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PubMed1999

Ectopic expression of non-catecholaminergic tyrosine hydroxylase in rat hypothalamic magnocellular neurons.

Hypothalamic magnocellular neurons constitute a good model of neurochemical plasticity, because a single neuron can express various combinations of neuropeptides and enzymes under different physiological conditions. Tyrosine hydroxylase has been shown to occur ectopically in various non-catecholaminergic neurons. We investigated the expression of tyrosine hydroxylase and its possible role in the magnocellular neurons of the supraoptic and paraventricular nuclei in salt-loaded and lactating rats, using in situ hybridization and immunohistochemistry, alone or combined, in light and electron microscopy. Our results demonstrated that almost 25% of the magnocellular neurons in the supraoptic nucleus and 15% in the paraventricular nucleus expressed tyrosine hydroxylase in salt-loaded rats, and 10% in the supraoptic nucleus of two-day lactating rats. Double labelling showed that this tyrosine hydroxylase was essentially synthesized in magnocellular neurons expressing vasopressin. The ultrastructural localization of tyrosine hydroxylase was less homogeneous in the cytoplasm of magnocellular neurons than in periventricular neurons. In lactating and salt-loaded rats, magnocellular neurons were devoid of the catecholamine biosynthesis markers aromatic L-amino acid decarboxylase, L-3,4 dihydroxyphenylalanine, dopamine and GTP-cyclohydrolase I. Tyrosine hydroxylase expression did not increase after rats were injected with reserpine. Our results indicate that the phenotype of the magnocellular neurons expressing tyrosine hydroxylase in lactating and salt-loaded rats is non-catecholaminergic, and suggest that this tyrosine hydroxylase might be involved in osmoregulation.

Animals↗
PubMed1999

Effect of pre- and postnatal ethanol exposure on protein tyrosine kinase activity and its endogenous substrates in rat cerebral cortex.

The rat brain contains high levels of tyrosine-specific protein kinases (PTKs) that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu4Tyr1). Using this peptide as a substrate, we have measured the protein tyrosine kinase activity in membrane and cytosolic fractions from the cerebral cortices of pre- and postnatal ethanol-exposed rats at time intervals of 8, 30, and 90 days. During the course of development of the cerebral cortex, PTK activity decreased both in the membrane and cytosolic fractions from 8 and 90 days of age. Maximum activity was associated at the age of 8 days and gradually declined in the later ages (30 and 90 days) of postnatal development. However, PTK activity in the ethanol exposed rat cerebral cortex was further decreased when compared to controls in all the ages of postnatal development in membrane as well as in cytosolic fractions. In the presence of vanadate, a specific inhibitor of protein tyrosine phosphatases (PTPs), the PTK activity increased, indicating that the balance between protein tyrosine kinase and protein tyrosine phosphatase might be lost during ethanol exposure. In addition, when using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were identified on an immunoblot of membrane and cytosolic fractions from the ethanol-exposed rat cerebral cortex. The immunoblot showed several phosphotyrosine-containing proteins with molecular weights of 114, 70, 36, 34, 32, 20, and 14 kDa that were present in the cerebral cortex. However, higher levels of immunoreactivity of these proteins were found in the ethanol-exposed membrane fractions when compared to control fractions-particularly at the age of 30 and 90 days. Two phosphotyrosine proteins with molecular weights of 38 and 40 kDa showed decreased immunoreactivity at the age of 90 days in the cytosolic fraction of an ethanol-exposed rat's cerebral cortex. The differences in tyrosine-specific protein kinase activity and in phosphotyrosine-containing proteins observed during pre- and postnatal ethanol exposure may reflect specific functional defects in the cerebral cortex which could possibly underlie the mechanism contributing to fetal alcohol syndrome (FAS).

Aging↗
PubMed1996

Schistosoma mansoni: effect of recombinant tumor necrosis factor alpha on fecundity and [14C]-tyrosine uptake in females maintained in vitro.

