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PubMed1994

C5a as a model for chemotactic factor-stimulated tyrosine phosphorylation in the human neutrophil.

Human neutrophils were exposed to the chemotactic factors C5a, FMLP, IL-8, leukotriene B4, and PAF, for 30 s, and subsequently analyzed for protein tyrosine phosphorylation by immunoblotting whole cell lysates with a polyclonal antiphosphotyrosine Ab. All chemotactic factors caused the rapid de novo tyrosine phosphorylation of a broad band of approximately 120 kDa and increased the phosphotyrosine content of several other proteins, including two with molecular masses of 60 and 56 kDa that were present in the unstimulated neutrophil. Tyrosine phosphorylation was evident as early as 10 s after stimulation and was maintained for 1 to 3 min before dephosphorylation occurred. The extent of tyrosine phosphorylation was dependent on the concentration of chemotactic factor, with stimulation observed at concentrations as low as 10 to 100 nM. To investigate the pathway used by chemotactic factors to transduce this signal, neutrophils were treated with PMA. PMA also stimulated tyrosine phosphorylation in the neutrophil but with a slower response time and a different pattern of affected proteins. Additional experiments suggested that tyrosine phosphorylation is involved in the regulation of the neutrophil respiratory burst because the tyrosine kinase inhibitor, herbimycin A, inhibited C5a-induced protein tyrosine phosphorylation and also prevented C5a- and FMLP-induced superoxide anion production. Herbimycin A also inhibited PMA-induced tyrosine phosphorylation and superoxide anion production. To confirm that the ability to stimulate tyrosine phosphorylation was intrinsic to the C5a receptor, tyrosine phosphorylation was examined in both undifferentiated U937 cells (C5a receptor negative) and cAMP differentiated U937 cells (C5a receptor positive). C5a induced tyrosine phosphorylation only in differentiated U937 cells. Analysis of the C5a receptor mRNA using the PCR confirmed its presence in differentiated and its absence in undifferentiated U937 cells. Therefore, C5a stimulates tyrosine phosphorylation via a receptor-mediated mechanism and U937 cells provide a system in which G-coupled receptor-mediated tyrosine phosphorylation can be investigated.

Benzoquinones↗
PubMed1996

Coupling between [arginine8]-vasopressin-activated increases in protein tyrosine phosphorylation and cellular calcium in A7r5 aortic smooth muscle cells.

Effects of genistein, a tyrosine kinase inhibitor, on increases in [Ca2+]i and protein tyrosine phosphorylation induced by 20 nM [arginine 8]vasopressin (AVP) were studied in A7r5 aortic smooth muscle cells. In fura-2-loaded cells, AVP induced a rapid (0.5-2 min) transient increase in [Ca2+]i that was followed by a smaller sustained increase in [Ca2+]i. In 66% of the cells, the transient response involved both influx of extracellular Ca2+ and release of intracellular Ca2+: influx accounted for 6% of the response, and release accounted for 40%. However, in 34% of the cells, the relative contribution of influx and release during the transient could not be assessed. In all cells, the smaller sustained response was entirely dependent on extracellular Ca2+. Genistein (148 microM) always blocked the transient and sustained components of the Ca2+ response showing that both influx and release were genistein-sensitive. Isobestic fluorescence analysis, in medium containing 0.5 mM Mn2+ in place of Ca2+, showed that the influx pathway was selective because it did not conduct Mn2+. It also confirmed that Ca2+ release was blocked by genistein. In contrast, 105 microM lavendustin A, a different tyrosine kinase inhibitor, suppressed the transient by only 30%. Another inhibitor, tyrphostin 47 (80 microM), did not alter the transient or sustained components of the Ca2+ response. No AVP-induced increases in tyrosine phosphorylation were detected unless special procedures were used. When cells were preincubated in 10 mM vanadate, a tyrosine phosphatase inhibitor, AVP induced a transient increase in tyrosine phosphorylation (5-60 s). The time course for AVP-induced phosphorylation was similar to that for increase in [Ca2+]i. Vanadate alone increased tyrosine phosphorylation and induced a slow small increase in [Ca2+]i that was dependent on extracellular Ca2+. Genistein blocked tyrosine phosphorylation induced by AVP and vanadate, and it blocked the increase in [Ca2+]i induced by vanadate alone. In contrast, lavendustin or tyrphostin unexpectedly enhanced tyrosine phosphorylation induced by vanadate alone and precluded assessment of AVP-induced tyrosine phosphorylation in the presence of vanadate. Lavendustin produced time-dependent enhancement of vanadate-induced increase in [Ca2+]i. These results underscored the need for measuring cellular changes in protein tyrosine phosphorylation to assess potential functions of tyrosine kinase activity. Under conditions where changes in phosphorylation could be measured, the results suggested that AVP-activated increases in tyrosine phosphorylation may be coupled to AVP-activated mechanisms that regulate influx of extracellular Ca2+ and release of intracellular Ca2+.

