Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: HUMANGGP:007536 (tyrosine hydroxylase)
 15,404 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated the role of serine 40 (Ser-40) in tyrosine hydroxylase (TH) catalysis of basal and activated enzymes by protein kinase A (PKA)-mediated phosphorylation. Wild type and mutant TH were transiently and stably expressed in AtT-20 cells, and the enzymatic activities of the recombinant enzymes were analyzed. The specific enzymatic activity of transiently expressed TH mutants Ser-40-->leucine or-->tyrosine (Leu-40m or Tyr-40m) was higher than that of the wild type enzyme or of other mutants in which Ser-8, -19, and -31 were replaced by leucine. The kinetic studies carried out with the stably expressed TH show that the Km for the cofactor 6-methyltetrahydropterine is lower and the Ki for dopamine is higher when the enzymatic hydroxylation is catalyzed by the Leu-40m or Tyr-40m than by the wild type enzyme. The kinetic parameters and the pH profile of the enzymatic hydroxylation catalyzed by the Leu-40m or Tyr-40m are similar to the enzyme activated by PKA-mediated phosphorylation. We suggest that Ser-40 in TH exerts an inhibitory influence on the enzymatic activity, and its replacement with another amino acid by site-directed mutagenesis or its modification by phosphorylation leads to a change in conformation with an increased enzymatic activity. The importance of Ser-40 in the activation of TH by PKA-mediated phosphorylation was investigated by comparing the activation of the wild type enzyme with that of Leu-40m or Tyr-40m. The findings that the enzymatic activity is increased by PKA-mediated phosphorylation of the wild type enzyme, but not of the Leu-40m or Tyr-40m, demonstrate that phosphorylation at Ser-40 is essential for activation of TH by PKA. The findings that addition of ATP plus cAMP to homogenates from transfected AtT-20 cells stimulates the recombinant wild type TH activity indicate that these cells contain endogenous cAMP-dependent protein kinase.
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PMID:Site-directed mutagenesis of tyrosine hydroxylase. Role of serine 40 in catalysis. 136 Nov 89

The effects of exogenous GM1 ganglioside on depolarization and ligand-induced Ca2+ signaling were investigated in PC12 cells. Cellular responses to K+ depolarization and bradykinin application in control and GM1-treated cells were examined with respect to: 1) changes in the intracellular Ca2+ concentration ([Ca2+]i) measured using fura-2 fluorescence in single cells, and 2) changes in Ca(2+)-dependent protein kinase activity as assayed by two-dimensional phosphopeptide analysis of the site-specific phosphorylation of tyrosine hydroxylase. Pretreatment of cells with GM1 (10 or 100 microM) enhanced K+ depolarization-stimulated increases in [Ca2+]i and in 32PO4 incorporation into tyrosine hydroxylase phosphopeptide T2, a Ca2+/calmodulin-dependent protein kinase II substrate. In contrast, GM1 treatment had no effect on the transient increases in [Ca2+]i evoked by bradykinin or on bradykinin-induced increases in the site-specific phosphorylation of tyrosine hydroxylase. The depolarization-induced and GM1-enhanced increases in [Ca2+]i and T2 phosphorylation were prevented by removal of external Ca2+ or pretreatment with 1 microM nitrendipine, suggesting that these increases result from Ca2+ entry through dihydropyridine-sensitive Ca2+ channels. The ability of exogenous gangliosides to potentiate increases in [Ca2+]i may underlie their diverse neuritogenic and neurotrophic actions in the nervous system.
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PMID:Modulation of a Ca2+ signaling pathway by GM1 ganglioside in PC12 cells. 144 16

We reported that one of the isoquinolinesulfonamide derivatives, KN-62, is a potent and specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M. and Hidaka, H. (1990) J. Biol. Chem. 265, 4315-4320). We have now investigated the inhibitory property of a newly synthesized methoxybenzenesulfonamide, KN-93, on CaMKII activity in situ and in vitro. KN-93 elicited potent inhibitory effects on CaMKII phosphorylating activity with an inhibition constant of 0.37 microM but this compound had no significant effects on the catalytic activity of cAMP-dependent protein kinase, Ca2+/phospholipid dependent protein kinase, myosin light chain kinase and Ca(2+)-phosphodiesterase. KN-93 also inhibited the autophosphorylation of both the alpha- and beta-subunits of CaMKII. Kinetic analysis indicated that KN-93 inhibits CaMKII, in a competitive fashion against calmodulin. To evaluate the regulatory role of CaMKII on catecholamine metabolism, we examined the effect of KN-93 on dopamine (DA) levels in PC12h cells. The DA levels decreased in the presence of KN-93. Further, the tyrosine hydroxylase (TH) phosphorylation induced by KCl or acetylcholine was significantly suppressed by KN-93 in PC12h cells while events induced by forskolin or 8-Br-cAMP were not affected. These results suggest that KN-93 inhibits DA formation by modulating the reaction rate of TH to reduce the Ca(2+)-mediated phosphorylation levels of the TH molecule.
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PMID:The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells. 166 7