It has been reported that recombinant tumor necrosis factor alpha (rTNF alpha) stimulates egg-laying in Schistosoma mansoni females. Because tyrosine requirement is increased in female undergoing sexual maturation in preparation for oogenesis and tyrosine is a major component of eggshell protein, we wanted to determine whether females treated with rTNF alpha would also incorporate more tyrosine. Adult females were first treated with 10, 20 or 40 ng/ml rTNF alpha for 1, 3 or 6 hr in RPMI 1640 containing 10% fetal calf serum (FCS). Another two groups of females were each exposed either to males or to male excretory-secretory (ES) products for 1 hr. They were then exposed to 20 microCi/ml-1 [14C]-tyrosine for 1 hr in RPMI 1640 containing 10% FCS. All females were incubated individually unless indicated otherwise. Females incorporated more tyrosine after exposure to males or their ES products. They incorporated significantly more tyrosine when treated with rTNF alpha for 1 hr; the increased uptake correlated with increasing amounts of rTNF alpha used. Although after a 3-hr treatment with 10 ng/ml rTNF alpha females incorporated slightly more tyrosine than controls, increasing amounts of rTNF alpha had an adverse effect. Females treated with rTNF alpha for 6 hr incorporated less tyrosine than controls and those treated for 1 hr. SDS-PAGE and fluorography did not reveal any differences in polypeptide profiles of untreated and rTNF alpha-treated females. These unexpected results led us to study the effect of rTNF alpha on fecundity in females. Contrary to the published report, we observed a sharp decline in egg-laying in females when exposed to increasing concentrations of rTNF alpha in vitro.

Animals↗
PubMed2002

Improved partitioning in aqueous two-phase system of tyrosine-tagged recombinant lactate dehydrogenase.

The partitioning of Bacillus stearothermophilus lactate dehydrogenase (LDH) in an aqueous two-phase system was studied. Particularly, the influence of tyrosine tags on the partitioning was evaluated. The hydrophobic effect, caused by the addition of tyrosine residues, was determined in a system based on dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer (EO30PO70). Five different LDH variants were constructed with N-terminal tags containing tyrosines (Y3 and Y6), tyrosines and prolines (Y3P2 and Y6P2), and only prolines (P2). LDH fused with tags containing tyrosines increased the partitioning coefficient, and the more tyrosines added to the protein, the larger improvement in partitioning. When prolines were added between the tyrosine-rich tag and the protein, a further increased partitioning was obtained. The enhanced partitioning was attributed to the rigid structure of the proline, which in turn led to an increase in the exposure of the tag to the surroundings. The best tyrosine tag, Y6P2, increased the partition coefficient four times and additionally, a higher thermostability was observed.

Amino Acid Sequence↗
PubMed1993

Formation of iron(III)-tyrosinate is the fastest reaction observed in ferritin.

Rapid mineralization of ferritin, characteristic of protein with H-type subunits, coincides with formation of a specific Fe(III)-tyrosinate complex. The pseudo-first-order rate constant for Fe(II) oxidation by H-subunit-type ferritin has now been shown to be 700-900 times greater than any previously reported for ferritin; kox = 1000 s-1 for formation of the specific Fe(III)-tyrosinate complex (A550nm) or formation of less defined Fe(III)-oxo multinuclear complexes (A420nm). Formation of multinuclear Fe(III)-oxo complexes and O2 consumption were biphasic. In the first phase, up to 50 Fe atoms/ferritin molecule were rapidly oxidized, accompanied by formation of the Fe(III)-tyrosinate complex; saturation of the sites which formed the Fe(III)-tyrosinate complex also required 50 Fe/ferritin molecule. The sigmoidal shape of the curve obtained by plotting the initial rate of oxidation during the rapid phase of mineralization versus added [Fe(II)] suggested a more complex reaction pathway of ferroxidation than previously described. During the second phase of mineralization, Fe(III)-tyrosinate decreased, but multinuclear Fe(III)-oxo complexes and O2 consumption continued to increase at a slower rate. Recovery of the rapid oxidation pathway paralleled recovery of the site for Fe(III)-tyrosinate formation; full regeneration of the Fe(III)-tyrosinate sites was gradual over a period of 12 h, as if the movement of Fe(III) along the biomineralization pathway in the protein was slow and was accompanied by conformational changes which affected the Fe(III)-tyrosinate site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates.

