosphorylation of eNOS and interaction with the tyrosine phosphatase SHP-1 were investigated by coimmunoprecipitation and immunoblotting. An in vitro immunoassay was used to determine eNOS activity together with the contribution of protein tyrosine phosphorylation. RESULTS: We found platelet eNOS was tyrosine phosphorylated under basal conditions. Thrombin induced a dose- and time-dependent increase in eNOS activity without altering overall level of tyrosine phosphorylation, although we did observe evidence of minor tyrosine dephosphorylation. In vitro tyrosine dephosphorylation of platelet eNOS using a recombinant protein tyrosine phosphatase enhanced thrombin-induced activity compared to thrombin alone, but had no effect on endothelial eNOS activity either at basal or after stimulation with bradykinin. Having shown that dephosphorylation could modulate platelet eNOS activity we examined the role of potential protein phosphatases important for platelet eNOS activity. We found SHP-1 protein tyrosine phosphatase, co-associated with platelet eNOS in resting platelets, but does not associate with eNOS in endothelial cells. Stimulation of platelets with thrombin increased SHP-1 association with eNOS, while inhibition of SHP-1 abolished the ability of thrombin to induce elevated eNOS activity. CONCLUSIONS: Our data suggest a novel role for tyrosine dephosphorylation in platelet eNOS activation, which may be mediated by SHP-1.

Animals↗
PubMed1996

Tumorigenic and mitogenic capacities are reduced in transfected fibroblasts expressing mutant insulin-like growth factor (IGF)-I receptors. The role of tyrosine residues 1250, 1251, and 1316 in the carboxy-terminus of the IGF-I receptor.

Regulation of ligand-mediated signal transduction through transmembrane tyrosine kinase growth factor receptors involves phosphorylation of tyrosine residues in the intracellular domain of the receptor. The insulin-like growth factor-I (IGF-I) receptor contains three tyrosine residues in the carboxy-terminal domain at positions 1250, 1251, and 1316. Of these, only the tyrosine at position 1316 is conserved in the homologous position of the insulin receptor. Mutational analysis was used to study the role of these tyrosines in specific outcomes of IGF-I-mediated signal transduction. Mutations in the human IGF-I receptor were either replacement of tyrosines 1250 and 1251 with phenylalanine and histidine (yyFH), respectively, or replacement of the conserved distal tyrosine (position 1316) with phenylalanine (yCF). The yyFH mutation results in an IGF-I receptor with the amino acids found in the homologous position of the human insulin receptor. Cells overexpressing mutated IGF-I receptors were compared with cells expressing only endogenous IGF-I receptors or overexpressing wild-type IGF-I receptors. The ability of yyFH mutant IGF-I receptors to autophosphorylate the beta-subunit or phosphorylate insulin receptor substrate-1 was not significantly different from wild-type type IGF-I receptors. However, one or both of the proximal tyrosine residues (positions 1250 and 1251) in the carboxy-terminus of the IGF-I receptor are essential for IGF-I-stimulation of mitogenic and tumorigenic pathways. IGF-I-induced mitogenesis, measured as thymidine incorporation and cellular proliferation, was abrogated in cells overexpressing mutant IGF-I receptors with replacement of the proximal double tyrosines (positions 1250 and 1251). Fibroblasts expressing this mutant IGF-I receptor formed fewer tumors than the negative control cells, whereas cells expressing wild-type IGF-I receptors formed large tumors in all recipient mice injected. Conversely, cells expressing mutant IGF-I receptors with only the conserved distal tyrosine (position 1316) replaced had slightly reduced IGF-I-stimulated beta-subunit autophosphorylation, thymidine incorporation, and cellular proliferation when compared with cells expressing wild-type receptors. Phosphorylation of insulin receptor substrate-1 by the yCF mutant receptors was not impaired. Despite the ability of these mutant receptors to stimulate mitogenic growth, fibroblasts expressing this mutant receptor were also incapable of forming tumors in recipient nude mice. The distal tyrosine (position 1316) of the IGF-I receptor is crucial for tumor formation but is not essential for IGF-I stimulated mitogenesis. Thus, the tyrosine moieties in the carboxy-terminus of the IGF-I receptor participate in the signal transduction pathways that affect the mitogenic and tumorigenic potentials of cells expressing mutant IGF-I receptors.

3T3 Cells↗
PubMed1992

High-affinity IgE receptor-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells induces early and late protein-tyrosine phosphorylations.

