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PubMed1997

Induction of tyrosine hydroxylase by forskolin: modulation with age.

With aging, circulating catecholamines are elevated in both humans and animals. This may be related to the increased basal levels of tyrosine hydroxylase messenger RNA (mRNA) levels and tyrosine hydroxylase enzyme activity in the adrenal medulla of senescent compared with younger animals. In addition, tyrosine hydroxylase gene expression in the senescent rat is resistant to further stimulation by cold exposure as compared with younger animals. Collectively, these observations suggest either that tyrosine hydroxylase expression is already maximally stimulated in senescent rats or that tyrosine hydroxylase gene induction pathways are impaired with senescence. To help distinguish between these possibilities, we examined the induction of tyrosine hydroxylase mRNA, tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity in the adrenal medulla following forskolin administration to young and old F-344 rats. Forskolin at doses of 1.8 and 3.5 mg/kg increased tyrosine hydroxylase mRNA levels 2.5-fold in adrenal medulla from young rats but did not increase either tyrosine hydroxylase immunoreactivity or tyrosine hydroxylase enzyme activity 5 h after administration. Prolonged treatment with forskolin (3 doses, 12 h apart) increased tyrosine hydroxylase mRNA levels and tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity. In senescent rats, the baseline level of tyrosine hydroxylase mRNA was more than 2-fold higher compared with young rats. A single injection of the lower dose of forskolin increased tyrosine hydroxylase mRNA levels by the same increment in senescent as compared with young rats. These data indicate that the tyrosine hydroxylase gene in the adrenal medulla from senescent rats is still capable of further stimulation.

Adrenal Medulla↗
PubMed2003

CagA tyrosine phosphorylation in gastric epithelial cells caused by Helicobacter pylori in patients with gastric adenocarcinoma.

BACKGROUND: Tyrosine phosphorylation of Helicobacter pylori cytotoxin-associated protein of in gastric epithelial cells is reported. The goals of this study are first to examine the occurrence of CagA tyrosine phosphorylation in H. pylori strains isolated from patients with gastric adenocarcinoma and gastritis, and second to clarify the relationship between the diversity of tyrosine phosphorylation motifs and the presence of CagA tyrosine phosphorylation. METHODS: Fifty-eight clinical isolates of H. pylori from patients with gastric adenocarcinoma (29 cases) and gastritis (29 cases) were studied for CagA tyrosine phosphorylation by Western blotting. Sequence diversity of tyrosine phosphorylation motifs was analysed among positive- or negative-CagA tyrosine phosphorylation isolates. RESULTS: Positive CagA tyrosine phosphorylation was found in 93.1% (27 of 29) of strains from gastric adenocarcinoma patients and 51.7% (15 of 29) of strains from gastritis patients (p < 0.001). Intact motifs were found in H. pylori isolates with CagA tyrosine phosphorylation. Of the 16 negative CagA tyrosine phosphorylation isolates, intact tyrosine phosphorylation motifs were found in 15 isolates. CONCLUSIONS: CagA tyrosine phosphorylation, which is significantly greater in strains from gastric adenocarcinoma patients, may play a role in gastric carcinogenesis, and could be a better marker of more virulent strains than the cag pathogenicity island in Asia, where the cag pathogenicity island is present in nearly all H. pylori strains. Sequence diversity of tyrosine phosphorylation motifs on CagA was not related to the presence of tyrosine phosphorylation. The absence of tyrosine phosphorylation motif might result in negative tyrosine phosphorylation phenotypes, but such motifs are not the sole factors associated with CagA tyrosine phosphorylation.

Adenocarcinoma↗
PubMed1996

Large daily fluctuations in plasma tyrosine in treated patients with phenylketonuria.

In patients with phenylketonuria (PKU), extra tyrosine supplementation is advocated in addition to tyrosine-enriched amino acid mixtures. PKU patients have low fasting plasma tyrosine concentrations, but little is known about tyrosine fluctuations during the day. Plasma tyrosine concentrations were studied in 12 PKU patients in response to a test without breakfast and to three tests with different tyrosine contents in breakfast and lunch: 0%/30%, 25%/30%, 50%/10%, and 75%/10% tests, reflecting the protein consumption at breakfast and lunch, respectively. Prolonged fasting resulted in a small decrease in the already low overnight fasting plasma tyrosine concentrations. Breakfast and lunch with 25% and 30% of the daily tyrosine intake resulted in both lower than normal and higher than normal tyrosine concentrations. The 50%/10% and 75%/10% tests resulted in excessively high plasma tyrosine concentrations in most patients. Therefore, both lower than normal and higher than normal postprandial plasma tyrosine concentrations were found in treated PKU patients, even if the daily tyrosine intake was distributed evenly. When there was a large fractional tyrosine intake from one meal, very high plasma tyrosine concentrations were found. Therefore, strict control of plasma tyrosine is necessary if tyrosine supplementation is considered in addition to the tyrosine-enriched amino acid mixtures.