The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.

Bacteriorhodopsins↗
PubMed2001

Inhibition of tyrosine phenol-lyase from Citrobacter freundii by 2-azatyrosine and 3-azatyrosine.

The interactions of 2-azatyrosine and 3-azatyrosine with tyrosine phenol-lyase (TPL) from Citrobacter freundii have been examined. 2-Aza-DL-tyrosine and 3-aza-DL-tyrosine were synthesized by standard methods of amino acid synthesis, while the L-isomers were prepared from 3-hydroxypyridine and 2-hydroxypyridine, respectively, with TPL (Watkins, E. B., and Phillips, R. S. (2001) Bioorg. Med. Chem. Lett. 11, 2099-2100). 3-Azatyrosine was examined as a potential transition state analogue inhibitor of TPL. Both compounds were found to be competitive inhibitors of TPL, with K(i) values of 3.4 mM and 135 microM for 3- and 2-aza-L-tyrosine, respectively. Thus, 3-azatyrosine does not act as a transition state analogue, possibly due to the lack of tetrahedral geometry at C-1. However, 2-aza-L-tyrosine is the most potent competitive inhibitor of TPL found to date. The K(i) value of 2-aza-L-tyrosine is half that of 2-aza-DL-tyrosine, indicating that the D-enantiomer is inactive as an inhibitor. Neither azatyrosine isomer was shown to be a substrate for beta-elimination, based on coupled assays with lactate dehydrogenase and on HPLC measurements. Both isomers of azatyrosine form equilibrium mixtures of external aldimine and quinonoid intermediates when they bind to TPL. However, 2-azatyrosine reacts about 10-fold faster to form a quinonoid intermediate than does 3-azatyrosine. Since 2-azatyrosine is in the zwitterion or phenolate ion form at all the pH values examined, the strong binding of this compound suggests that L-tyrosine may be bound to the active site of TPL as the phenolate anion.

Alanine↗
PubMed1996

Ligand effects on the fluorescence properties of tyrosine-9 in alpha 1-1 glutathione S-transferase.

A conserved tyrosine plays a critical role in catalysis by mammalian glutathione S-transferases (GSTs) of the alpha-, mu-, and pi-classes, by forming a hydrogen bond to and stabilizing the thiolate form of glutathione. The hydrogen bonding properties of this tyrosine in the rat A1-1 GST (Tyr-9), in the absence and presence of ligands, have been studied by steady state and time-resolved fluorescence spectroscopy. In order to achieve this, the single tryptophan (Trp 21) found in the rat A1-1 GST has been replaced with the fluorometrically silent phenylalanine (W21F). Additionally, a double mutant lacking this tryptophan and the catalytic tyrosine (W21F:Y9F) has been constructed, and these mutants have been used as probes of ligand effects at Tyr-9. A comparison of the correlated excitation--emission spectra of the W21F mutant and the W21F-Y9F indicates that a red-shifted emission component is contributed by Tyr-9 with excitation bands at 255 and 300 nm, in the ligand-free enzyme. The pH-dependence of the intensity of these spectral cross-peaks is consistent with an active site tyrosine with a pKa of 8.1-8.3. Upon addition of GSH, the red-shifted component is quenched. Multifrequency phase/modulation fluorescence experiments qualitatively demonstrate that GSH causes a decrease in the average excited state lifetime on the red-edge of the spectrum of W21F but not of the W21F:Y9F spectrum. Steady state correlated difference spectra (W21F-W21F:Y9F) have been used to obtain a model for the excitation-emission correlated spectrum of Tyr-9, which indicates that Tyr-9 is heterogeneous at pH 7.5, with properties of both tyrosinate and "normal tyrosine". The tyrosinate fraction is eliminated, and the blue-shifted component becomes more intense upon addition of GSH conjugates, indicating that the weak hydrogen bond between Tyr-9 and thioethers has little charge-transfer character. The S-methyl GSH yields an "anomalous" spectrum at pH 7.5, which retains cross-peaks consistent with ionized tyrosinate. These results indicate that, in the absence of ligand, Tyr-9 forms a strongly polarized hydrogen bond or a fraction of the phenolic hydroxyl group is partially deprotonated. However, when a GSH conjugate with a sufficiently large hydrophobic group occupies the H-site, Tyr-9 is fully protonated, with little charge-transfer character.