We reported previously that stimulation of RBL-2H3 cells through the high-affinity IgE receptor resulted in tyrosine phosphorylation of a 72-kDa protein (pp72) that was coupled to signal transduction. In the present study, although pp72 tyrosine phosphorylation was induced only by antigen triggering, stimulation of RBL-2H3 cells by either antigen or the calcium-ionophore A23187 led to increased tyrosine phosphorylation of a 110-kDa protein (pp110). This tyrosine phosphorylated protein was also observed when RBL-2H3 cells were transfected with the G protein-coupled m3 muscarinic receptor and then stimulated to secrete with carbachol. In contrast to tyrosine phosphorylation of pp72, antigen-induced pp110 tyrosine phosphorylation required extracellular calcium, was absent in cells depleted of protein kinase C, and was detected between 1 and 5 min after stimulation. The protein-tyrosine kinase inhibitor genistein blocked both histamine release and tyrosine phosphorylation induced by A23187. Altogether, the data suggest a role for pp110 in secretion. However, protein kinase C activation induced pp110 tyrosine phosphorylation but not histamine release demonstrating that pp110 tyrosine phosphorylation alone is not sufficient for degranulation. We conclude that tyrosine phosphorylation of pp72 is associated with the early steps of IgE receptor-generated signaling, whereas pp110 tyrosine phosphorylation occurs secondary to calcium influx and protein kinase C activation.

Animals↗
PubMed1990

Signal transduction of the human granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors involves tyrosine phosphorylation of a common set of cytoplasmic proteins.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) exert multiple effects on the proliferation, differentiation, and function of myeloid lineage cells through their interaction with specific cell-surface receptors. There is a considerable degree of overlap in the biological effects of these two growth factors, but little is known about the mechanisms of postreceptor signal transduction. We have investigated the effects of GM-CSF and IL-3 on protein tyrosine-kinase activity in a human cell line, MO7E, which proliferates in response to either factor. Tyrosine-kinase activity was detected using immunoblotting with a monoclonal antibody (MoAb) specific for phosphotyrosine. GM-CSF and IL-3 were found to induce a nearly identical pattern of protein tyrosine phosphorylation using both one- and two-dimensional gel electrophoresis. Tyrosine phosphorylation of two cytosolic proteins in particular was increased more than 10-fold, a 93-Kd protein (pp93) and a 70-Kd protein (pp70). Tyrosine phosphorylation of pp93 and pp70 was observed within 1 minute, reached a maximum at 5 to 15 minutes, and gradually decreased thereafter. Other proteins of 150, 125, 63, 55, 42, and 36 Kd were also phosphorylated on tyrosine in response to both GM-CSF and IL-3, although to a lesser degree. Tyrosine phosphorylation was dependent on the concentration of GM-CSF over the range of 0.1 to 10 ng/mL and on IL-3 over the range of 1 to 30 ng/mL. Stimulation of MO7E cells with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) or cytokines such as G-CSF, M-CSF, interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon gamma, tumor necrosis factor (TNF), or transforming growth factor-beta (TGF-beta) did not induce tyrosine phosphorylation of pp93 or pp70, suggesting that these two phosphoproteins are specific for GM-CSF-or IL-3-induced activation. The extent and duration of phosphorylation of all the substrates were increased by pretreatment of cells with vanadate, an inhibitor of protein-tyrosine phosphatases. Importantly, culture of MO7E cells with vanadate (up to 10 mumol/L) resulted in a dose-dependent increase in GM-CSF-or IL-3-induced proliferation of up to 1.8-fold. These results suggest that tyrosine phosphorylation may be important for GM-CSF and IL-3 receptor-mediated signal transduction and that cell proliferation may be, at least partially, regulated by a balance between CSF-induced protein-tyrosine kinase activity and protein-tyrosine phosphatase activity.

Antibodies, Monoclonal↗
PubMed1994

Tyrosine phosphorylation of the gamma subunit of Fc gamma receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages.