Adolescent↗
PubMed1996

Activation of the epidermal growth factor receptor signal transduction pathway stimulates tyrosine phosphorylation of protein kinase C delta.

The expression of an oncogenic rasHa gene in epidermal keratinocytes stimulates the tyrosine phosphorylation of protein kinase C delta and inhibits its enzymatic activity (Denning, M. F., Dlugosz, A. A., Howett, M. K., and Yuspa, S. H. (1993) J. Biol. Chem. 268, 26079-26081). Keratinocytes expressing an activated rasHa gene secrete transforming growth factor alpha (TGFalpha) and have an altered response to differentiation signals involving protein kinase C (PKC). Because the neoplastic phenotype of v-rasHa expressing keratinocytes can be partially mimicked in vitro by chronic treatment with TGF alpha and the G protein activator aluminum fluoride (AlF4-), we determined if TGF alpha or AlF4- could induce tyrosine phosphorylation of PKCdelta. Treatment of primary keratinocyte cultures for 4 days with TGFalpha induced tyrosine phosphorylation of PKCdelta, whereas AlF4- only slightly stimulated PKCdelta tyrosine phosphorylation. The PKCdelta that was tyrosine-phosphorylated in response to TGFalpha had reduced activity compared with the nontyrosine-phosphorylated PKCdelta. Treatment of keratinocytes expressing a normal epidermal growth factor receptor (EGFR) with TGFalpha or epidermal growth factor for 5 min induced PKCdelta tyrosine phosphorylation. This acute epidermal growth factor treatment did not induce tyrosine phosphorylation of PKCdelta in keratinocytes isolated from waved-2 mice that have a defective epidermal growth factor receptor. In addition, the level of PKCdelta tyrosine phosphorylation in v-rasHa-transduced keratinocytes from EGFR null mice was substantially lower than in v-rasHa transduced wild type cells, suggesting that activation of the EGFR is important for PKC delta tyrosine phosphorylation in ras transformation. However, purified EGFR did not phosphorylate recombinant PKC delta in vitro, whereas members of the Src family (c-Src, c-Fyn) and membrane preparations from keratinocytes did. Furthermore, clearing c-Src or c-Fyn from keratinocyte membrane lysates decreased PKCdelta tyrosine phosphorylation, and c-Src and c-Fyn isolated from keratinocytes treated with TGFalpha had increased kinase activity. Acute or chronic treatment with TGFalpha did not induce significant PKCdelta translocation in contrast to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which induced both translocation and tyrosine phosphorylation of PKCdelta. This suggests that TGFalpha-induced tyrosine phosphorylation of PKC delta results from the activation of a tyrosine kinase rather than physical association of PKCdelta with a membrane-anchored tyrosine kinase. Taken together, these results indicate that PKCdelta activity is inhibited by tyrosine phosphorylation in response to EGFR-mediated signaling and activation of a member of the Src kinase family may be the proximal tyrosine kinase acting on PKCdelta in keratinocytes.

Alleles↗
PubMed1993

Modulation of the activity and subcellular distribution of protein tyrosine kinases in human neutrophils by phorbol esters.

Although several tyrosine kinases are present in human neutrophils, little is known regarding the biochemical basis for their activation. We have identified two tyrosine kinase activities in 0.1 and 1% Triton cell extracts of human neutrophils using a non-denaturing gel assay. The first protein tyrosine kinase activity of a faster mobility was associated exclusively with the 0.1% Triton cell extract. The second activity, of slower mobility, was mainly associated with the 0.1% Triton cell extract and to a lesser extent with the 1% Triton cell extract. A modulation of the activities and the distribution of these two tyrosine kinase activities was observed upon stimulation of neutrophils with PDBu (phorbol 12,13-dibutyrate), a direct PKC (protein kinase C) activator. The addition of 1 microM PDBu induced a time-dependent decrease of both tyrosine kinases in the 0.1% Triton cell extract. Although the fast mobility tyrosine kinase activity disappeared completely, the slow mobility tyrosine activity decreased only partially. Concomitantly, an increase in the latter activity was detected in the 1% Triton cell extract. The pattern of tyrosine phosphorylation upon PDBu stimulation was also examined and the results showed that the phorbol ester induced time-dependent increases in the level of phosphotyrosine-containing proteins in at least 10 distinct bands. Two lines of evidence indicated that the effects of PDBu were mediated by PKC: 1) The stereo-isomer of PDBu, 4 alpha-PDBu, did not affect the activities and distribution of the tyrosine kinases, and 2) The PKC inhibitor, RO 318220, prevented the redistribution of the tyrosine kinase activities and inhibited the stimulation of tyrosine phosphorylation induced by PDBu. These results show that the activity and distribution of at least two human neutrophil tyrosine kinases are modulated after the activation of PKC and that the low mobility tyrosine kinase activity is the most sensitive to PDBu. Based on previous studies, the fast mobility tyrosine kinase activity was likely to be a member of the pp60src tyrosine kinase family and the slower one may be related to the pp93fes. Furthermore, these results begin to define the nature of the relationships among the PKC- and the tyrosine kinase-signaling pathways.