Animals↗
PubMed2005

Effect of nitration on protein tyrosine phosphatase and protein phosphatase activity in neuronal cell membranes of newborn piglets.

Protein tyrosine phosphatase predominantly determines the status of protein tyrosine kinase-dependent phosphorylation of specific proteins and controls the survival and death of neurons. Previous studies have shown that protein tyrosine phosphatase activity is decreased during hypoxia in cortical membranes of the newborn piglet. We have also shown that nitric oxide (NO) free radicals are generated during hypoxia, and may result in modification of protein tyrosine phosphatase via peroxynitrite-mediated modification. The present study tests the hypothesis that the hypoxia-induced decrease in protein tyrosine phosphatase activity is NO-mediated. To test this hypothesis, in vitro experiments were conducted by measuring protein tyrosine phosphatase activity in the presence of an NO donor, sodium nitroprusside (SNP), or peroxynitrite. Since 3-nitrotyrosine is produced as a consequence of peroxynitrite reactions, we have also examined the effect of 3-nitrotyrosine on protein phophatase activity. Cerebral cortical P(2) membranes were prepared from seven normoxic newborn piglets and each sample was divided into three aliquots: a control group, a SNP group (exposed to 200 microM SNP), and a peroxynitrite group (exposed to 100 microM peroxynitrite). Protein tyrosine phosphatase activity was determined spectrophotometrically in the presence or absence of 2 microM bpV(phen), a highly selective inhibitor of protein tyrosine phosphatase. The protein tyrosine phosphatase activity was 198+/-25 nmol/mg protein/h in the normoxic group, 177+/-30 nmol/mg protein/h in the SNP group (p=NS versus normoxic) and 77+/-20 nmol/mg protein/h in the peroxynitrite group (p<0.001 versus normoxic). The results show that peroxynitrite but not SNP exposure results in decreased protein tyrosine phosphatase activity in vitro. Furthermore 3-nitrotyrosine (100 microm), a product of peroxynitrite, decreased the enzyme activity from 926+/-102 to 200+/-77 (p<0.001). We conclude that protein tyrosine phosphatase regulation is mediated by peroxynitrite. We propose that hypoxia-induced NO production leading to peroxynitrite formation is a potential mechanism of protein tyrosine phosphatase inactivation in vivo. The NO-induced decrease in protein tyrosine phosphatase and protein phosphatase activity, leading to Bcl-2 protein phosphorylation and loss of its antiapoptotic activity may be a NO-mediated mechanism of programmed cell death in the hypoxic brain.

Animals↗
PubMed2001

Protein tyrosine phosphatase genes downregulated in melanoma.

Phospho-tyrosine levels are increased in melanoma, apparently consistent with reports of elevated protein tyrosine kinase activity. Some protein tyrosine kinases are encoded by oncogenes and have been implicated in melanoma genesis. Decreased protein tyrosine phosphatase activity may also increase phospho-tyrosine. Protein tyrosine phosphatase genes are candidate tumor suppressors and loss of expression may contribute to melanoma genesis. Here we survey protein tyrosine phosphatase expression in pigment cells. Protein tyrosine phosphatase genes were cloned by reverse transcriptase polymerase chain reaction using degenerate primers based upon conserved sequences within the phosphatase catalytic domain. Reaction products were cloned and sequenced: 118 and 113 partial protein tyrosine phosphatase products were isolated from normal melanocytes and melanoma cells, respectively. Northern blotting analysis was used to study expression of 15 protein tyrosine phosphatase genes. Expression of PTP-kappa and PTP-pi was absent or downregulated in more than 20% of melanoma cell lines and in some unmanipulated melanoma biopsies. These closely related enzymes are members of the 2B receptor protein tyrosine phosphatase family previously implicated in contact inhibition. Loss of protein tyrosine phosphatase expression may contribute to the abnormal tyrosine phosphorylation seen in melanoma; these genes are candidate tumor suppressors.