Fc receptor-mediated phagocytosis in mouse macrophages occurs by a tyrosine kinase-dependent pathway (Greenberg, S., Chang, P., and Silverstein, S.C. (1993) J. Exp. Med. 177, 529-534). To identify proteins that are phosphorylated on tyrosine residues during phagocytosis, we used anti-phosphotyrosine antibodies to perform immunoblotting and immunoprecipitation of lysates derived from Fc receptor-stimulated macrophages. Proteins of 26, 30, 35, 37, 40, 43, 47, 56, 60, 68, 83, 116, and 150 kDa displayed enhanced tyrosine phosphorylation during Fc receptor-mediated phagocytosis. Tyrosine phosphorylation of these proteins was not a consequence of actin polymerization since treatment with cytochalasin D did not alter the pattern of Fc receptor-stimulated protein tyrosine phosphorylation. The 68-kDa tyrosine phosphoprotein was identified as paxillin, a cytoskeletal-associated tyrosine kinase substrate previously identified in fibroblasts and shown to localize to focal adhesions (Turner, C.E., Glenney, J.R., and Burridge, K. (1990) J. Cell Biol. 111, 1059-1068). Paxillin colocalized with F-actin beneath nascent phagosomes. In addition to the above proteins detected by anti-phosphotyrosine immunoblotting, the gamma subunit of FcRI and III was shown to undergo tyrosine phosphorylation during Fc receptor-mediated phagocytosis. Of several candidate tyrosine kinases that may be activated during Fc receptor stimulation, p72syk, but not p125FAK, displayed enhanced tyrosine phosphorylation during Fc receptor aggregation. The coordinated tyrosine phosphorylation of the gamma subunit of macrophage Fc receptors, the tyrosine kinase syk, and the cytoskeletal-associated protein, paxillin, may be important steps in integrating signals between Fc receptors and the underlying cytoskeleton.

Actins↗
PubMed1994

rel Is rapidly tyrosine-phosphorylated following granulocyte-colony stimulating factor treatment of human neutrophils.

Stimulation of neutrophils with granulocyte-colony stimulating factor (G-CSF) results in an enhanced respiratory burst, prolonged survival, and increased tumor cell killing. The effects of G-CSF are mediated by binding to specific, high affinity receptors. G-CSF receptors lack intrinsic tyrosine kinase activity, but activation of the receptor results in the rapid induction of tyrosine kinase activity. Antiphosphotyrosine immunoblots of whole cell lysates prepared from neutrophils show that the G-CSF rapidly induces prominent tyrosine phosphorylation of a protein of a relative molecular mass of 80 kDa. Using monospecific antibodies, the 80-kDa tyrosine-phosphorylated protein has been shown to be p80c-rel, a proto-oncogene belonging to a family of transcriptional regulators which include NF-kB. The induction of tyrosine phosphorylation of p80c-rel was unique to G-CSF in that granulocyte-macrophage colony stimulating factor which also stimulates neutrophils and induces tyrosine phosphorylation does not result in tyrosine phosphorylation of p80c-rel. The consequences of p80c-rel tyrosine phosphorylation are not yet known; however, tyrosine-phosphorylated p80c-rel is capable of binding to DNA, and G-CSF stimulation results in an increase in the amount of p80c-rel which binds to DNA. These results demonstrate that one of the first biochemical events which occurs in neutrophils following G-CSF stimulation, activation of a tyrosine kinase, leads directly to the tyrosine phosphorylation of p80c-rel. Thus, the tyrosine kinase activated by G-CSF appears to directly transduce a signal to a protein which functions as a transcriptional regulator.

Adult↗
PubMed1997

Interleukin-2 induces tyrosine phosphorylation of SHP-2 through IL-2 receptor beta chain.

Coupling of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) induces rapid increase in tyrosine phosphorylation of cellular substrates through activation of non-receptor protein tyrosine kinases. Here, we report that stimulation through the IL-2R induced tyrosine phosphorylation of the SH2-containing protein-tyrosine phosphatase SHP-2 in F7, a hematopoietic BAF-B03 transfectant clone expressing the IL-2Rbeta chain. The tyrosine phosphorylation of SHP-2 was specific since another protein-tyrosine phosphatase SHP-1, which is structurally homologous to SHP-2, was not tyrosine phosphorylated. The IL-2-induced tyrosine phosphorylation of SHP-2 required the acidic region within the IL-2Rbeta chain where Src-family PTKs interact. Though the serine-rich region within IL-2Rbeta chain was also required for the phosphorylation of SHP-2, Jak3 activation was dispensable. In COS-7 cells, co-expression of SHP-2 with Lyn resulted in increased tyrosine phosphorylation levels of SHP-2, whereas co-expression of SHP-2 with Fyn failed to alter the levels significantly. Considering that Lyn and Fyn are major Src-family PTKs expressed in BAF-B03 cells, our data suggest that Lyn may be principally responsible for the tyrosine phosphorylation of SHP-2 in F7 cells. Furthermore, the IL-2 stimulation also induced tyrosine phosphorylation of SHP-2 in the human IL-2-dependent T-cell line ILT-Mat. Taken together, these studies demonstrate an involvement of SHP-2 in the IL-2-mediated signaling events through the activation of specific PTKs.