Adult↗
PubMed2000

Tyrosine kinase and phosphatidylinositol 3-kinase activation are required for cyclic adenosine 3',5'-monophosphate-dependent potentiation of deoxyribonucleic acid synthesis induced by insulin-like growth factor-I in FRTL-5 cells.

In previous studies, we showed that pretreatment of rat FRTL-5 thyroid cells with TSH, or other agents that increased intracellular cAMP, markedly potentiated DNA synthesis in response to insulin-like growth factor-I (IGF-I). In addition, we found that TSH pretreatment caused an increase in tyrosine phosphorylation of intracellular proteins including an unidentified 125-kDa protein that was well correlated with the TSH-potentiating effect on DNA synthesis induced by IGF-I. These results suggested that cAMP amplified IGF-I-dependent signals for cell growth through changes of cAMP-dependent tyrosine phosphorylation. The present studies were undertaken to determine how tyrosine kinase activation followed by an increase in tyrosine phosphorylation is required for cAMP-dependent potentiation of DNA synthesis induced by IGF-I in this cell line. First of all, we measured tyrosine kinase or protein-tyrosine phosphatase activities in the cell lysates by the in vitro assay. Chronic treatment with TSH or (Bu)2-cAMP stimulated tyrosine kinase activity in the particulate fraction and protein-tyrosine phosphatase activity in the soluble fraction, suggesting that tyrosine kinase plays more important roles for a cAMP-dependent increase in tyrosine phosphorylation of intracellular proteins. The increased tyrosine kinase activity was sensitive to genistein, a potent tyrosine kinase inhibitor. Genistein abolished both the cAMP-dependent increase in tyrosine phosphorylation of the 125-kDa protein and the enhanced DNA synthesis induced by IGF-I in a similar concentration-dependent manner. The only tyrosine-phosphorylated protein associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase in response to cAMP was 125 kDa. In addition, we found that PI 3-kinase activity bound to p85 subunit significantly increased after (Bu)2cAMP treatment. These results suggested that cAMP stimulates PI 3-kinase through tyrosine phosphorylation of the 125-kDa protein. We then measured DNA synthesis in cells pretreated for 24 h with TSH or (Bu)2cAMP in the absence or presence of LY294002, a PI 3-kinase inhibitor, followed by treatment with IGF-I for 24 h. Presence of LY294002 during TSH or (Bu)2cAMP pretreatment completely abolished cAMP-dependent potentiation of DNA synthesis induced by IGF-I. These results suggest that in FRTL-5 cells cAMP activates genistein-sensitive tyrosine kinases that in turn activate PI 3-kinase activity. These mechanisms appear to be necessary for cAMP-dependent potentiation of the DNA synthesis induced by IGF-I.

Animals↗
PubMed1983

The contribution of phenylalanine to tyrosine metabolism in vivo. Studies in the post-absorptive and phenylalanine-loaded rat.