Blotting, Northern↗
PubMed1981

The activity of rat pineal and brain tyrosine hydroxylase during the daily cycle of light and darkness as determined by the modified 14CO2 assay method.

A previous published assay method for tyrosine hydroxylase by the evolution of 14CO2 was modified to a two-step procedure to allow reliable measurement of large numbers of samples containing low tyrosine hydroxylase activity. The reliability of the method was examined in detail. Properties of rat brain and pineal tyrosine hydroxylase solubilized with 0.2% Triton X-100 were as follows. The apparent Km values of the brain enzyme for L-tyrosine with 1 mM-(6-DL)-5,6,7,8-tetrahydro-L-erythro-biopterin (BPH4) as cofactor and for BPH4 with 62 microM-L-tyrosine as substrate were approximately 25 microM and 85 microM, respectively. The Km's for L-tyrosine with 1 mM-(6-DL)-5,6,7,8-tetrahydro-6-methylpterin (6MPH4) as cofactor and for 6MPH4 with 210 microM-L-tyrosine as substrate were 68 microM and 270 microM, respectively. The marked substrate inhibition by high concentrations of L-tyrosine was observed only when BPH4 was used as cofactor. High concentrations of BPH4 inhibited the reaction slightly. The kinetic properties of tyrosine hydroxylase in the pineal extract were similar to those of the brain enzyme, except that a Lineweaver-Burk plot of reciprocal velocity versus the reciprocal concentration of BPH4 with 62 microM-L-tyrosine as substrate deviated downward at a BPH4 concentration of about 100 microM. Analyses of the plot indicated that the peculiar kinetic property may represent either the reaction occurring at two independent sites or with two forms (6L- and 6D-isomers) of the tetrahydrobiopterin cofactor, with apparent Km for BPH4 of 23 microM and 1025 microM, respectively, or the negatively cooperative ligand binding with a Hill coefficient of 0.72. Based on the results obtained as reported above the standard assay conditions of tyrosine hydroxylase in tissue extracts were established. Using the assay method and conditions, the absence of the daily rhythmicity of tyrosine hydroxylase in rat pineal glands and three discrete brain areas was demonstrated. The findings, especially on pineal tyrosine hydroxylase, are discussed in relation to the daily change of noradrenaline turnover.

Animals↗
PubMed2006

Cell surface nucleolin on developing muscle is a potential ligand for the axonal receptor protein tyrosine phosphatase-sigma.

Reversible tyrosine phosphorylation, catalyzed by receptor tyrosine kinases and receptor tyrosine phosphatases, plays an essential part in cell signaling during axonal development. Receptor protein tyrosine phosphatase-sigma has been implicated in the growth, guidance and repair of retinal axons. This phosphatase has also been implicated in motor axon growth and innervation. Insect orthologs of receptor protein tyrosine phosphatase-sigma are also implicated in the recognition of muscle target cells. A potential extracellular ligand for vertebrate receptor protein tyrosine phosphatase-sigma has been previously localized in developing skeletal muscle. The identity of this muscle ligand is currently unknown, but it appears to be unrelated to the heparan sulfate ligands of receptor protein tyrosine phosphatase-sigma. In this study, we have used affinity chromatography and tandem MS to identify nucleolin as a binding partner for receptor protein tyrosine phosphatase-sigma in skeletal muscle tissue. Nucleolin, both from tissue lysates and in purified form, binds to receptor protein tyrosine phosphatase-sigma ectodomains. Its expression pattern also overlaps with that of the receptor protein tyrosine phosphatase-sigma-binding partner previously localized in muscle, and nucleolin can also be found in retinal basement membranes. We demonstrate that a significant amount of muscle-associated nucleolin is present on the cell surface of developing myotubes, and that two nucleolin-binding components, lactoferrin and the HB-19 peptide, can block the interaction of receptor protein tyrosine phosphatase-sigma ectodomains with muscle and retinal basement membranes in tissue sections. These data suggest that muscle cell surface-associated nucleolin represents at least part of the muscle binding site for axonal receptor protein tyrosine phosphatase-sigma and that nucleolin may also be a necessary component of basement membrane binding sites of receptor protein tyrosine phosphatase-sigma.