Animals↗
PubMed1998

Reactive oxygen species signaling through regulation of protein tyrosine phosphorylation in endothelial cells.

Tyrosine phosphorylation of proteins, controlled by tyrosine kinases and protein tyrosine phosphatases, plays a key role in cellular growth and differentiating. A wide variety of hormones, growth factors, and cytokines modulate cellular tyrosine phosphorylation to transmit signals across the plasma membrane to the nucleus. Recent studies suggest that reactive oxygen species (ROS) also induce cellular protein tyrosine phosphorylation through receptor or nonreceptor tyrosine kinases. To determine whether protein tyrosine phosphorylation by ROS regulates endothelial cell (EC) metabolism and function, we exposed vascular ECs to H2O2 or H2O2 plus vanadate. This resulted in a time- and dose-dependent increase in protein tyrosine phosphorylation of several proteins (M(r) 21-200 kDa), as determined by immunoprecipitation and Western blot analysis with antiphosphotyrosine antibody. Immunoprecipitation with specific antibodies identified increased tyrosine phosphorylation of mitogen-activated protein kinases (42-44 kDa), paxillin (68 kDa), and FAK (125 kDa) by ROS. An immediate signaling response to increased protein tyrosine phosphorylation by ROS was activation of phospholipases such as A2, C, and D. Suramin pretreatment inhibited ROS stimulation of phospholipase D (PLD), suggesting a role for growth factor receptors in this activation. Further, PLD activation by ROS was attenuated by N-acetylcysteine, indicating that intracellular thiol status is critical to ROS-mediated signal transduction. These results provide evidence that ROS modulate EC signal transduction via a protein tyrosine phosphorylation-dependent mechanism.

Animals↗
PubMed1993

Activation of the respiratory burst and tyrosine phosphorylation of proteins in human neutrophils: no direct relationship and involvement of protein kinase C-dependent and -independent signaling pathways.

Human neutrophils maximally stimulated with the optimal concentration (100 ng/ml) of phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), for 5 min at 37 degrees C did not respond with superoxide (O2-) release to the later addition of PMA itself or the Ca2+ ionophore ionomycin. However, these cells did respond with enhanced release of O2- to the later addition of N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A (Con A). In these PMA-pretreated cells, an increase in cytoplasmic free Ca2+ ([Ca2+]i) induced by ionomycin was unaffected, whereas that induced by FMLP was inhibited by 50-60% and that induced by Con A was completely abolished. A 42-kDa protein was predominantly and consistently tyrosine-phosphorylated by FMLP, PMA and ionomycin with the different kinetics according to the stimuli. The dose-response curves showed that tyrosine phosphorylation and O2- release were stimulated in parallel by PMA, whereas tyrosine phosphorylation and an increase in [Ca2+]i, but not O2- release, were stimulated in parallel by FMLP or ionomycin. The potency of inducing tyrosine phosphorylation was ionomycin > FMLP = PMA, whereas the potency of triggering of O2- release was PMA > ionomycin = FMLP. UCN-01, a PKC inhibitor, inhibited O2- release and tyrosine phosphorylation induced by PMA, but not by FMLP or ionomycin. In contrast, pertussis toxin inhibited O2- release and tyrosine phosphorylation induced by FMLP, but not by PMA. Tyrosine kinase inhibitors (erbstatin and genistein) inhibited O2- release induced by FMLP, but not by PMA. However, both tyrosine kinase inhibitors did not impair FMLP- or PMA-induced tyrosine phosphorylation of a 42-kDa protein. Increased tyrosine phosphorylation of a 42-kDa protein was also detected in immature myeloid cells (HL-60 cells) stimulated by PMA, but not by ionomycin. These findings suggest that FMLP and Con A trigger the respiratory burst in human neutrophils by activating the definite pathway which include other signals than activation of PKC and an increase in [Ca2+]i; tyrosine phosphorylation of a 42-kDa protein is induced by the PKC-dependent and independent mechanisms according to the stimuli, and the PKC-independent and ionomycin-sensitive mechanism is inoperative in HL-60 cells; and tyrosine phosphorylation of a 42-kDa protein is unlikely to be causally related to activation of the respiratory burst.