1. Rates of appearance and oxidation of plasma L-leucine, L-phenylalanine and L-tyrosine, as well as conversion of plasma phenylalanine into plasma tyrosine, were determined in 90-120 g rats after overnight starvation and while receiving 115-120 mumol of L-phenylalanine/h. 2. In the post-absorptive state, plasma tyrosine and phenylalanine appearances were similar, despite the fact that 22% of plasma tyrosine appearance could be attributed to the hydroxylation of phenylalanine. 3. A constant infusion of 115-120 mumol of L-phenylalanine/h did not significantly alter plasma leucine kinetics, but increased appearance of plasma phenylalanine and tyrosine. The percentage of phenylalanine and tyrosine appearance that was oxidized increased from 12.1% and 24.4% to 37.3% and 48.0% respectively. In phenylalanine-loaded rats, 72% of plasma tyrosine appearance could be attributed to the conversion of phenylalanine. 4. Whole-body tyrosine oxidation measured from a continuous infusion of either L-[14C]tyrosine or L-[14C]phenylalanine differed by 165%. 5. It can be concluded that, in the post-absorptive state, phenylalanine hydroxylation makes a substantial contribution to the plasma appearance of tyrosine and is significantly increased when phenylalanine is administered. The disposal of excess infused phenylalanine is a result of a greater percentage of plasma phenylalanine being converted into tyrosine and a greater proportion of tyrosine being further oxidized. However, apparent tyrosine oxidation rates estimated from plasma tyrosine specific radioactivities and appearance of expired 14CO2 during administration of [14C]tyrosine are underestimates of true rates, in part because tyrosine generated from phenylalanine hydroxylation is catabolized without freely equilibrating with the plasma compartment.

Amino Acids↗
PubMed1995

The unique amino-terminal domain of p56lck regulates interactions with tyrosine protein phosphatases in T lymphocytes.

The catalytic activity of p56lck is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue (tyrosine 505). Accumulating data show that this phosphorylation is mediated by the tyrosine protein kinase p50csk and that it is reversed by the transmembrane tyrosine protein phosphatase CD45. Recent studies have indicated that dephosphorylation of tyrosine 505 in resting T cells is necessary for the initiation of antigen-induced T-cell activation. To better understand this phenomenon, we have characterized the factors regulating tyrosine 505 phosphorylation in an antigen-specific T-cell line (BI-141). As is the case for other T-cell lines, Lck molecules from unstimulated BI-141 cells exhibited a pronounced dephosphorylation of the inhibitory carboxyl-terminal tyrosine. This state could be corrected by incubation of cells with the tyrosine protein phosphatase inhibitor pervanadate, suggesting that it reflected the unrestricted action of tyrosine protein phosphatases. In structure-function analyses, mutation of the site of Lck myristylation (glycine 2) partially restored phosphorylation at tyrosine 505 in BI-141 cells. Since the myristylation-defective mutant also failed to stably associate with cellular membranes, this effect was most probably the consequence of removal of p56lck from the vicinity of membrane phosphatases like CD45. Deletion of the unique domain of Lck, or its replacement by the equivalent sequence from p59fyn, also increased the extent of tyrosine 505 phosphorylation in vivo. This effect was unrelated to changes in Lck membrane association and therefore was potentially related to defects in crucial protein-protein interactions at the membrane. In contrast, deletion of the SH3 or SH2 domain, or mutation of the phosphotransfer motif (lysine 273) or the site of autophosphorylation (tyrosine 394), had no impact on phosphate occupancy at tyrosine 505. In combination, these results indicated that the hypophosphorylation of the inhibitory tyrosine of p56(lck) in T lymphocytes is likely the result of the predominant action of tyrosine protein phosphatases. Moreover, they showed that both the amino-terminal myristylation signal and the unique domain of p56(lck) play critical roles in this process.

3T3 Cells↗
PubMed1996

Thapsigargin-induced calcium influx in the absence of detectable tyrosine phosphorylation in human platelets.

Tyrosine phosphorylation is a potential mechanism for mediating store-operated calcium (SOC) influx in platelets and other nonexcitable cells. Thapsigargin induces calcium-dependent tyrosine phosphorylation and SOC influx in platelets. We prevented thapsigargin-induced tyrosine phosphorylation by buffering cytosolic calcium rise with the calcium chelator 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetomethoxyester (BAPTA-AM). Calcium influx, induced by thapsigargin and measured by 45Ca2+ accumulation, persisted in BAPTA-loaded platelets in the absence of tyrosine phosphorylation. This calcium influx was blocked by the SOC influx inhibitor SKF-96365. Tyrosine kinase inhibitors have been used to demonstrate a role for tyrosine phosphorylation in SOC influx. We compared the effects of four tyrosine kinase inhibitors genistein, methyl-2, 5-dihydroxycinnamate (erbstatin analog), tyrphostin A47, and lavendustin A, on thapsigargin-induced tyrosine phosphorylation in control platelets and on thapsigargin-induced SOC influx into BAPTA-loaded platelets in absence of tyrosine phosphorylation. Tyrphostin A47 prevented all measurable tyrosine phosphorylation in control platelets, but did not decrease calcium influx into BAPTA-loaded platelets. Genistein and the erbstatin analog were poor inhibitors of tyrosine phosphorylation but decreased SOC influx into BAPTA-loaded platelets to 55.8 +/- 3% and 51.9 +/- 7.5% of control, respectively. Lavendustin A did not decrease tyrosine phosphorylation or calcium influx. Thus, thapsigargin-induced SOC influx can occur without detectable tyrosine phosphorylation and the inhibition of SOC influx by tyrosine kinase inhibitors does not correlate with their ability to prevent tyrosine phosphorylation.