Amino Acid Sequence↗
PubMed2001

Modulation of excitability in Aplysia tail sensory neurons by tyrosine kinases.

Tyrosine kinases have recently been shown to modulate synaptic plasticity and ion channel function. We show here that tyrosine kinases can also modulate both the baseline excitability state of Aplysia tail sensory neurons (SNs) as well as the excitability induced by the neuromodulator serotonin (5HT). First, we examined the effects of increasing and decreasing tyrosine kinase activity in the SNs. We found that tyrosine kinase inhibitors decrease baseline SN excitability in addition to attenuating the increase in excitability induced by 5HT. Conversely, functionally increasing cellular tyrosine kinase activity in the SNs by either inhibiting opposing tyrosine phosphatase activity or by direct injection of an active tyrosine kinase (Src) induces increases in SN excitability in the absence of 5HT. Second, we examined the interaction between protein kinase A (PKA), which is known to mediate 5HT-induced excitability changes in the SNs, and tyrosine kinases, in the enhancement of SN excitability. We found that the tyrosine kinases function downstream of PKA activation since tyrosine kinase inhibitors reduce excitability induced by activators of PKA. Finally, we examined the role of tyrosine kinases in other forms of 5HT-induced plasticity in the SNs. We found that while tyrosine kinase inhibitors attenuate excitability produced by 5HT, they have no effect on short-term facilitation (STF) of the SN-motor neuron (MN) synapse induced by 5HT. Thus tyrosine kinases modulate different forms of SN plasticity independently. Such differential modulation would have important consequences for activity-dependent plasticity in a variety of neural circuits.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1993

Prostaglandin F2 alpha-induced mitogenesis in MC3T3-E1 osteoblasts: role of protein kinase-C-mediated tyrosine phosphorylation.

Prostaglandin F2 alpha (PGF2 alpha) stimulates DNA synthesis in osteoblasts through phospholipase-C-dependent increases in intracellular calcium and protein kinase-C (PKC) activity. We present evidence that stimulation of protein tyrosine phosphorylation by PKC is an additional component of the signaling pathways involved in PGF2 alpha-stimulated DNA synthesis in MC3T3-E1 osteoblast-like cells. Mitogenic doses of PGF2 alpha (42 nM) rapidly induced tyrosine phosphorylation of multiple substrates in these osteoblast-like cells. PGF2 alpha stimulated tyrosine phosphorylation of new proteins with apparent mol wt of 87, 80, 50, 47, 36, and 33 kilodaltons and up-regulated phosphorylation of preexisting tyrosine components with mol wt of 123, 112, 68, and 56 kilodaltons. Stimulation of PKC by 1.6 microM phorbol 12-myristate 13-acetate mimicked the pattern of PGF2 alpha-induced protein tyrosine phosphorylation, whereas PKC-deficient cells (induced by overnight pretreatment with 16 microM phorbol 12-myristate 13-acetate) were refractory to PGF2 alpha-stimulated protein tyrosine phosphorylation and DNA synthesis. The tyrosine kinase inhibitors tyrphostin and genistein blocked PGF2 alpha-stimulated DNA synthesis and protein tyrosine phosphorylation, and the tyrosine phosphatase inhibitor orthovanadate prolonged PGF2 alpha-stimulated tyrosine phosphorylation; these findings are consistent with activation of a putative tyrosine kinase. Calcium/calmodulin antagonists also inhibited PGF2 alpha-stimulated DNA synthesis, but the calcium-signaling pathway played no role in PGF2 alpha-induced tyrosine phosphorylation. Our findings suggest that cross-talk between receptor-mediated activation of PKC and protein tyrosine phosphorylation is an important distal signaling pathway necessary for PGF2 alpha-induced DNA synthesis in osteoblast-like cells.

Animals↗
PubMed1998

Direct association of protein-tyrosine phosphatase PTP-PEST with paxillin.

