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PubMed1983

Different acute effects of the tyrosine hydroxylase inhibitors alpha-methyl-p-tyrosine and 3-iodo-L-tyrosine on hypothalamic noradrenaline activity and adrenocorticotrophin release in the rat.

Computerized gas chromatography-mass spectrometry techniques using selected ion monitoring and deuterated internal standards were used to assay simultaneously the medial basal hypothalamic concentrations of dopamine (DA) and noradrenaline (NA) and their major metabolites in individual rats 30 min after the administration of two different inhibitors of tyrosine hydroxylase, alpha-methyl-p-tyrosine (alpha-MT) and 3-iodo-L-tyrosine (MIT). Consistent with inhibition of DA synthesis, administration of both alpha-MT and MIT resulted in marked reductions (P less than 0.005) in the hypothalamic concentrations of DA and its metabolite homovanillic acid as well as in highly significant increases in prolactin secretion. alpha-MT administration, but not MIT, resulted in a highly significant decrease in NA concentration and a highly significant increase in the concentration of the NA metabolite 3,4-dihydroxyphenylethyleneglycol (DHPG). The hypothalamic ratio DHPG/NA was thus markedly increased (P less than 0.005) by alpha-MT indicating increased NA neuronal activity. alpha-MT administration also resulted in increased ACTH secretion (P less than 0.0005), an effect not observed following MIT. It is proposed that the effects on hypothalamic NA activity and ACTH secretion caused by alpha-MT are stress-mediated and unrelated to tyrosine hydroxylase inhibition. MIT is devoid of these effects but exhibits blockade activity, thus indicating it to be a preferable drug for the acute inhibition of tyrosine hydroxylase in neuroendocrine investigations.

Adrenocorticotropic Hormone↗

PubMed1994

Comparative study of three protein-tyrosine phosphatases. Chicken protein-tyrosine phosphatase lambda dephosphorylates c-Src tyrosine 527.

To examine the substrate preference of protein tyrosine phosphatases (PTPs), we compared the activity of three transmembrane PTPs on dephosphorylation and regulation of c-Src and v-Src: chicken PTP lambda (ChPTP lambda), chicken PTP alpha (ChPTP alpha), and human leukocyte common antigen-related molecule (HLAR). In vitro, all three PTPs dephosphorylated v-Src, but only ChPTP lambda dephosphorylated c-Src. Their activities were also compared in Cos cells coexpressing Src and the phosphatase domains of three PTPs. These domains were fused with peptides for myristylation, so they associated with the cellular membrane. When c-Src was coexpressed with myrPTP lambda, its kinase activity was elevated 3-4-folds. This activation was less obvious when c-Src was coexpressed with myrPTP alpha or myrLAR. Analysis by cyanogen bromide cleavage showed that ChPTP lambda and myrPTP lambda dephosphorylated Tyr-527 of c-Src. Our data demonstrated the different activities of three PTPs on phosphoproteins, suggesting that Src Tyr-527 may require more specific PTP(s) than Src Tyr-416 for dephosphorylation in vivo.

Amino Acid Sequence↗

PubMed1998

Formation and fate of tyrosine. Intracellular partitioning of newly synthesized tyrosine in mammalian liver.

Tyrosine in an hepatocyte is transported from the plasma, synthesized from phenylalanine, or released during protein turnover. Effects of phenylalanine and tyrosine on the formation and fate (partitioning) of tyrosine from the different sources were examined in primary rat hepatocyte cultures. Rates of tyrosine degradation, transport, incorporation into and release from protein, and synthesis from phenylalanine were measured as well as the intracellular dilution of labeled tyrosine and phenylalanine incorporated into protein. We found tyrosine had little effect on phenylalanine hydroxylation over a wide range of conditions, that transported tyrosine and tyrosine from phenylalanine are in different metabolic pools, and that there appears to be channeling of newly synthesized tyrosine during degradation. In addition, under some conditions, intracellular partitioning of tyrosine is determined by tyrosine concentration. Specifically, if extracellular tyrosine is low and phenylalanine is at a normal plasma level, tyrosine use in protein synthesis takes precedence over tyrosine degradation or export. It is proposed that the mechanism controlling this is kinetic, based on relative rates of tyrosyl-tRNA formation and tyrosine degradation and export. A quantitative model of tyrosine and phenylalanine in-flow and out-flow in hepatocytes is given, incorporating tyrosine synthesis, degradation, plasma membrane transport, and tyrosine and phenylalanine use and release during protein turnover.