Animals↗
PubMed1997

Tyrosyl interactions in the folding and unfolding of bovine pancreatic ribonuclease A: a study of tyrosine-to-phenylalanine mutants.

Three tyrosine-to-phenylalanine mutants of ribonuclease A (Y25F, Y92F, and Y97F) are investigated for their enzymatic activities, molecular stabilities, and unfolding/refolding kinetics. These mutants exhibit 80, 90, and 80%, respectively, of the catalytic activity of the wild-type enzyme. Thermal, Gdn.HCl, and pH transition measurements indicate that Y25F and Y97F are less stable than the wild-type protein, whereas the bulk of the thermodynamic and kinetic evidence indicates that Y92F is as stable as the wild-type protein. Differences in molar extinction coefficients indicate that tyrosines 25, 92, and 97 contribute 38, 13, and 39%, respectively, to the absorption difference between the folded and unfolded states, in general agreement with previous studies but possibly indicating the contribution of a fourth tyrosine residue to account for the remaining 10%. Stopped-flow single- and double-jump kinetic experiments were carried out on the wild-type and three mutant proteins. At least one tyrosine residue besides tyrosine 92 contributes to the slow fluorescence-unfolding phase; the likely candidate for this residue is tyrosine 115 which monitors the cis-trans isomerization of the X-Pro114 peptide bond. Tyrosines 25 and 97 are involved in interactions that retard conformational unfolding and accelerate conformational refolding as well as the cis-trans proline isomerization of the slow-refolding phases, presumably by stabilizing the major beta-hairpin structure of RNase A. These interactions may contribute to the strong pH dependence of the folding and unfolding of ribonuclease A. In contrast, tyrosine 92 does not affect the folding and unfolding rates significantly. An improved "box" model of proline isomerization under unfolding conditions was derived from exhaustive fitting of all possible box models. The kinetic data support the identification of Pro93 as the proline whose isomerization distinguishes the slow-refolding species (USII and USI) from the other, faster-refolding species (Uvf, Uf, and Um), implying that Pro93 isomerizes in the slow-refolding reactions USI --> N and IN --> N. Similarly, Pro114 seems to distinguish between the very fast-refolding species Uvf and the fast-refolding species Uf. Lastly, Pro117 seems to distinguish the major slow-refolding species USII from the minor slow-refolding species USI and the medium-refolding species Um from the fast- and very fast-refolding species.

Animals↗
PubMed1999

CCKA receptor activation stimulates p130(Cas) tyrosine phosphorylation, translocation, and association with Crk in rat pancreatic acinar cells.