Concanavalin A↗
PubMed1988

Neuropeptide tyrosine in the rat adrenal gland--immunohistochemical and in situ hybridization studies.

The adrenal gland of the rat was analysed with immunohistochemistry and antisera to neuropeptide tyrosine, to the catecholamine-synthesizing enzymes tyrosine hydroxylase, phenyl-ethanolamine-N-methyltransferase, and to acetylcholinesterase and with in situ hybridization using a nick-translated 280 base pair deoxyribonucleic acid probe coding for exon 2 of the rat neuropeptide tyrosine gene. Neuropeptide tyrosine-like immunoreactivity was observed in three structures: chromaffin cells, medullary ganglion cells and nerve fibers. The chromaffin cells were of both the noradrenaline- and adrenaline-type. The ganglion cells did not seem to contain any catecholamine-synthesizing enzymes but exhibited a strong immunoreaction for acetylcholinesterase. They were thus in all probability cholinergic neurons. In situ hybridization using the nick-translated deoxyribonucleic acid probe to rat neuropeptide tyrosine messenger ribonucleic acid revealed a very high-grain density over the ganglion cells, a moderate density over the chromaffin cells and a low background over cortex, in agreement with the immuno-histochemical demonstration of neuropeptide tyrosine-like immunoreactivity both in chromaffin and ganglion cells. The intense neuropeptide tyrosine-like immunoreactivity and low content of neuropeptide tyrosine messenger ribonucleic acid suggest that the chromaffin cells have fairly large peptide stores but that the peptide turnover is low. In contrast, the ganglion cell bodies seem to contain low amounts of neuropeptide tyrosine-like immunoreactivity but exhibit a high neuropeptide tyrosine synthesis rate. Preliminary studies with the amine-depleting drug reserpine revealed an increase in messenger ribonucleic acid both in ganglion cells and medullary cells. In the chromaffin cells the highest activity was seen 3 and 4 days after injection, and the levels were down to normal after 8 days. The present findings demonstrate neuropeptide tyrosine synthesis and storage in two cell populations in the adrenal medulla. In situ hybridization with its cellular resolution can provide information on possible differential effects of drugs and experimental procedures on these two neuropeptide tyrosine stores.

Acetylcholinesterase↗
PubMed2004

Development of infrared optical sensor for selective detection of tyrosine in biological fluids.

In this paper, a new and simple evanescent wave type of infrared biosensor is described for the selective detection of tyrosine in biological fluids. This sensor is based on the formation of copper complexes between the sensing phase and tyrosine. To demonstrate that this principle was applicable to the selective detection of tyrosine, a proline-modified sensing phase was synthesized on the surface of the internal reflection elements. This sensing phase was saturated with copper ions to allow it to interact with tyrosine units in aqueous solution through the formation of stable proline-Cu2+-tyrosine complexes. Tyrosine exhibits a unique spectral feature in its absorption band at 1515 cm-1. This band significantly differs from those of other amino acids and provides a further method for the discrimination of tyrosine. By investigating the signals from 12 amino acids, only three amino acids, each containing a phenyl group, could be sensed selectively by this sensing phase. Based on the unique absorption of tyrosine located at 1515 cm-1, tyrosine can be selectively detected. To perform quantitative analyses of tyrosine using this sensing phase, a theoretical working equation was developed and correlated with the experimental data. The analytical results indicated that the developed equations do explain and predict the detection behaviors of the proposed sensing scheme. Using the optimal conditions, the regression coefficients for standard curves of tyrosine recorded in the region of concentrations below 600 microM were higher than 0.996 under either equilibrium or non-equilibrium conditions. Detection limit of tyrosine when using this method was ca. 3 microM.

Biosensing Techniques↗
PubMed2004

Effect of hypoxia on protein tyrosine kinase activity in cortical membranes of newborn piglets--the role of nitric oxide.