Animals↗

PubMed1989

Phenylalanine and tyrosine metabolism in renal failure: dipeptides as tyrosine source.

Several lines of evidence suggest that tyrosine formation is impaired in renal failure. The concentration of tyrosine is decreased and the phenylalanine/tyrosine ratio is increased in plasma and in skeletal muscle cells. After an oral or intravenous load, the rise of plasma phenylalanine is augmented, the clearance is decreased, oxidation is diminished and the corresponding rise of plasma tyrosine level is blunted. Tyrosine elimination and oxidation are not altered in uremia. The defect in tyrosine formation may be especially important in uremic patients on a low protein diet supplemented with tyrosine-free essential amino acid preparations and in subjects on artificial nutritional support. Thus, tyrosine should be regarded as a conditionally essential amino acid in renal failure and should be supplied exogenously, at least in these patient groups. Oral tyrosine supplementation was shown to replete plasma and intracellular pools and improve nitrogen balance in chronic renal failure patients on a low protein diet. However, because of poor solubility in aqueous solutions, tyrosine cannot be included in the free form in amino acid solutions for parenteral nutrition. To circumvent stability or solubility problems, tyrosine containing dipeptides and/or N-acetyl-tyrosine may serve as tyrosine sources for parenteral supply. Renal failure does not affect alanyl-tyrosine hydrolysis, and there is an immediate increase of plasma tyrosine concentration after peptide infusion. Elimination and hydrolysis of glycine-tyrosine is retarded in renal failure, but the clearance exceeds clinically relevant infusion rates. After infusion of N-acetyl-tyrosine, no increase in plasma tyrosine is seen, and the half-life N-acetyl-tyrosine is grossly prolonged in uremia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Tyrosine availability and brain noradrenaline synthesis in the fetus: control by maternal tyrosine ingestion.

In spite of substantial evidence that tyrosine availability modulates catecholamine synthesis, under a variety of conditions, in adult brains, its possible influence during brain development has received little attention. In the present paper, the effect of tyrosine when administered to pregnant rats on tyrosine and noradrenaline brain levels in the fetus was investigated. In the first experiment noradrenaline (NA) was detected in the fetal brain by day 13 of development. NA levels increased from days 13 to 19. Administration of tyrosine to the mother raises both tyrosine and NA levels in the fetal brain. The increase in tyrosine was similar on days 13, 15, 17 and 19 of pregnancy. However, the increase of NA was more pronounced on days 17 and 19 than on days 13 or 15. Tyrosine and NA enhancement was detected 30 min after tyrosine administration, persisted for at least 2 h and disappeared completely 6 h after its administration. Both tyrosine and NA increase are tyrosine dose-related. The present studies suggest that there are no important barriers for tyrosine transport between the maternal blood and the fetal brain. Furthermore, during prenatal life the rate of NA synthesis is regulated by tyrosine brain concentration and therefore maternal intake of tyrosine is an important factor for noradrenaline synthesis regulation in the fetal brain. The possible functional significance of noradrenaline brain increase after tyrosine administration are unknown. However, the NA modification could be the cause of the persistent behavioral modification that we have found in adult rats whose mothers were treated with tyrosine during pregnancy.

Animals↗

PubMed1989

Brain tyrosine increases after treating with prodrugs: comparison with tyrosine.

After mice had been treated with L-tyrosine, O-phospho-L-tyrosine, L-tyrosine methyl ester or N-acetyl-L-tyrosine, tyrosine was assayed by HPLC coupled with fluorometric detection. O-Phospho-L-tyrosine behaved as a tyrosine prodrug after its hydrolysis by acid and alkaline phosphatases. After the intraperitoneal administration of O-phospho-L-tyrosine or the methyl ester, there was a substantial increase in bioavailability in terms of the effect of tyrosine. The two prodrugs were as powerful as tyrosine following oral administration. N-Acetyl-L-tyrosine was the least effective prodrug tested. The stability, solubility and bioavailability of O-phospho-L-tyrosine are consistent with proposing it for use as a tyrosine prodrug. In addition, it can be used parenterally. The use of a tyrosine aminotransferase inhibitor is necessary for limiting the hepatic breakdown of tyrosine and for increasing its bioavailability.