The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP-dependent protein kinase as well as other protein kinases. We determined the Km and Vmax of rat pheochromocytoma tyrosine hydroxylase for cyclic AMP-dependent protein kinase and obtained values of 136 microM and 7.1 mumol/min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high Km of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydroxylase phosphorylation sites. We found that the best substrates for cyclic AMP-dependent protein kinase were those peptides corresponding to serine 40. Tyrosine hydroxylase (36-46), for example, exhibited a Km of 108 microM and a Vmax of 6.93 mumol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine 19 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme.
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PMID:Phosphorylation of rat tyrosine hydroxylase and its model peptides in vitro by cyclic AMP-dependent protein kinase. 167 83

Physiological stress induces tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, via trans-synaptic mechanisms within the adrenal medulla. Previous studies have implicated cAMP as a second messenger capable of inducing tyrosine hydroxylase; however, it is unclear whether any receptor coupled to adenylate cyclase mediates tyrosine hydroxylase induction. Recently, vasoactive intestinal polypeptide, whose receptor is coupled to adenylate cyclase in many tissues, has been shown to meet many of the criteria for a neuromodulator within the adrenal medulla. We therefore undertook a series of studies to determine whether vasoactive intestinal polypeptide may induce tyrosine hydroxylase in PC12 cells, a cell line derived from rat adrenal medulla. Here we report that vasoactive intestinal polypeptide produces a transient, time- and concentration-dependent increase in tyrosine hydroxylase mRNA levels which is followed by a stable increase in tyrosine hydroxylase protein. The increase in tyrosine hydroxylase mRNA does not occur in a mutant PC12 cell line deficient in cAMP-dependent protein kinase activity, indicating that the effect of vasoactive intestinal polypeptide is mediated through the cAMP second messenger pathway. This is the first report demonstrating that a neuromodulator which acts on an adenylate cyclase-coupled receptor can induce tyrosine hydroxylase.
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PMID:Vasoactive intestinal polypeptide induces tyrosine hydroxylase in PC12 cells. 167 10

Trypsin proteolysis of tyrosine hydroxylase (TH) produces a 34-kDa fragment that is catalytically active but does not contain the regulatory phosphorylation sites. In this report, activation of TH by proteolysis was characterized further. Proteolysis results in a decrease in Kms for both substrate and cofactor. The increase in affinity for cofactor was identical to that produced by phosphorylation with cAMP-dependent protein kinase. Additionally, proteolysis of an N-terminal region containing the regulatory phosphorylation sites was sufficient to produce a decrease in Km for cofactor. Activation of substrate binding required more extensive proteolysis but also corresponded to N-terminal digestion. Moreover, this activation was coincident with a broadened substrate specificity. In combination, these data indicate that the N-terminus of tyrosine hydroxylase regulates cofactor binding and directs substrate specificity.
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PMID:Limited proteolysis of rat brain tyrosine hydroxylase defines an N-terminal region required for regulation of cofactor binding and directing substrate specificity. 167 92

We have investigated the ability of exogenous gangliosides to modulate nerve growth factor (NGF) signal transduction in PC12 cells. The effects of exogenous ganglioside GM1 on multiple protein kinase activities were assayed by analyzing site-specific serine phosphorylation of tyrosine hydroxylase (TyrOHase) by two-dimensional phosphopeptide mapping. In the presence of NGF, exogenous GM1 (1-10 microM) increased 32P incorporation into TyrOHase phosphopeptide T2, a Ca2+/calmodulin-dependent protein kinase substrate whose phosphorylation is not normally affected by NGF treatment. In the absence of NGF, GM1 treatment had no significant effects on TyrOHase phosphorylation. The removal of extracellular Ca2+ or blockade of dihydropyridine-sensitive Ca2+ channels prevented the GM1-induced increases in 32P incorporation into phosphopeptide T2. Exogenous GM1 also potentiated K+ depolarization-induced increases in the phosphorylation of TryOHase. These results suggest that the stimulatory effects of exogenous GM1 ganglioside on NGF actions may be due to its ability to potentiate a Ca(2+)-dependent signaling pathway.
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PMID:Stimulation of a Ca(2+)-dependent protein kinase by GM1 ganglioside in nerve growth factor-treated PC12 cells. 167 13