Tyrosine phosphorylation of focal adhesion-associated proteins may be involved in the regulation of the cytoskeleton and in the control of signals for growth and survival. The focal adhesion kinase (FAK) functions in regulating tyrosine phosphorylation of several of these proteins, including paxillin, tensin, and p130(cas). Protein- tyrosine phosphatases, the counterparts of protein-tyrosine kinases, also presumably regulate phosphorylation of these proteins. We have tested the hypothesis that FAK intimately associates with a protein-tyrosine phosphatase. Protein-tyrosine phosphatase activity associated with the recombinant C-terminal domain of FAK in vitro and could be coimmunoprecipitated with both FAK and paxillin from lysates of chicken embryo cells. However, the interaction with FAK appeared to be indirect and mediated via paxillin. The protein-tyrosine phosphatase was subsequently identified as protein-tyrosine phosphatase-PEST, a nonreceptor protein-tyrosine phosphatase. The C-terminal noncatalytic domain of protein-tyrosine phosphatase-PEST directly bound to paxillin in vitro. The association of both a protein-tyrosine kinase and a protein-tyrosine phosphatase with paxillin suggests that paxillin may play a critical role in the regulation of the phosphotyrosine content of proteins in focal adhesions.

3T3 Cells↗
PubMed1998

Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP-dependent pathway.

Changes of protein tyrosine phosphorylation in ejaculated boar sperm incubated in vitro were examined with the use of antiphosphotyrosine antibodies and immunoblotting. The intracellular levels of cAMP were modulated by treatment with various combinations of caffeine, 3-isobutyl-1-methylxanthine (IBMX), and dibutyryl cyclic AMP (dbcAMP), and acrosome reactions (ARs) were induced via treatment with divalent cation ionophore A23187. Proteins of Mr 34, 38, 40, and 44 (p34 ... p44) were strongly phosphorylated on tyrosine residues in freshly prepared sperm samples and at the same level during all subsequent treatments. Incubation of sperm in vitro for various periods of time induced an increase of tyrosine phosphorylation of p20, p93, and p175. The tyrosine phosphorylation of p93, p175, and several other sperm proteins was up-regulated in a concentration-dependent manner following treatment of the sperm with dbcAMP, caffeine, or IBMX alone, or with combinations of caffeine and IBMX, respectively, with dbcAMP; the tyrosine phosphorylation of p20 was not correlated with treatment of sperm with cAMP-elevating reagents. The percentage of sperm cells undergoing spontaneous ARs was not affected by the manipulation of cAMP levels and was not correlated with protein tyrosine phosphorylation. In contrast, the addition of calcium to the incubation media decreased protein tyrosine phosphorylation and elevated percentage of spontaneous ARs. The induction of ARs with A23187 caused a significant decrease of tyrosine phosphorylation of p93, p175, and p220/230, indicating that dephosphorylation on protein tyrosine residues might be associated with calcium influx during physiological ARs as well. Proteins p93 and p175 were effectively solubilized in greater than 9M urea/1% triton and in SDS sample buffer, but to only a small extent in triton, while p20 was virtually completely extractable with triton. In conjunction with the previously reported isolation of active tyrosine kinase sp42 from triton extracts of noncapacitated boar sperm cells (Berruti and Porzio, 1992: Biochim Biophys Acta 1118: 149-154), our results suggest that a cAMP-dependent event is required for tyrosine phosphorylation of triton-insoluble proteins such as p93 and p175. On the other hand, the tyrosine phosphorylation of p20 (and potentially other triton-soluble substrates) might not strictly require such cAMP up-regulation. We discuss the differences in the regulation of cAMP-dependent tyrosine phosphorylation in mouse, human, and boar sperm, and suggest that sensitivity to calcium and distinct basal levels of cyclic nucleotide PDE might correspond to species-specific reproduction strategies in mammals.

Animals↗
PubMed1993

Rapid and reversible tubulin tyrosination in human neutrophils stimulated by the chemotactic peptide, fMet-Leu-Phe.

Neutrophil activation by specific stimuli, such as the oligopeptide chemotactic factor fMet-Leu-Phe (fMLF), is associated with an increased enzymatic addition of tyrosine to tubulin alpha-subunits, as measured by 14C tyrosine uptake. In studies using immunoblots we have found that this increased tyrosine uptake into tubulin in activated neutrophils reflects an increase in the proportion of cellular tubulin that is tyrosinated rather than simply an increase in the turnover of tyrosinated subunits. However, the increased accumulation of tyrosinated tubulin was also found to follow an initial depletion of tyrosinated tubulin and concomitant increase in detyrosinated tubulin between 0 and 60 sec following stimulation of neutrophils with fMLF. Immunogold electron microscopy studies of intact microtubules recovered from activated neutrophils demonstrated that these rapid changes in the relative content of tubulin isoforms in the cells were not associated with the formation or disappearance of microtubule microdomains composed of only one form of tubulin. Previously, we have shown that under conditions of fMLF-stimulated exocytosis there is an increased binding of neutrophil granules to endogenous microtubules. Since neutrophil activation by fMLF is associated with increased tyrosination of alpha-tubulin subunits, we speculated that rapid changes in the levels of tyrosinated tubulin in the microtubules of activated neutrophils might have a role in the regulation of granule-microtubule interactions. When the binding of purified neutrophil granules to reconstituted rat brain microtubules containing approximately 50% tyrosinated tubulin was measured by electron microscopy and compared with granule binding to microtubules that contained no detectable tyrosinated tubulin, granule-microtubule associations were found to be significantly favored by detyrosinated vs. tyrosinated tubulin. These findings indicate that interactions between cytoplasmic granules and microtubules in activated neutrophils may be modulated by rapid changes in the relative content of detyrosinated and tyrosinated tubulin in the microtubule network of the cells.