Animals↗

PubMed1985

N-acetyl-L-tyrosine as a tyrosine source during total parenteral nutrition in adult rats.

The tyrosine content of parenteral solutions is limited by poor tyrosine solubility. N-acetyl-L-tyrosine has excellent solubility and is a potential source of intravenous tyrosine. Infusion of N-acetyl-U-14C-L-tyrosine as part of a total parenteral nutrition regimen in the rat at a level of 0.5 mmol/kg/day resulted in rapid labeling of tissue tyrosine pools, production of 14CO2, incorporation of 14C-labeled tyrosine into protein, and modest urinary losses (8.3%). Plasma tyrosine levels, however, remained at fasting values (73.8 +/- 5.40 microM). Infusion of N-acetyl-L-tyrosine at 2 mmol/kg/day increased plasma tyrosine above fasting levels (141 +/- 16.1 microM), resulted in a rapid labeling of tissue tyrosine pools, production of 14CO2, and incorporation of 14C-labeled tyrosine into protein. However, urinary losses were higher (16.8%). Rapid utilization of N-acetyl-L-tyrosine was noted at both infusion levels. Plasma- and tissue-free tyrosine pools were rapidly labeled, as was tissue protein. Radioactivity incorporated in tissue protein was shown to be tyrosine after acid hydrolysis.

Amino Acids↗

PubMed1989

Cardiovascular effects of L-tyrosine: influence of blockade of tyrosine metabolism.

Tyrosine is the precursor of catecholamines. Small doses of tyrosine produce tachycardia and hypertension while higher doses produce bradycardia and hypotension in anaesthetised rats. The mechanism of these effects has not been established. An increased synthesis and release of catecholamines has been suggested to be the mechanism. Various pretreatments were given to anaesthetised Wistar rats to study the influence of a blockade of L-tyrosine metabolism and thus a blockade of catecholamine synthesis, on these cardiovascular effects: valine, which inhibits tyrosine uptake into brain, alpha-methyl-p-tyrosine, which blocks the rate-limiting enzyme, tyrosine hydroxylase, carbidopa and benserazide, which both inhibit dopa decarboxylase, and desipramine, which blocks catecholamine re-uptake. Benserazide and alpha-methyl-p-tyrosine partially blocked the stimulatory effects of tyrosine. None of the pretreatments were able to block effectively the inhibitory effects of L-tyrosine. Therefore, the metabolism of tyrosine to form catecholamines may be involved in the stimulatory but not in the inhibitory cardiovascular effects of L-tyrosine. Valine pretreatment did not antagonize the depressant effects of tyrosine. Since valine blocks the uptake of L-tyrosine into the brain, the depressant effects of L-tyrosine might be peripheral rather than central in origin.

Animals↗

PubMed1997

Tyrosine kinetics and requirements during total parenteral nutrition in the neonatal piglet: the effect of glycyl-L-tyrosine supplementation.

Tyrosine may be a conditionally indispensable amino acid in the neonate; however, the provision of aromatic amino acids to neonates receiving total parenteral nutrition (TPN) is complicated by the poor solubility of crystalline tyrosine. In the present study, we investigated tyrosine kinetics and requirements during TPN, when tyrosine was supplied as the soluble dipeptide, glycyl-L-tyrosine in a neonatal piglet model. Fifteen 3-d-old male Yorkshire piglets were fitted with external jugular and femoral catheters and randomized to one of five tyrosine intakes: 0.11, 0.31, 0.41, 0.51 and 0.71 g.kg-1.d-1. Total parenteral amino acid and energy intakes were 15.0 g.kg-1.d-1 and 1.1 MJ.kg-1.d-1, respectively. Piglets were maintained on TPN for 6 d, with nitrogen balance measured over the final 3 d of the study. On the final study day, tyrosine kinetics were measured during a 4-h primed-constant infusion of L-[l14C]tyrosine. Nitrogen retention was 67% at the lowest tyrosine intake and increased significantly (p < 0.05) at intakes of 0.31 g.kg-1.d-1 and above (84, 86, 87, and 88% for intakes of 0.31, 0.41, 0.51, and 0.71 g.kg-1.d-1, respectively). Plasma tyrosine concentrations and tyrosine oxidation (expressed as either a percentage of the dose oxidized or when corrected for flux) were low and similar at the two lowest intakes, but increased significantly at the higher intakes. Two-phase regression analysis of the data (plasma tyrosine, tyrosine oxidation) yielded estimates of a mean tyrosine requirement of 0.31 and 0.35, respectively, with estimated safe intakes (upper 95% confidence limit) of 0.44 and 0.42 g.kg-1.d-1. The present work also indicates that oxidation techniques may be suitable for the estimation of amino acid requirements during TPN in the neonate.