p130(Cas) (Crk-associated substrate), because of its structure as an adapter protein, can interact when tyrosine-phosphorylated with a large number of cellular proteins and therefore be an important modulator of downstream signals. A number of growth factors, lipids, and a few G protein-coupled receptors can stimulate p130(Cas) tyrosine phosphorylation. Recent studies show that tyrosine phosohorylation of intracellular proteins by the hormone/neurotransmitter cholecystokinin (CCK) in rat pancreatic acinar cells may be an important signaling cascade. In this study, we show in rat dispersed pancreatic acini CCK-8 rapidly stimulates tyrosine phosphorylation of p130(Cas), reaching a maximum (6.6 +/- 1. 4)-fold increase with a half-maximal effect at 0.3 nM. Activation of protein kinase C by TPA or increases in [Ca2+]i by the calcium ionophore A23187 stimulated p130(Cas) phosphorylation. Blockade of CCK increases in [Ca2+]i or PKC activity did not alter CCK-8-stimulated p130(Cas) phosphorylation; however, simultaneous blockage of both cascades caused a 50% inhibition. Partial inactivation by C. botulinum toxin of the small GTP-binding protein Rho caused a 41 +/- 12% decrease in the CCK-stimulated p130(Cas) phosphorylation. Disruption of the actin cytoskeleton with cytochalasin D, but not the microtubule network with colchicine, completely inhibited CCK-8-stimulated p130(Cas) phosphorylation. Total p130(Cas) under basal conditions was largely localized (70 +/- 2%) in the membrane fraction, and stimulation with CCK-8 induced total p130(Cas) translocation from the cytosolic fraction. CCK stimulation also caused a (5 +/- 1)-fold increase in p130(Cas) tyrosine phosphorylated in the plasma membrane. Treatment with tyrphostin B44 inhibited CCK-8-stimulated p130(Cas) phosphorylation, but it had no effect on p130(Cas) translocation. CCK-8 caused rapid formation of a p130(Cas)-Crk complex. In conclusion, our results demonstrate CCKA receptor activation causes rapid tyrosine phosphorylation of p130(Cas) through PLC-dependent and -independent mechanisms that require the participation of the small GTP-binding protein Rho and the integrity of the actin cytoskeleton, but not the microtubule network. Moreover, CCKA receptor activation causes translocation of p130(Cas) to the membrane and an increase in membrane tyrosine-phosphorylated p130(Cas). The translocation to the membrane does not require antecedent tyrosine phosphorylation. CCKA activation promotes the rapid formation of a p130(Cas)-Crk complex. These results suggest that p130(Cas) is likely an important modulator of downstream signals activated by CCK-8, possibly involved in regulating numerous cellular effects, such as effects on cell growth or cell shape.

Actin Cytoskeleton↗
PubMedDate not supplied

Tyrosine-bearing polyphosphazenes.

Tyrosine-functionalized polyphosphazenes were synthesized, and their hydrolytic stability, pH-sensitive behavior, and hydrogel-forming capabilities were investigated. The physical and chemical properties of the polymers varied with the type of linkage between the tyrosine unit and phosphazene backbone. Poly[(ethyl glycinat-N-yl)(ethyl tyrosinat-N-yl)phophazenes] (linkage via the amino group of tyrosine) were found to be hydrolytically erodible. The rate of hydrolysis was dependent on the ratio of the two side groups, the slowest rate being associated with the highest concentration of tyrosine. The hydrolysis products were identified as phosphates, tyrosine, glycine, ammonia, and ethanol derived from the ester group. The hydrolytically stable phenolic-linked tyrosine derivatives were prepared from N-t-BOC-L-tyrosine methyl ester and alkoxy-based cosubstituents. Polyphosphazenes with both propoxy and phenolic-linked tyrosine side groups showed a pH-sensitive solubility behavior, which was dependent on the ratio and nature of the two side groups. The polymer was soluble in aqueous media below pH 3 and above pH 4. From pH 3-4, the polymer was insoluble. Replacement of propoxy by trifluoroethoxy units yielded a polymer that was insoluble in aqueous media at all pH values. Replacement of propoxy by methoxyethoxyethoxy groups gave a polymer that was soluble at all pH values. Exposure of both the propoxy and methoxyethoxyethoxy polymers to calcium ions in aqueous media caused gel formation due to ionic cross-linking through the carboxylate groups.

Biocompatible Materials↗
PubMed2003

Redox-active tyrosine residues: role for the peptide bond in electron transfer.