Amino Acid Sequence↗
PubMed1996

Beta2 integrins mediate protein tyrosine phosphorylation in human neutrophils.

Tyrosine kinases play a prominent role in various intracellular signal transduction pathways. In this study, beta2 integrin (CD11/CD18)-mediated adhesive interactions of human neutrophils (PMN) were mimicked by antibody cross-linking of CD11/CD18. Cross-linking of CD18 induced tyrosine phosphorylation of several proteins with apparent molecular masses of approximately 120, 78, 72, 65, and 56 kDa, respectively, as shown by anti-phosphotyrosine immunoblotting of whole cell lysates. Cross-linking of the alpha-subunits of the beta2 integrins demonstrated that only cross-linking of Mac-1 but not LFA-1 or gp150/95 triggered tyrosine signaling. The tyrosine phosphorylations showed a rapid and transient time course. Comparison of the CD18-mediated tyrosine phosphorylation patterns with those induced by chemoattractants gave similar results. The observed tyrosine phosphorylation was specific, since binding and non-binding irrelevant primary antibodies had no effect. Furthermore, F(ab')2 fragments of the anti-CD18 antibody IB4 and addition of F(ab')2 fragments of secondary antibody were sufficient to induce tyrosine phosphorylation. Inhibition of tyrosine kinases by herbimycin A resulted in partial inhibition of the CD18-mediated tyrosine phosphorylation of the 120- and 65-kDa proteins and in complete inhibition of tyrosine phosphorylation of the 78- and 56-kDa proteins. In unstimulated PMN, the tyrosine phosphatase inhibitor sodium orthovanadate had no effect on tyrosine phosphorylation of the 120-kDa protein, but induced phosphorylation of the 78-, 72-, 65-, and 56-kDa proteins. These results indicate that the beta2 integrin-mediated intracellular signaling cascade involves different tyrosine phosphorylation (and dephosphorylation) events, which may play a role in the regulation of PMN functions during inflammation.

Animals↗
PubMed1981

Further evidence for translational regulation of tyrosine aminotransferase synthesis by dibutyryl cyclic AMP in Reuber H35 hepatoma cells.

Cyclic AMP derivatives increase the rate of synthesis of tyrosine aminotransferase in Reuber H35 hepatoma cells. Various studies lend support to the hypothesis that cyclic AMP increases the synthesis of tyrosine aminotransferase by acting at a posttranscriptional site. The presence of a limited non-translatable pool of tyrosine aminotransferase mRNA prior to the formation of the translatable tyrosine aminotransferase mRNA implicates a possible site of action of cyclic AMP. We compared the capacity of N6,O2'-dibutyryl cyclic AMP to induce tyrosine aminotransferase synthesis when untranslatable tyrosine aminotransferase mRNA sequences are present or absent. The transition of a condition in which non-translatable tyrosine aminotransferase mRNA sequences were present to a condition in which they were absent was established by preinduction of Reuber H35 cells with dexamethasone, followed by addition of actinomycin D. In the time period thereafter, the amount of non-translatable mRNA decreased and 1.5-2 h after addition of actinomycin D, only translatable tyrosine aminotransferase mRNA was present. It can be seen that the induction of tyrosine aminotransferase synthesis by dibutyryl cyclic AMP follows the normal decrease of tyrosine aminotransferase mRNA. We present evidence that dibutyryl cyclic AMP in Reuber H35 hepatoma cells regulates tyrosine aminotransferase synthesis at a posttranscriptional site independent of the pool of non-translatable tyrosine aminotransferase mRNA sequences, but influencing the efficiency of translation of active tyrosine aminotransferase mRNA.