Amino Acids↗

PubMed1988

Alterations in retinal tyrosine and dopamine levels in rats consuming protein or tyrosine-supplemented diets.

The ad libitum ingestion of casein diets varying in protein content altered serum and retinal levels of tyrosine. The serum tyrosine level rose when protein ingestion was increased from 6 to 24% casein. In rats consuming high-protein diets (40% casein), no further increase in serum tyrosine level occurred, although the levels of other large neutral amino acids, which compete with tyrosine for retinal uptake, continued to rise. The activity of the liver enzyme tyrosine aminotransferase varied directly with the percentage of protein in the diet and may partially explain the failure of chronic high-protein feeding to increase serum tyrosine levels. The retinal tyrosine concentration was significantly correlated with the serum tyrosine level and with the serum tyrosine ratio at all levels of protein intake. Retinal 3,4-dihydroxyphenylalanine synthesis and dopamine (DA) level varied in parallel with the level of the precursor, tyrosine. Addition of pure L-tyrosine (1, 2, or 4%) to normal protein diets resulted in a stepwise increase in serum and retinal tyrosine levels and retinal DA turnover. Alterations of retinal tyrosine level as a result of change in amount of dietary protein or by its addition to the normal diet can influence retinal DA synthesis and release.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1986

Site-directed mutagenesis of polyomavirus middle-T antigen sequences encoding tyrosine 315 and tyrosine 250.

Tyrosine residues of middle-T and tyrosine phosphorylation are thought to be important in the transformation of cultured rodent cells by polyomavirus. Of the potential tyrosine sites in the carboxyl-terminal half of middle-T, tyrosines 297, 315, and 322 have been studied previously, whereas tyrosine 250 has not. Two mutant plasmids, XD121 and pT250, encode polyomavirus middle-T species in which the tyrosine 250 residue is affected. XD121 is a deletion mutant in which the region encoding tyrosine 250, together with three adjacent amino acids, is deleted, whereas pT250 is a point mutant in which the tyrosine 250 codon has been converted to a phenylalanine codon. The plasmids were handicapped in transforming ability, as judged by focus formation on a monolayer of Rat-1 cells. Both demonstrated a reduction in the number of foci produced and a lag in the time of appearance of foci when compared with wild-type plasmid. The importance of residue 250 in this phenotype was indicated by the observation that plasmids containing multiple mutations proximal to the tyrosine 250 codon were wild type in their transforming ability. Furthermore, a revertant of pT250 (pT250-w.t.), which utilized the alternative tyrosine codon of TAC, was shown to regain full transforming activity. A combined-mutant plasmid, pTH, encodes a middle-T species in which both tyrosines 250 and 315 are converted to phenylalanine. This plasmid was totally defective in the transformation of rodent cells in a focus formation assay; however, it did impart a small measure of anchorage-independent growth when the encoded protein was expressed in NIH 3T3 cells. The in vitro kinase activity and pp60c-src association of the mutant middle-T antigens were examined. These assays demonstrated a reduction in phosphate acceptor activity for the middle-T species encoded by pT250 and pTH. Quantitative kinase assays showed that all of the tyrosine-mutant middle-T species, encoded by pAS131 (containing the tyrosine 315 codon-to-phenylalanine codon mutation), pT250, and pTH, were able to enhance pp60c-src kinase activity but only at levels which were intermediate and which reflected their transforming abilities relative to wild type.

Amino Acid Sequence↗

PubMed1994

Synthesis of phosphatidylinositol 3,4-bisphosphate is regulated by protein-tyrosine phosphorylation but the p85 alpha subunit of phosphatidylinositol 3-kinase may not be a target for tyrosine kinases in thrombin-stimulated human platelets.