Photosystem II (PSII) catalyzes the light-driven oxidation of water and reduction of plastoquinone. In PSII, redox-active tyrosine Z conducts electrons between the primary chlorophyll donor and the manganese cluster, which is the catalytic site. In this report, difference FT-IR spectroscopy is used to show that oxidation of redox-active tyrosine Z causes perturbations of the peptide bond. PSII data were acquired on control samples, as well as samples in which tyrosine was 2H4 (ring)-labeled. Comparison to model compound data, acquired both from tyrosinate and its 2H4 isotopomer, was performed. The PSII FT-IR spectrum exhibited vibrational bands that are assignable to imide and amide vibrational modes. In previous work, we have shown that oxidation of tyrosinate perturbs the terminal amino group of tyrosinate (Ayala, I.; Range, K.; York, D.; Barry, B. A. J. Am. Chem. Soc. 2002, 124, 5496-5505). Density functional calculations on tyrosinate supported the interpretation that the perturbation is due to spin delocalization onto the amino group. In tyrosine-containing dipeptides, perturbations of the peptide bond were observed. Therefore, the imide and amide perturbations observed here are attributed to spin delocalization into the peptide bond in PSII. Migration of the electron hole in PSII may be consistent with peptide bond involvement in tyrosyl radical-based electron-transfer reactions.

Amides↗
PubMed2006

Photoactivated h/d exchange in tyrosine: involvement of a radical anion intermediate.

The aromatic hydrogen nuclei of tyrosine are photochemically labile and exchange with deuterons in neutral D(2)O solution. The site meta to the ring hydroxyl substituent is preferentially deuterated, exhibiting a meta/ortho deuteration rate of approximately 4:1. In contrast with acid-catalyzed H/D exchange and with nearly all of the reported photoactivated H/D exchange studies, the UV-induced H/D exchange of tyrosine is optimal at pH 9 and is effectively quenched at acid pH. Photochemical H/D exchange is strongly stimulated by the alpha-amino group (the aromatic hydrogens of p-cresol are far less subject to exchange) and by imidazole or phosphate buffers. On the basis of the results obtained here and on the previously identified cyclohexadienyl radical (Bussandri, A.; van Willigen, H. J. Phys. Chem. A 2002, 106, 1524-1532), we conclude that the exchange reaction involves a radical intermediate and results from two distinct roles of tyrosine: (1) as a phototransducer of light energy into solvated electrons (e(aq)(-)), and (2) as an acceptor of an electron to create a radical anion intermediate which is rapidly protonated, yielding a neutral cyclohexadienyl radical. Regeneration of the tyrosine can occur via a bimolecular redox reaction of the cyclohexadienyl and phenoxyl radicals to yield a carbocation/phenoxide pair, followed by deprotonation of the carbocation. The oxidation step is pH dependent, requiring the deprotonated form of the cyclohexadienyl radical. The H/D exchange thus results from a cyclic one-electron (Birch) reduction/protonation/reoxidation (by phenoxyl radical)/deprotonation cycle. Consistent with these mechanistic conclusions, the aromatic hydrogens of tyrosine O-methyl ether are photochemically inert, but become labile in the presence of tyrosine at high pH. The deuteration rate of O-methyl tyrosine is lower than that of tyrosine and shows a preference for the ortho positions. This difference is proposed to result from a variation in the oxidation step, characterized by a preferential oxidation of a cyclohexadienyl resonance structure with the unpaired electron localized on the oxygen substituent.

Amino Acids↗
PubMed2000

Inactivation of wild-type BCR/ABL tyrosine kinase in hematopoietic cells by mild hyperthermia.