Previous studies have shown that cerebral hypoxia results in increased tyrosine phosphorylation of cerebral cortical cell membrane proteins as well as nuclear membrane anti-apoptotic protein, Bcl-2. The present study tests the hypothesis that hypoxia results in increased protein tyrosine kinase activity in cortical cell membranes of newborn piglets and that the inhibition of neuronal NOS by administration of 7-nitroindazole sodium salt (7-NINA), a selective inhibitor of nitric oxide synthase (NOS), will prevent the hypoxia-induced increase in protein tyrosine kinase activity. To test this hypothesis, protein tyrosine kinase activity was determined in cerebral cortical membranes of 2- to 4-day-old newborn piglets divided into normoxic (n=6), hypoxic (n=5) and 7-NINA-treated hypoxic (n=5) (7-NINA, 1mg/kg, i.p., prior to hypoxia) groups. Tissue hypoxia was achieved by exposing the animals to an FiO(2) of 0.07 for 60 min and was documented biochemically by determining tissue ATP and phosphocreatine (PCr) levels. Cortical P(2) membranes were isolated and protein tyrosine kinase activity determined by (33)P incorporation into a specific peptide substrate for 15 min at 37 degrees C in a medium containing 100 mM HEPES, pH 7.0, 1mM EDTA, 125 mM MgCl(2), 25 mM MnCl(2), 2mM DTT, 0.2 mM sodium orthovanadate, 2mM EGTA, 150 microM tyrosine kinase peptide substrate [Lys 19] cdc2(6-20)-NH(2), (33)P-ATP, and 10 microg of membrane protein. Protein tyrosine kinase activity was determined by the difference between (33)P incorporation in the presence and absence of specific peptide substrate and expressed as pmol/mg protein/h. The ATP values in the normoxic, hypoxic and 7-NINA-treated hypoxic animals were ATP: 4.57+/-0.45 micromol/g, 1.29+/-0.23 micromol/g (p<0.05 versus normoxic) and 1.50+/-0.14 micromol/g brain (p<0.05 versus normoxic), respectively. The PCr values in the normoxic, hypoxic and 7-NINA-treated hypoxic animals were: 3.77+/-0.36 micromol/g, 0.77+/-0.13 micromol/g (p<0.05 versus normoxic) and 1.02+/-0.24 micromol/g brain (p<0.05 versus normoxic), respectively. Protein tyrosine kinase activity in the normoxic, hypoxic and the 7-NINA-treated groups was 378+/-77 pmol/mg protein/h, 854+/-169 pmol/mg protein/h (p<0.05 versus normoxic) and 464+/-129 pmol/mg protein/h (p<0.05 versus hypoxic), respectively. The data show that cerebral tissue hypoxia results in increased protein tyrosin kinase activity in cortical membranes of newborn piglets and pretreatment with 7-NINA prevents the hypoxia-induced increase in protein tyrosine kinase activity. We conclude that the hypoxia-induced increase in protein tyrosine kinase activity is NO-mediated. We propose that the hypoxia-induced increase in protein tyrosine kinase activity leading to increased phosphorylation of Bcl-2 is a critical link to hypoxic neuronal injury pathway.

Animals↗
PubMed2006

Tyrosine kinase receptor immunoreactivity in trigeminal mesencephalic and motor neurons following transection of masseteric nerve of the rat.

Neurotrophins are known to promote survival after neural injury. To determine the relative importance of tyrosine kinase receptors on the survival of axotomized trigeminal nuclear neurons, we examined the temporal expression profile of tyrosine kinase A, tyrosine kinase B and tyrosine kinase C receptors in the mesencephalic trigeminal nucleus and the motor trigeminal nucleus following transection of the masseteric nerve in rats. Axotomized neurons in these nuclei were retrogradely identified with FluoroGold. We found increase in tyrosine kinase A-immunoreactive mesencephalic trigeminal nucleus neurons in the second week after axotomy but no change in the number of tyrosine kinase A-immunoreactive motor trigeminal nucleus neurons. There was no change in the number of tyrosine kinase B-immunoreactive mesencephalic trigeminal nucleus neurons but the significant increase of tyrosine kinase B-immunoreactive motor trigeminal nucleus neurons throughout the period of observation (3 weeks) peaked at approximately 1 week after axotomy. There was no alteration in the number of tyrosine kinase C-immunoreactive mesencephalic trigeminal nucleus neurons but significant increase in tyrosine kinase C-immunoreactive motor trigeminal nucleus neurons observable by 4 days post-axotomy was followed by decline to levels lower than the control in 2 weeks. Temporal changes in the expression of individual tyrosine kinase receptors in mesencephalic trigeminal nucleus and motor trigeminal nucleus neurons following transection of the masseteric nerve suggest differential contribution of tyrosine kinase-specific neurotrophins to the survival of these neurons after axotomy.