To elucidate the mechanism involving synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), which is the main species of 3-phosphorylated phosphoinositides in activated blood platelets, we observed a correlation among protein-tyrosine phosphorylation, protein kinase C (PKC) activation, and PtdIns(3,4)P2 synthesis in these anucleate cells. Thrombin (1 U/ml) elicited marked protein-tyrosine phosphorylation, PKC activation, and PtdIns(3,4)P2 synthesis. In contrast, 1 microM 12-O-tetrade-canoylphorbol 13-acetate barely induced tyrosine phosphorylation and PtdIns(3,4)P2 synthesis although it strongly activated PKC. A variety of kinase inhibitors were tested for their ability to inhibit the thrombin effects. Both staurosporine and tyrphostin inhibited thrombin-stimulated tyrosine phosphorylation and PtdIns(3,4)P2 synthesis. H-7, which specifically, although weakly, inhibited PKC activation, had no effect on tyrosine phosphorylation and PtdIns(3,4)P2 production. Among the various kinase inhibitors tested, staurosporine was the most potent inhibitor of protein tyrosine phosphorylation and PtdIns(3,4)P2 synthesis, and there was a good correlation of the inhibition between these two parameters, although it also inhibited PKC activation. To examine the involvement of PtdIns 3-kinase, which is believed to play an important role in 3-phosphorylated phosphoinositide synthesis, we studied tyrosine phosphorylation and the association with tyrosine-phosphorylated proteins of the p85 alpha subunit of PtdIns 3-kinase in thrombin-stimulated platelets. We did not detect tyrosine-phosphorylated protein by Western blotting where p85 alpha was located. Similarly, when platelet lysates were precipitated with anti-p85 alpha antibodies and then blotted with anti-phosphotyrosine antibodies, tyrosine-phosphorylated p85 alpha was undetectable. Furthermore, when the cell lysates were precipitated with anti-phosphotyrosine antibodies, no p85 alpha was found in the immunoprecipitates. These results show that PtdIns(3,4)P2 synthesis in stimulated platelets is mediated by tyrosine phosphorylation, as it is in proliferating cells, but the p85 alpha subunit of PtdIns 3-kinase may not be a target for tyrosine kinases and that staurosporine, though non-specific, would be a useful tool for elucidating signal transduction involving D-3-phosphorylated phosphoinositide generation and protein-tyrosine phosphorylation in blood platelets.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

Parenteral glycyl-L-tyrosine maintains tyrosine pools and supports growth and nitrogen balance in phenylalanine-deficient rats.

Poor solubility hampers the addition of sufficient amounts of free tyrosine to parenteral amino solutions. We investigated the use of a highly soluble synthetic dipeptide, glycyl-L-tyrosine, as a parenteral tyrosine source in 18 male Wistar rats (body weight 180-200 g). The animals were randomized into three equal groups and catheterized to facilitate isoenergetic (1.2 MJ.kg-1.d-1) and isonitrogenous (1.25 g nitrogen.kg-1.d-1) total parenteral nutrition for 7 d. Controls (Group 1) received a complete amino acid solution, Group 2 received the same solution deficient in phenylalanine (nitrogen replaced with glycine), and group 3 received the phenylalanine-deficient solution supplemented with glycyl-L-tyrosine. Between d 4 and 7, weight gain and nitrogen retention were lower in Group 2 and in Group 1 or 3. In plasma and organ samples obtained at the end of the study, amino acids and dipeptides were analyzed by means of reversed phase-HPLC. In Group 2, phenylalanine and tyrosine concentrations were lower than in controls in plasma, muscle and kidney; in liver, only the tyrosine concentration was lower compared with controls. With glycyl-L-tyrosine supplementation, plasma, liver and kidney tyrosine concentrations and the phenylalanine:tyrosine ratio were normal. Intact glycyl-L-tyrosine was not detectable, suggesting a virtually quantitative elimination or utilization of the infused dipeptide. The results indicate that in phenylalanine-deficient rats, parenteral glycyl-L-tyrosine rapidly provides free tyrosine to facilitate normal growth, promote nitrogen metabolism and maintain intra- and extracellular tyrosine pools.