Temperature-sensitive mutants of BCR/ABL tyrosine kinase have been extensively used to study the mechanisms of cell transformation and signal transduction. However, little is known about the effect of temperature on the activity of wild-type BCR/ABL gene product. In this study, we demonstrate that in vivo tyrosine kinase activity of p210, p190 BCR/ABL and v-abl are temperature-sensitive when expressed in hematopoietic cells and decline when temperature is raised 2 degrees C above normal range. In vitro tyrosine kinase activities of purified recombinant Abl and immunoprecipitated p210 BCR/ABL were also sensitive to increased temperature. Tyrosine phosphorylation of cellular proteins was markedly reduced in BCR/ABL transformed cells after 16 h at 39 degrees C, whereas the expression of BCR/ABL was unchanged. Temperature-induced downregulation of BCR/ABL kinase activity was reversible when cells were shifted back to 37 degrees C. The downregulation of Abl tyrosine kinase activity was not influenced by mutation or deletion of SH2 or SH3 domains or mutation of the GRB2 binding site. No increase in functional activity or expression of protein-tyrosine phosphatases, PTP-1B, SH-PTP1 or SH-PTP2 was detected in cells grown at 39 degrees C. Temperature-induced downregulation in tyrosine kinase activity correlated with decline in phosphotyrosine-associated PI 3-kinase whereas there was no change in growth factor independence of transformed hematopoietic cells. In conclusion, Abl tyrosine kinase has intrinsic sensitivity to temperature and BCR/ABL expressed in hematopoietic cells is downregulated by increasing temperature 2 degrees C. These observations provide a unique opportunity to identify cellular factor(s) which regulate BCR/ABL kinase in vivo and suggests possible novel treatment of CML by a mild hyperthermia.

3T3 Cells↗
PubMed2006

Effect of acute tyrosine depletion in using a branched chain amino-acid mixture on dopamine neurotransmission in the rat brain.

Central dopamine function is reduced by decreasing the availability of the catecholamine precursor, tyrosine, using a tyrosine-free amino acid mixture containing multiple large neutral as well as branched chain amino-acids, which compete with tyrosine for uptake into the brain. Current mixtures are cumbersome to make and administer, and unpalatable to patients and volunteers. Here, we investigate whether individual or limited amino-acid combinations could reduce brain tyrosine levels and hence dopamine function. Measurements of regional brain tyrosine levels, catecholamine and indoleamine synthesis (L-DOPA and 5-HTP accumulation, respectively) were used to identify an effective paradigm to test in neurochemical, behavioral and fos immunocytochemical models. Administration of leucine or isoleucine, or a mixture of leucine, isoleucine, and valine reduced tyrosine and 5-HTP, but not L-DOPA accumulation. A mixture of leucine, valine, and isoleucine supplemented with tryptophan reduced brain tyrosine and L-DOPA, but not 5-HTP. In microdialysis experiments this amino-acid mixture reduced basal and amphetamine-evoked striatal dopamine release, as well as amphetamine-induced hyperactivity. This mixture also reduced amphetamine-induced fos expression in striatal areas. In conclusion, the present study identified a small combination of amino acids that reduces brain tyrosine and dopamine function in a manner similar to mixtures of multiple amino acids. This minimal mixture may have use as a dopamine reducing paradigm in patient and volunteer studies.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2000

Cell-cell adhesion-mediated tyrosine phosphorylation of nectin-2delta, an immunoglobulin-like cell adhesion molecule at adherens junctions.

We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, an immunoglobulin-like cell adhesion molecule, and afadin, an actin filament-binding protein which connects nectin to the actin cytoskeleton. Among the members of the nectin family, we have found here that nectin-2delta is tyrosine-phosphorylated in response to cell-cell adhesion. Expression of E-cadherin induced tyrosine phosphorylation of nectin-2delta, while disruption of cell-cell adhesion by an anti-E-cadherin antibody reduced the tyrosine phosphorylation of nectin-2delta. An inhibitor specific for Src family kinase or expression of Csk reduced tyrosine phosphorylation of nectin-2delta. In addition, Src kinase tyrosine phosphorylates the recombinant cytoplasmic region of nectin-2delta in vitro. The major tyrosine phosphorylation site of nectin-2delta was Tyr505 in the cytoplasmic region, because the mutant nectin-2delta, of which Tyr505 was replaced by Phe, showed a loss of tyrosine phosphorylation in vivo and in vitro. These results, together with our recent observations, indicate that the cadherin-catenin system and the nectin-afadin system are closely connected to each other. The cadherin-mediated cell-cell adhesion system may link to the activation of a Src family kinase, that is, at least in part, responsible for the tyrosine phosphorylation of the cytoplasmic region of nectin-2delta. Oncogene (2000) 19, 4022 - 4028.