Blood Platelets↗
PubMed1996

Tyrosine phosphorylation of the Kv1.3 potassium channel.

Kv1.3, a voltage-dependent potassium channel cloned from mammalian brain and T lymphocytes, contains multiple tyrosine residues that are putative targets for tyrosine kinases. We have examined the tyrosine phosphorylation of Kv1.3, expressed transiently in human embryonic kidney (or HEK) 293 cells, by endogenous and coexpressed tyrosine kinases. Tyrosine phosphorylation is measured by a strategy of immunoprecipitation followed by. Western blot analysis, using antibodies that specifically recognize Kv1.3 and phosphotyrosine. Coexpression of the constitutively active tyrosine kinase v-src, together with Kv1.3, causes a large increase in the tyrosine phosphorylation of the channel protein. This phosphorylation of Kv1.3 can be reversed by treatment with alkaline phosphatase before Western blot analysis. Coexpression with a receptor tyrosine kinase, the human epidermal growth factor receptor, also causes an increase in tyrosine phosphorylation of Kv1.3. The effects of endogenous tyrosine kinases were examined by treating Kv1.3-transfected cells with the specific membrane-permeant tyrosine phosphatase inhibitor pervanadate. Pervanadate treatment causes a time- and concentration-dependent increase in the tyrosine phosphorylation of Kv1.3. This increased tyrosine phosphorylation of Kv1.3 is accompanied by a time-dependent decrease in Kv1.3 current, measured by patch-clamp analysis with cell-attached membrane patches. The pervanadate-induced suppression of current and much of the channel tyrosine phosphorylation are eliminated by mutation of a specific tyrosine residue, at position 449 of Kv1.3, to phenylalanine. Thus, there is a continual phosphorylation and dephosphorylation of Kv1.3 by endogenous kinases and phosphatases, and perturbation of this constitutive phosphorylation/dephosphorylation cycle can profoundly influence channel activity.

Animals↗
PubMed1995

Immunocytochemical localization of the striatal enriched protein tyrosine phosphatase in the rat striatum: a light and electron microscopic study with a complementary DNA-generated polyclonal antibody.

The present study concerns the immunocytochemical localization of the striatal enriched protein tyrosine phosphatase in the rat striatum. A novel molecular biology technique allowed us to produce a complementary DNA-generated polyclonal antibody raised against the non-catalytic domain of the striatal enriched protein tyrosine phosphatase, which selectively recognized the striatal enriched protein tyrosine phosphatase protein with 46,000 mol. wt on western blots. Immunocytochemical analysis with the specific antibody revealed strong striatal enriched protein tyrosine phosphatase immunoreactivity in the striatum. Light microscopy showed striatal striatal enriched protein tyrosine phosphatase-immunopositive neurons to be of medium size (mean diameter of 14.4 microns), and to comprise approximately 80% of the total neuronal population in the striatum. These cells had round, triangular or polygonal cell bodies with relatively little cytoplasm. Nerve fibers stained positively for striatal enriched protein tyrosine phosphatase were also present in the globus pallidus and the substantia nigra, and the nigral labeling on the ipsilateral side almost disappeared subsequent to cerebral hemitransection, suggesting these immunolabeled structures to be striatal projections. Double-immunofluorescence analysis demonstrated separate populations of striatal enriched protein tyrosine phosphatase-positive cells and neurons stained for parvalbumin. Also, ultrastructural study showed that the striatal enriched protein tyrosine phosphatase-positive neurons (n = 50) possessed no nuclear indentations or intranuclear inclusions. Thus, most striatal striatal enriched protein tyrosine phosphatase-positive neurons were thought to be of the medium-sized spinous type. At the light microscopic level, stained striatal neurons exhibited striatal enriched protein tyrosine phosphatase immunolabeling in their somata, dendrites and axonal processes, but not in their nuclei. Electron microscopic observation showed strong striatal enriched protein tyrosine phosphatase-immunoreactivity on the inner surface of the plasmalemma, on the outer surfaces of mitochondria and on microtubules, particularly of dendrites. A heavy deposit of immunoreaction product was also present on postsynaptic densities in labeled dendrites, while a light deposit was seen on the synaptic vesicles of nerve terminals. The characteristic distribution profile of striatal enriched protein tyrosine phosphatase suggested that the enzyme may play a role in a variety of functional properties of striatal neurons, especially in postsynaptic signaling processes and in regulation of microtubular functions. On the basis of the present findings, we propose the following conclusions: (i) a protein tyrosine phosphorylation system regulated by striatal enriched protein tyrosine phosphatase is involved in certain specialized cellular processes (e.g. signal transduction cascades) of medium-sized spinous neurons distinct from those of other neuronal subsets in the striatum; (ii) a striatal medium spiny neuron is characterized by its expression of striatal enriched protein tyrosine phosphatase and, therefore, the enzyme is useful for detection of the distinct subset of striatal cells or for tracing their axonal projection fibers in the basal ganglia.