Amino Acids↗

PubMed1979

L-alanyl-L-tyrosine as a tyrosine source during total parenteral nutrition. Infusion at 0.5 and 2 mmoles/kg/day in adult rats.

Tyrosine peptides, such as L-alanyl-L-tyrosine, have excellent solubility and are potential sources of iv tyrosine. Infusion of L-alanyl-L-U-14C-tyrosine as part of a total parenteral nutrition regimen in the rat at a level of 0.5 mmole/kg/day resulted in rapid labeling of tissue tyrosine pools, production of 14CO2, incorporation of 14C-labeled tyrosine into protein, and minimal urinary losses (7.7%). Plasma tyrosine levels, however, remained at fasting. Infusion of L-alanyl-L-tyrosine at 2 mmole/kg/day increased plasma tyrosine above fasting levels and maintained tissue tyrosine at levels seen in orally fed control animals without increasing the percent lost in urine (5.5%). Rapid utilization of L-alanyl-L-tyrosine was noted at both infusion levels with no accumulation of peptide noted in plasma. Plasma and tissue free tyrosine pools were rapidly labeled, as was tissue protein. Radioactivity incorporated in tissue protein was shown to be tyrosine after acid hyrolysis.

Alanine↗

PubMed1983

Effects of dietary protein level and ascorbic acid supplementation on the contents of tyrosine metabolites in droppings and plasma of chicks fed a diet containing excess tyrosine.

A study on chickens was conducted to investigate whether or not: a) excess dietary tyrosine increases the content of tyrosine metabolites in plasma and excreta, b) these elevations of tyrosine metabolites are presented by increasing dietary protein level or supplementing with ascorbic acid (AA), and c) urine is a major excretory route of tyrosine metabolites. Chicks fed a 10% protein diet with excess tyrosine developed external foot lesions accompanied by retarded growth and depressed feed intake. These adverse effects were alleviated by elevating dietary protein level or supplementing with AA. Excreta and plasma of chicks fed the 10% protein diet contained small or undetectable amounts of free tyrosine, 4-hydroxyphenylpyruvate (4-HPP), 4-hydroxyphenylacetate (4-HPL), and 4-hydroxyphenylacetate (4-HPA), while these metabolites were markedly increased by the addition of excess tyrosine to the 10% protein diet. From the results with colostomized cocks, the major source of 4-HPP, 4-HPL, and 4-HPA excreted by chicks fed a tyrosine excess diet was considered more likely to be of urinary than fecal origin. Elevated contents of tyrosine and its metabolites in plasma were partially counteracted by increasing dietary protein level or AA supplementation. In excreta, elevated contents of tyrosine and its metabolites caused by excess tyrosine were reduced by increasing dietary protein level and supplementing with AA when expressed in the proportion of tyrosine intake. These results suggest that the beneficial effects of increased dietary protein level and supplementation with AA are related to enhanced ability of chicks to degrade excessively ingested tyrosine.

Animals↗

PubMed1992

Regulation of tyrosine hydroxylase gene transcription rate and tyrosine hydroxylase mRNA stability by cyclic AMP and glucocorticoid.

Tyrosine hydroxylase mRNA is induced in rat pheochromocytoma PC18 cells by cAMP analogs and glucocorticoids. Previous studies have shown that these increases in tyrosine hydroxylase mRNA are due at least in part to stimulation of the tyrosine hydroxylase gene. However, the involvement of post-transcriptional mechanisms in the regulation of tyrosine hydroxylase mRNA by these inducing agents has not been investigated. In the present study, using nuclear run-on assays we show that the relative transcription rate of the tyrosine hydroxylase gene is stimulated 2-5-fold within 20 min after treatment of PC18 cells with cAMP analogs or dexamethasone and that the rate of transcription remains elevated 2-3-fold for at least 24 hr in the continual presence of these inducing agents. Pulse-labeling experiments using 4-thiouridine indicate that the rate of synthesis of tyrosine hydroxylase mRNA is increased approximately 3-fold or 10-fold after treatment with either a cyclic AMP analog or dexamethasone, respectively. These increases in rates of synthesis agree well with the fold increases in tyrosine hydroxylase mRNA levels after treatment with these inducers. Treatment of the cells with cycloheximide lowers the basal relative transcription rate of the tyrosine hydroxylase gene 2-3-fold; however, the relative transcription rate of the tyrosine hydroxylase gene is still elevated in cells treated with either dexamethasone or cAMP analogs in the presence of cycloheximide, compared with the transcription rate of the gene in cells treated with cycloheximide alone. These results indicate that protein synthesis is not required for the short term regulation of the gene by these inducing agents. The apparent t1/2 for tyrosine hydroxylase mRNA has been estimated by two different procedures, approach to steady state kinetics and pulse-chase analysis. Both procedures yield an estimated apparent t1/2 of approximately 6-9 hr for tyrosine hydroxylase mRNA under basal culture conditions. Dexamethasone does not substantially alter this apparent t1/2 value; however, cAMP appears to lower this apparent t1/2 value transiently. Our results suggest that cAMP and glucocorticoid regulate tyrosine hydroxylase mRNA levels primarily by stimulating the transcription rate of the tyrosine hydroxylase gene; however, cAMP may also regulate the stability of the mRNA for a short period of time, such that it is induced more rapidly in the cells.