Animals↗
PubMed2000

Tyrosine kinases and gastric cancer.

Carcinoma of the stomach is one of the most prevalent cancer types in the world today. Two major forms of gastric cancer are distinguished according to their morphological and clinicopathological classifications (well differentiated/intestinal type and poorly differentiated/diffuse type), characteristics that could also be attributed to the altered expression of different types of oncogenes or tumor suppressor genes. Significant differences exist for gastric cancer incidence comparing people of different ethnic origins, implicating various genetic and epigenetic factors for gastric oncogenesis. There are only a limited number of molecular markers available for gastric cancer detection and prognostic evaluation, among which are tyrosine kinases. There is convincing evidence that tyrosine kinases are involved in oncogenesis and disease progression for many human cancers. Amplifications of certain tyrosine kinases (c-met, k-sam and erbB2/neu) have been associated with human gastric cancer progression. Alternatively spliced transcripts and enhanced protein-expression levels for some of these tyrosine kinases are correlated with clinical outcomes for gastric cancer patients. With advent of high throughput techniques, it is now possible to detect nearly all expressed tyrosine kinases in a single screen. This increases the chance to identify additional tyrosine kinases as predictive markers for gastric cancers. In this article, we will first review the literature data concerning certain tyrosine kinases implicated in gastric carcinogenesis and then summarize more recent work which provide comprehensive tyrosine kinase profiles for gastric cancer specimens and cell lines. Two new gastric cancer molecular markers (tie-1 and mkk4) have been identified through the use of these profiles and demonstrated effective as clinical prognostic indicators.

Humans↗
PubMed2006

Minimal features of paxillin that are required for the tyrosine phosphorylation of focal adhesion kinase.

Tyrosine phosphorylation of FAK (focal adhesion kinase) regulates signalling that results from the interaction of integrins with extracellular matrix and growth factor receptors. A critical step in this process is the phosphorylation of Tyr397 of FAK, which creates a binding site for Src family kinases, PI3K (phosphoinositide 3-kinase) and Shc (Src homology and collagen homology). An intact Tyr397 site is required for FAK-mediated regulation of cell migration, survival signals and full responsiveness to soluble growth factors. We showed previously that the adaptor protein paxillin is required for the overall tyrosine phosphorylation of FAK in embryonic stem cells [Wade, Bohl and Vande Pol (2002) Oncogene 21, 96-107]. In the present paper, we identify the minimal structural features of paxillin that are required to support overall FAK tyrosine phosphorylation and Tyr397 phosphorylation. Paxillin contains N-terminal leucine-rich LD motifs that bind directly to FAK and four LIM (Lin-11, Isl-1 and Mec-3) domains in the C-terminus. We show that paxillin LIM domains 1, 2 and 3 are each required for FAK tyrosine phosphorylation, while LIM4 is dispensable. In addition to paxillin LIM domains 1, 2 and 3, a single LD motif on paxillin is required to support FAK tyrosine phosphorylation in embryonic stem cells. Both sequence and spatial requirements exist for LD motifs to support FAK tyrosine phosphorylation. Interestingly, synthetic LD motifs that fail to bind FAK in vitro are able to fully support FAK tyrosine phosphorylation, indicating that minimal interactions of LD motifs with FAK suffice. Our results demonstrate at least four distinct structural domains of paxillin support at least three distinct functions that are each required for FAK tyrosine phosphorylation.

Amino Acid Motifs↗