Animals↗
PubMed2001

Tyrosine levels regulate the melanogenic response to alpha-melanocyte-stimulating hormone in human melanocytes: implications for pigmentation and proliferation.

Melanocyte-stimulating hormone (alpha-MSH) increases cytosolic levels of cAMP as well as tyrosinase activity in murine melanocytes. These activities depend upon the presence of melanin precursors and may differ in human melanocytes. In this study, we demonstrate that high levels of tyrosine (3.7 mM), the chief melanin precursor, reduced the proliferative effect of alpha-MSH and altered human melanocyte morphology as compared to treatment with low (25-30 microM, half-physiological) levels of tyrosine. The anti-proliferative effect of high levels of tyrosine was not restricted to alpha-MSH; tyrosine also reduced proliferation induced by forskolin, a direct activator of the cAMP pathway. Exposure to low tyrosine levels and alpha-MSH induced a dendritic morphology; in the presence of high tyrosine and alpha-MSH, melanocytes displayed large, pigmented cell bodies and less dendricity. Exposure to alpha-MSH in the presence of low tyrosine for up to 9 days did not appreciably increase melanin levels, but culturing the human melanocytes in high levels of tyrosine with alpha-MSH increased melanin levels 10-50-fold, depending on the pigmentation background of the donor. A greater induction of melanin accumulation was observed in melanocytes derived from light-skinned donors than was observed in cells obtained from dark-skinned donors. The poor ability of alpha-MSH to stimulate melanin synthesis was not caused by a lack of induction of melanogenic proteins, as alpha-MSH increased the expression of microphthalmia (MITF), tyrosinase, dopachrome tautomerase (DCT), and Pmel-17, compared to untreated cells or cells stimulated by phorbol ester alone, regardless of tyrosine levels. DCT levels were greatly induced by low tyrosine with alpha-MSH, but were dramatically decreased by high tyrosine with alpha-MSH. Interestingly, in this same medium (high tyrosine), MITF levels also decreased after 2 weeks and were barely detectable by the third week. Despite the absence of MITF at 3 weeks of treatment in high tyrosine medium, tyrosinase levels remained high, thereby suggesting that additional factors must be responsible for tyrosinase transcription in human melanocytes. Our results indicate that tyrosine levels can regulate the proliferative activity induced by alpha-MSH, as well as the extent of melanogenesis in normal human melanocytes. The significance of this work is that tyrosine levels may be part of the mechanism that switches melanocytes out of a proliferative status and into a melanin-synthesizing, terminally differentiated phenotype.

Antibodies, Monoclonal↗
PubMed2000

Tyrosine phosphorylation of the alpha subunit of transducin and its association with Src in photoreceptor rod outer segments.

Recent evidence indicates that tyrosine phosphorylation may play important roles in retinal photoreceptor rod outer segments (ROS). We investigated the tyrosine phosphorylation of endogenous proteins in isolated bovine ROS. Several proteins with apparent molecular masses of 31, 39, 60, 83, 90, 97, 120, 140, and 180 kDa were tyrosine-phosphorylated in ROS incubated with Mg(2+), ATP, and orthovanadate. Several tyrosine kinase inhibitors significantly inhibited tyrosine phosphorylation of these proteins in ROS. The 39- and 60-kDa tyrosine-phosphorylated proteins were identified as the alpha subunit of the G protein transducin (Talpha) and the tyrosine kinase Src, respectively. The presence of Src and tyrosine kinase activity in bovine ROS was confirmed by their cofractionation with rhodopsin and Talpha on continuous sucrose gradients. Several tyrosine-phosphorylated proteins, including Src, coimmunoprecipitated with Talpha. The association of Src with Talpha was detected in the absence of tyrosine phosphorylation, but was enhanced with increased tyrosine phosphorylation of ROS. Moreover, tyrosine kinase activity also associated with Talpha was sevenfold higher under tyrosine-phosphorylating conditions. The recovery of transducin by hypotonic GTP extraction from tyrosine-phosphorylated ROS was significantly less than that from nonphosphorylated ROS. We localized the site on Talpha phosphorylated by Src to the amino-terminal half by limited tryptic digests, and further mapped it by ion trap mass spectrometry to Tyr(142) in the helical domain of Talpha. Talpha was also tyrosine-phosphorylated in vivo in rat retina, but this phosphorylation was not affected by light.