Animals↗

PubMed1996

Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis.

Erythropoietin (Epo), the primary in vivo stimulator of erythroid proliferation and differentiation, acts, in part, by altering the tyrosine phosphorylation levels of various intracellular signaling molecules. These phosphorylation levels are tightly regulated by both tyrosine kinases and tyrosine phosphatases. We have recently shown that the SH2 containing tyrosine phosphatase, Syp, binds directly to both the tyrosine phosphorylated form of the Epo receptor (EpoR) and to Grb2 after Epo stimulation of M07e cells engineered to express high levels of human EpoRs (T. Tauchi, et al: J Biol Chem 270:5631, 1995). To determine which tyrosine within the EpoR is responsible for binding Syp, we examined DA-3 cell lines expressing full-length mutant EpoRs bearing tyrosine to phenylalanine substitutions for each of the eight tyrosines within the intracellular domain of the EpoR. We found that: (1) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, coimmunoprecipitated with Syp; (2) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, bound to a GST-fusion protein containing both SH2 domains of Syp; (3) Jak2 could phosphorylate GST-Syp in vitro after Epo stimulation of wild-type (wt) EpoR expressing DA-3 cells; (4) Epo-stimulated tyrosine phosphorylation of Syp in vivo was markedly reduced in Y425F EpoR expressing DA-3 calls; and (5) DA-3 cells expressing the Y425F EpoR grow less well in response to Epo than wt EpoR expressing cells. These results suggest that Syp binds via its SH2 domains to phosphorylated Y425 within the EpoR and is then phosphorylated on tyrosine residues by Jak2. Moreover, Y425 in the EpoR reduces the Epo requirement for Syp tyrosine phosphorylation and promotes proliferation.

Animals↗

PubMed2000

H(2)O(2)-induced tyrosine phosphorylation of protein kinase cdelta by a mechanism independent of inhibition of protein-tyrosine phosphatase in CHO and COS-7 cells.

It has been proposed that H(2)O(2) increases tyrosine phosphorylation of cellular proteins by inhibiting protein-tyrosine phosphatase through oxidation of the cysteine residue of the enzyme essential for its catalytic activity. Tyrosine phosphorylation of the delta isoform of protein kinase C (PKC) was induced by H(2)O(2) in CHO and COS-7 cells. H(2)O(2) also induced activation of mitogen-activated protein kinase. Vanadate and molybdate, which inhibit protein-tyrosine phosphatase by binding to its active site, did not induce tyrosine phosphorylation of PKCdelta, but enhanced H(2)O(2)-induced tyrosine phosphorylation of PKCdelta in the cell. The oxoanions, however, generated the active form of mitogen-activated protein kinase. Another protein-tyrosine phosphatase inhibitor, phenylarsine oxide, which bridges the thiol residues of the enzyme, induced tyrosine phosphorylation of PKCdelta, and the reaction was enhanced by vanadate. These results suggest that inhibition of protein-tyrosine phosphatase is insufficient for induction of tyrosine phosphorylation of PKCdelta in the cells, and that presumably activation of protein-tyrosine kinase may be essential for tyrosine phosphorylation of the PKC isoform.

3T3 Cells↗