Tyrosine hydroxylase was maximally phosphorylated by protein kinase C, with a stoichiometry of 0.43 mol of phosphate/mol of tyrosine hydroxylase subunit at Ser40, and by calmodulin-dependent protein kinase II, with stoichiometries of 0.43 mol/mol at Ser40 and 0.76 mol/mol at Ser19, respectively, without undergoing any significant direct activation. In contrast, the enzyme was maximally phosphorylated with a stoichiometry of 0.78 mol of phosphate/mol of subunit at Ser40 by cAMP-dependent protein kinase, which resulted in a large activation of the enzyme (about 3-fold activation under the assay conditions). Incubation of the enzyme, which had previously been maximally phosphorylated by calmodulin-dependent protein kinase II, with protein kinase C under phosphorylating conditions resulted in no additional incorporation of phosphate into the enzyme, suggesting that both protein kinases phosphorylated Ser40 of the same subunits of the enzyme. Since tyrosine hydroxylase is thought to be composed of four identical subunits, the results may indicate that calmodulin-dependent protein kinase II or protein kinase C phosphorylates only two of the four subunits of the enzyme at Ser40 without affecting the enzyme activity and that cAMP-dependent protein kinase phosphorylates Ser40 of all four subunits of the enzyme molecule, causing a marked activation. Based on a linear relationship between phosphorylation and the resulting activation of the enzyme by cAMP-dependent protein kinase, possible mechanisms for the activation of the enzyme by the protein kinase are discussed.
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PMID:Different effects on activity caused by phosphorylation of tyrosine hydroxylase at serine 40 by three multifunctional protein kinases. 167 38

The single human tyrosine hydroxylase (TH) gene generates four different mRNA species through alternative splicing events. TH-1 and TH-2 mRNAs are expressed mostly in the brain. We have produced large amounts of the corresponding proteins in Escherichia coli to analyze their respective molecular characteristics. The polypeptides have molecular weights similar to those of TH expressed in Xenopus oocytes and react with antibodies to TH. The two isoforms were purified with a purity of 90% using a three-step procedure. The phosphorylation sites have been determined in the two isoforms after labeling with [gamma-32P]ATP in the presence of cAMP-dependent protein kinase (PKA) or calmodulin-dependent protein kinase II (CaM-PK II). In both isoforms, Ser-40 was found to be phosphorylated by PKA, and Ser-19 and Ser-40 were found to be phosphorylated by CaM-PK II. The putative phosphorylation site generated by alternative splicing (Ser-31) was phosphorylated specifically by CaM-PK II in TH-2 only. The kinetic properties of the two isoforms in the presence of various concentrations of the substrate (tyrosine) and of the natural cofactor [6R)-tetrahydrobiopterin) were also analyzed. TH produced in E. coli is unphosphorylated but nevertheless active. At 50 microM tyrosine and 300 microM (6R)-tetrahydrobiopterin, the specific activities of TH-1 and TH-2 are 1300 and 620 nmol of dihydroxyphenylalanine/min/mg, respectively. Phosphorylation of TH-1 and TH-2 by PKA activates both isoenzymes as shown by the increase in the affinity for the cofactor. No changes in kinetic parameters of the isoenzymes were observed after phosphorylation by CaM-PK II. Dopamine was found to inhibit both TH isoenzymes to the same extent as shown by their similar Ki values for dopamine. These values were increased after phosphorylation of each enzyme by PKA. Unlike TH-1, phosphorylation of TH-2 by CaM-PK II resulted in an increase of the Ki value for dopamine. This property may be related to the presence of the additional phosphorylated residue in TH-2 isoform.
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PMID:Phosphorylation of human recombinant tyrosine hydroxylase isoforms 1 and 2: an additional phosphorylated residue in isoform 2, generated through alternative splicing. 168 Jan 28

A rat cDNA containing the complete coding sequence for rat tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) was isolated from a rat PC12 cDNA library and subcloned in a bacterial expression plasmid, and large amounts of functional enzyme were produced in Escherichia coli. The recombinant enzyme was purified approximately 20-fold to a final specific activity of 1.8 mumol/min per mg of protein, with a yield of 30%. As much as 1 mg of pure protein could be obtained from 1 g of wet bacterial cells. The purified hydroxylase was shown to be homogeneous by denaturing polyacrylamide electrophoresis and isoelectric focusing. Amino acid analysis of the N terminus (25 residues) revealed 100% identity with rat PC12 tyrosine hydroxylase, as deduced from its cDNA sequence. Several of the kinetic properties of the recombinant enzyme resembled those of the native PC12 hydroxylase. However, in contrast to the native enzyme, the purified recombinant hydroxylase was shown to be in an activated form. Phosphorylation with cAMP-dependent protein kinase resulted in stoichiometric incorporation of phosphate, but the kinetic profile of the recombinant enzyme was unaffected. Several clues to these differences are considered that may provide insight into the structural features important to the regulation of tyrosine hydroxylase.
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PMID:High-level expression of rat PC12 tyrosine hydroxylase cDNA in Escherichia coli: purification and characterization of the cloned enzyme. 168 42


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