Animals↗
PubMed1986

Cholinergic receptor-mediated phosphorylation and activation of tyrosine hydroxylase in cultured bovine adrenal chromaffin cells.

We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phosphorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one- and two-dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Two-dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+-dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP-dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+-dependent protein kinase. After 2 min phosphorylation by a cyclic AMP-dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.

Adrenal Medulla↗
PubMed2005

Urinary ortho-tyrosine excretion in diabetes mellitus and renal failure: evidence for hydroxyl radical production.

BACKGROUND: Phenylalanine is converted to para- and ortho-tyrosine by hydroxyl free radical, or to para-tyrosine by the phenylalanine hydroxylase enzyme. The aim of this study was to measure para- and ortho-tyrosine in the urine and plasma of patients with chronic renal disease and/or diabetes, to obtain information on the renal handling of the different tyrosine isomers and, furthermore, to measure urinary levels of 8-epi-prostaglandin-F(2alpha), a marker of lipid peroxidation. METHODS: In our cross-sectional study we measured para-, ortho-tyrosine, and phenylalanine levels, using high performance liquid chromatography and 8-epi-prostaglandin-F(2alpha) with enzyme-linked immunosorbent assay (ELISA). We compared 4 groups: (1) controls (CONTR, N = 14), (2) patients with chronic kidney disease (CKD, N = 12), (3) patients with type 2 diabetes mellitus (DIAB, N = 17), (4) patients with chronic kidney disease and type 2 diabetes (DIAB-CKD, N = 19). RESULTS: We found a decreased plasma para-tyrosine level and decreased urinary para-tyrosine excretion in CKD patients, while the fractional excretion of para-tyrosine was similar in all 4 groups, approximately 1%. There was no difference in the plasma ortho-tyrosine levels between the groups. However, urinary ortho-tyrosine excretion was higher in all 3 groups of patients than in the CONTR group, and higher in DIAB and in DIAB-CKD patients than in CKD patients. The fractional excretion of ortho-tyrosine was significantly higher in DIAB and in DIAB-CKD patients than in the CONTR group. The fractional excretion of ortho-tyrosine exceeded 100% in the 2 diabetic groups. Urinary 8-epi-prostaglandin-F(2alpha)/creatinine ratio did not correlate with urinary ortho-tyrosine excretion. CONCLUSION: The difference between para-tyrosine levels of the groups is probably due to renal impairment, while there is indirect evidence for an increased tubular secretion or production of ortho-tyrosine in the kidney in diabetic patients with or without CKD.

Aged↗
PubMed1993

A factor(s) from a trophoblast cell line increases tyrosine hydroxylase activity in fetal hypothalamic cell cultures.

We previously reported that a factor(s) from rat choriocarcinoma (Rcho-1) cells suppresses circulating PRL levels and increases tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons in vivo. The purposes of this study were to determine whether this factor(s) increases tyrosine hydroxylase activity in fetal hypothalamic cells in vitro and to evaluate its chemical nature. The Rcho-1 cells are of placental origin and have the capacity to differentiate into giant cells and produce members of the placental PRL family. MMQ cells, a pituitary cell line that secretes PRL, and HRP-1, a placental cell line that does not produce any known members of the PRL family, were used as control cells. Tyrosine hydroxylase activity was assessed by incubation of hypothalamic cells for 1 h with 100 microM brocresine, an inhibitor of aromatic L-amino acid decarboxylase. Tyrosine hydroxylase activity was increased in a density-dependent manner when Rcho-1, but not HRP-1 or MMQ, cells were cocultured with hypothalamic cells for 24 h. Control and Rcho-1-stimulated tyrosine hydroxylase activities were markedly reduced with 1 mM alpha-methyl-p-tyrosine, a specific inhibitor of tyrosine hydroxylase. Tyrosine hydroxylase activity was not altered when hypothalamic cells were incubated for 24 h with rat PRL or recombinant rat placental lactogen-I, whereas a 24-h stimulation with 100,000 Rcho-1 cells and a 1-h stimulation with 5 mM (Bu)2cAMP increased tyrosine hydroxylase activity 3.7- and 3-fold, respectively. The magnitudes of the increase in tyrosine hydroxylase activity were similar when hypothalamic cells were cocultured with Rcho-1 cells for 1 and 24 h. Acetic acid extracts of Rcho-1, but not HRP-1 or MMQ, cells increased tyrosine hydroxylase activity within 1 h in a concentration-dependent manner. The 3-fold increase in tyrosine hydroxylase activity observed with 500,000 Rcho-1 cell equivalents was markedly reduced with 1 mM alpha-methyl-p-tyrosine. The mol wt range of the tyrosine hydroxylase-activating factor(s) (THAF) was estimated using ultrafiltration membranes. The majority of activity was found in the eluate from a 1,000 mol wt cut-off membrane. THAF activity in Rcho-1 cell extracts was decreased by preincubation with pronase, a nonspecific proteolytic enzyme, suggesting that the factor(s) is a peptide. THAF was resistant to inactivation by trypsin or chymotrypsin pretreatment. However, both enzymes destroyed the ability of pituitary adenylate cyclase-activating peptide, either alone or with Rcho-1 cell extracts, to increase tyrosine hydroxylase activity. Oxidation of Rcho-1 cell extracts with performic acid abolished THAF activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Involvement of tyrosine kinase and cAMP-dependent kinase cross-talk in the regulation of human sperm motility.