Animals↗
PubMed1984

The use of synthetic peptides for defining the specificity of tyrosine protein kinases.

The tyrosine protein kinases are a large family of enzymes that may be involved in regulating cell growth and differentiation. An important property of these enzymes is their substrate specificity. Defining the specificity of these enzymes will contribute to a greater understanding of their biological functions. Synthetic peptides provide a useful means for studying the specificities of protein kinases. The utility of synthetic peptides for specificity studies is exemplified by the results obtained with the cyclic nucleotide-dependent protein kinases. A large number of synthetic peptides have been tested as substrates for these enzymes. There are three important conclusions from this work. First, in terms of primary sequence, the two cyclic nucleotide-dependent protein kinases both require the presence of a pair of basic residues on the N-terminal side of the phosphorylatable residue. Second, recognition of a specific secondary structure in the substrate is an equally important factor in the substrate specificities of these enzymes. Third, the specificities of the two cyclic nucleotide-dependent protein kinases are remarkably similar in terms of both primary and secondary structure recognition. Sequences at the sites of tyrosine phosphorylation often show the presence of numerous acidic residues on the N-terminal side of the tyrosine. This result led to the suggestion that these acidic residues might be important for the recognition of these sites by the tyrosine protein kinases. Specificity studies using synthetic peptides have provided some experimental verification of this concept. In the case of four tyrosine protein kinases, LSTRA cell tyrosine protein kinase, epidermal growth factor receptor kinase, insulin receptor kinase and pp60gag-yes tyrosine protein kinase, the presence of acidic residues on the N-terminal side of the tyrosine in a synthetic peptide was a favorable determinant. In the case of a fifth member of the tyrosine protein kinase family, namely pp60src kinase, the data are less clear that the presence of acidic residues is involved in substrate recognition. This enzyme readily phosphorylated several peptides that did not have acidic residues on the N-terminal side of the tyrosine. Although there is some indication that the presence of acidic residues on the N-terminal side of the tyrosine is a factor in substrate recognition by several of the tyrosine protein kinases, the changes in kinetic parameters with the various peptide substrates were rather small. In addition, some sites phosphorylated in proteins do not have any acidic residues on the N-terminal side of the phosphorylated tyrosine residue.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2004

Simultaneous determination of tyrosine, phenylalanine and deoxyguanosine oxidation products by liquid chromatography-tandem mass spectrometry as non-invasive biomarkers for oxidative damage.

We developed an isotope dilution HPLC-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the simultaneous determination of p-tyrosine, phenylalanine, o,o'-dityrosine, m-tyrosine, o-tyrosine, 3-chlorotyrosine and 3-nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHdG) that requires no extensive sample pre-treatment. p-[(2)H(4)]Tyrosine and o,o'-[(2)H(6)]dityrosine were used as internal standards. Calibration curves of the method were linear (r(2)=0.990-0.999) over a concentration range of 0.03-10 microM for o-tyrosine; 0.04-10 microM for 3-nitrotyrosine and 3-chlorotyrosine; 0.05-10 microM for o,o'-dityrosine; and for m-tyrosine; 1.0-100 microM for p-tyrosine and for phenylalanine; and 0.01-10 microM for 8-OHdG. The detection limits were from 0.025 to 0.05 microM for the tyrosine derivatives; 0.01 microM for 8-OHdG; and 0.5 microM for p-tyrosine and for phenylalanine, respectively. Within-day coefficients of variation (CV) for spiked human urine samples ranged from 2.7 to 7.0%, except for 8-OHdG (13.7%). Between-day variations ranged from 7.9 to 13.0%, except for o-tyrosine (CV = 18.2%), and for 8-OHdG (CV = 24.7%). The background levels of p-tyrosine, phenylalanine, o,o'-dityrosine, and o-tyrosine in morning urine of eight healthy volunteers were 3890+/-590, 3420+/-730, 5.8+/-0.3, and 9.2+/-1.5 micromol/mol creatinine, respectively. Using the present HPLC-APCI-MS/MS method, the urinary background levels of m-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine and 8-OHdG were below the limit of detection.

Calibration↗
PubMed2006

Misincorporation of free m-tyrosine into cellular proteins: a potential cytotoxic mechanism for oxidized amino acids.