Animals↗
PubMed2003

Mutational analysis of the mouse aryl hydrocarbon receptor tyrosine residues necessary for recognition of dioxin response elements.

Tyrosine phosphorylation of the aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix/PER-ARNT-SIM transcription factor family, has been shown to regulate its dioxin response elements (DRE) binding ability, although no specific residues have been directly demonstrated to be phosphorylated. Of the 23 tyrosines in the mouse AhR, 19 are conserved across all mammalian species sequenced thus far. The studies presented here were conducted to examine tyrosine residue(s) that are both likely candidates of phosphorylation and necessary for DNA binding and/or transcriptional activity of the AhR. Two-dimensional gel electrophoresis of phosphatase-treated AhR indicated that the receptor is phosphorylated on serine/threonine and tyrosine residues. Computational analysis predicted several highly conserved tyrosine residues to be phosphorylated. Both the N terminus (amino acids 1-399) and the C terminus (amino acids 399-805) of the mouse receptor synthesized in vitro using a rabbit reticulocyte lysate system are tyrosine phosphorylated as detected by antiphosphotyrosine antibodies. Furthermore, the N-terminal AhR bound DRE in a ligand-dependent manner similar to that by the full-length receptor, suggesting that phosphorylated tyrosines involved in DNA binding are likely located in the region between residues 1 and 399. Mouse AhR tyrosine (Y) residues were evaluated by phenylalanine (F) mutational analysis for both DNA binding (electrophoretic mobility shift assays; EMSAs) and ability to induce a DRE-driven reporter gene in transiently transfected AhR-deficient cells. Of the 12 tyrosine residues in the N-terminal AhR, only a tyrosine 9 mutant (AhRY9F) significantly decreased DRE binding as determined by EMSA. Similarly, only the AhRY9F mutant decreased the DRE-driven luciferase expression in AhR-deficient cells. Overall, these data strongly suggest that the putative posttranslational modification at, or mediated by, tyrosine 9, and not any other individual mouse AhR tyrosine residue, is necessary for AhR DRE binding and transcriptional activity.

Animals↗
PubMed1999

Evaluation of phenylalanine and tyrosine metabolism in late human pregnancy.

The [2H5]-phenylalanine method for measurement of protein metabolism requires the phenylalanine hydroxylation to tyrosine to be calculated from the tyrosine flux. Although this can be estimated, for pregnancy, we made a direct measurement of the molar ratio of the fluxes of tyrosine and phenylalanine from protein breakdown (Pt/Pp) using [2H2]-tyrosine infusion. Six normal pregnant women were studied at 37 weeks' gestation. While fasting, they were administered a 3-hour primed-constant infusion with [13C]-leucine, [2H5]-phenylalanine, and [2H2]-tyrosine. Leucine (alpha-ketoisocaproic acid [KIC]) flux was 136.2+/-15.1 micromol/kg/h (mean +/- SD), phenylalanine flux 41.2+/-5.6, and tyrosine flux 25.0+/-6.0, and phenylalanine hydroxylation was 3.3+/-2.1 micromol/kg/h. The mean tyrosine to phenylalanine molar flux ratio (Pt/Pp) was 0.52+/-0.10, lower than the ratio of 0.65 to 0.85 reported in normal nonpregnant subjects and 0.73 estimated from animal studies. We studied protein metabolism in six additional pregnant women and six nonpregnant women using [13C]-leucine and [2H5]-phenylalanine infusions only and applied the lower Pt/Pp ratio to the former group. Tyrosine flux (42.0+/-7.2 micromol/kg/h) and phenylalanine hydroxylation (9.2+/-4.2 micromol/kg/h) were significantly higher in nonpregnant subjects than in both groups of pregnant subjects. The percent contribution of phenylalanine hydroxylation to total tyrosine flux was reduced from 20% to 14%. When using [2H5]-phenylalanine to study whole-body protein metabolism in pregnancy and tyrosine flux is not measured directly by infusion of [2H2]-tyrosine, the lower Pt/Pp ratio is required. The phenylalanine model shows that tyrosine flux derived from protein breakdown and phenylalanine hydroxylation are both reduced in pregnancy.

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