Tyrosine phosphorylation and its upregulation by cAMP have been associated with capacitation and motility changes of spermatozoa. In the present study, washed spermatozoa were incubated for 6 h in protein-supplemented complete medium with or without kinase inhibitors to verify whether upstream activation of protein kinase A is indispensable for tyrosine phosphorylation and motility changes to occur in capacitating human spermatozoa. H89, a specific protein kinase A inhibitor, significantly inhibited the activity of sperm protein kinase A. However, this inhibition did not alter capacitation-related tyrosine kinase activation. Tyrosine phosphorylated proteins, motion parameters and the incidence of phosphotyrosine-immunoreactive spermatozoa were decreased only slightly. Conversely, genistein, a tyrosine kinase inhibitor which inhibited sperm tyrosine kinase but not protein kinase A, significantly reduced all the parameters studied. Spermatozoa incubated with cAMP and pentoxifylline showed a rapid enhancement of tyrosine phosphorylation and some of the sperm motion parameters, particularly hyperactivation. Inclusion of H89 reduced cAMP stimulation of tyrosine kinase, and tyrosine phosphorylation and motion parameters were reduced almost to basal values. Treatment with genistein reduced tyrosine kinase activity, especially in the soluble fraction of sperm extracts. A decrease in tyrosine phosphorylation of soluble proteins, 105, 81, 55 and 48 kDa, correlated with a significant reduction in sperm motion parameters. Hyperactivation was reduced by tenfold. Tyrosine phosphorylated proteins in the insoluble fraction and the incidence of tyrosine phosphorylated-positive spermatozoa were not reduced markedly. Upstream protein kinase A activation may be a facilitatory rather than an indispensable step in the capacitation-induced tyrosine phosphorylation mediating motility changes in human spermatozoa. Triton-x100 soluble tyrosine phosphorylated proteins, more than their insoluble counterparts, appear to be involved in the modulation of human sperm motion characteristics.

Cells, Cultured↗
PubMed1985

Effect of dietary level of protein or methionine and threonine on the amino acids and catecholamines in brain of rats fed a high tyrosine diet.

When rats were fed a high tyrosine diet (5% in diet), plasma tyrosine, brain tyrosine, norepinephrine (NE), and 3-methoxy-4-hydroxyphenylethyleneglycol-sulfate (MHPG-SO4) were increased. The concentrations of brain tyrosine were directly correlated with those of plasma tyrosine and tyrosine ratios (tyrosine/large neutral amino acid (LNAA)) in plasma and brain. When rats were fed a high tyrosine diet with increasing amounts of the dietary protein, the contents of brain tyrosine and MHPG-SO4 decreased stepwise. By the supplementation of both methionine and threonine to the high tyrosine diet, the enhancement of brain tyrosine and MHPG-SO4 caused by tyrosine was significantly reduced. These results suggest that effects of tyrosine toxicity such as growth retardation or external pathological lesions may be due to the accelerated NE-turnover in the brain accompanied by the enhancement of brain tyrosine concentration. The supplemented methionine and threonine may cause an increase of tyrosine utilization, because methionine and threonine are the most limiting amino acids in casein, and also may inhibit the uptake of tyrosine into the brain via the blood-brain barrier.

Amino Acids↗