In vitro studies demonstrate that the hydroxyl radical converts L-phenylalanine into m-tyrosine, an unnatural isomer of L-tyrosine. Quantification of m-tyrosine has been widely used as an index of oxidative damage in tissue proteins. However, the possibility that m-tyrosine might be generated oxidatively from free L-phenylalanine that could subsequently be incorporated into proteins as an L-tyrosine analogue has received little attention. In the present study, we demonstrate that free m-tyrosine is toxic to cultured CHO (Chinese-hamster ovary) cells. We readily detected radiolabelled material in proteins isolated from CHO cells that had been incubated with m-[14C]tyrosine, suggesting that the oxygenated amino acid was taken up and incorporated into cellular proteins. m-Tyrosine was detected by co-elution with authentic material on HPLC and by tandem mass spectrometric analysis in acid hydrolysates of proteins isolated from CHO cells exposed to m-tyrosine, indicating that free m-tyrosine was incorporated intact rather than being metabolized to other products that were subsequently incorporated into proteins. Incorporation of m-tyrosine into cellular proteins was sensitive to inhibition by cycloheximide, suggesting that protein synthesis was involved. Protein synthesis using a cell-free transcription/translation system showed that m-tyrosine was incorporated into proteins in vitro by a mechanism that may involve L-phenylalanine-tRNA synthetase. Collectively, these observations indicate that m-tyrosine is toxic to cells by a pathway that may involve incorporation of the oxidized amino acid into proteins. Thus misincorporation of free oxidized amino acids during protein synthesis may represent an alternative mechanism for oxidative stress and tissue injury during aging and disease.

Animals↗
PubMed2003

Pharmacokinetics of systemically administered tyrosine: a comparison of serum, brain tissue and in vivo microdialysate levels in the rat.

Tyrosine uptake has been reported to differ across brain regions. However, such studies have typically been conducted over brief intervals and in anesthetized rats; anesthesia itself affects amino acid transport across the blood-brain barrier. To address these concerns, serum, brain tissue and in vivo microdialysate tyrosine levels were compared for 0-3 h after administration of tyrosine [0.138-1.10 mmol/kg intraperitoneally (i.p.)] to groups of awake rats. Serum and brain tissue tyrosine levels increased linearly with respect to dose. Basal tissue tyrosine levels varied significantly across brain regions [medial prefrontal cortex (MPFC), striatum, hypothalamus, and cerebellum], but the rate of tyrosine uptake was similar for hypothalamus, striatum and MPFC. For brain regions in which tyrosine levels in both microdialysate and tissue were assayed, namely MPFC and striatum, there was a high degree of correlation between tyrosine levels in tissue and in microdialysate. Increasing brain tyrosine levels had no effect on DA levels in MPFC microdialysate. We conclude that (i) regional differences in the response of dopamine neurons to systemic tyrosine administration cannot be attributed to pharmacokinetic factors; (ii) in vivo microdialysate provides an excellent index over time and across a wide range of tyrosine doses, of brain tissue tyrosine levels; and (iii) increases in brain tyrosine levels do not affect basal DA release in the MPFC.

Animals↗
PubMed1998

Tyrosine supplementation in the treatment of maternal phenylketonuria.

Lower than average concentrations of tyrosine could be a factor in the fetal damage produced by maternal phenylketonuria (PKU). Dietary supplementation with L-tyrosine has been inconsistent in these reported pregnancies. Consequently, we studied the response to supplementation with L-tyrosine in five maternal PKU pregnancies. Before supplementation the mean plasma tyrosine concentration during midpregnancy was only 34 micromol/L compared with the expected value of 45 micromol/L in the normal population. A single dose of 1.3 g L-tyrosine was sufficient to raise plasma tyrosine concentrations well above the recommended minimum concentration of 45 micromol/L. The response was rapid and sustained over a 3-h study period in each subject and was associated with a markedly increased ratio of tyrosine to large neutral amino acids. These results indicate that the plasma tyrosine concentration can be increased to normal or above-normal values in maternal PKU pregnancies for a period of > or = 3 h by supplementation of the diet with L-tyrosine. The high ratio of tyrosine to large neutral amino acids indicates that tyrosine might readily cross the placenta and provide sufficient tyrosine to the fetus to support normal protein and catecholamine synthesis in what otherwise might be a tyrosine-poor environment.

Female↗