Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

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

The chiral specificities of bovine striatal tyrosine hydroxylase (TH) (unphosphorylated and phosphorylated by cAMP-dependent protein kinase) and rat liver phenylalanine hydroxylase (PH) were examined at physiological pH using the pure C6 stereoisomers of 6-methyl- and 6-propyl-5,6,7,8-tetrahydropterin (6-methyl-PH4 and 6-propyl-PH4) and (6R)- and (6S)-tetrahydrobiopterin (BH4). Both PH and phosphorylated TH have substantially higher Vmax values with the unnatural (6R)-propyl-PH4 than the natural (6S)-propyl-PH4 (approximately 6- and 11-fold, respectively). However, the Km's are also higher such that Vmax/Km is almost unaffected by C6 chirality. Unphosphorylated TH has equal Km values for both isomers of 6-propyl-PH4, but has about a 6 times greater Vmax with the unnatural isomer, making it the fastest cofactor yet for this form of the enzyme. With the shorter 6-methyl group, chiral differences are still recognized by phosphorylated TH but hardly at all by PH. Inhibition of both PH and TH by amino acid substrate which occurs with (6R)-BH4 as cofactor is also observed with (6S)-propyl-PH4 but not with (6S)-BH4, (6R)-propyl-PH4, or (6R)- or (6R,S)-methyl-PH4. The Km for (6S)-BH4 with phosphorylated TH is nearly 3 times higher than with (6R)-BH4, but Vmax is unchanged. With unphosphorylated TH, (6S)-BH4 produces very low decelerating rates, which was shown not to be due to irreversible inactivation of the enzyme. The Km for (6R)-BH4 with either hydroxylase is 10 times higher than for the equivalently configured (6S)-propyl-PH4. Comparison of these two cofactors reveals that the 1' and 2' side-chain hydroxyl groups of the natural cofactor promote different regulatory functions in PH than in TH.
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PMID:Role of C6 chirality of tetrahydropterin cofactor in catalysis and regulation of tyrosine and phenylalanine hydroxylases. 168 99

We investigated the involvement of second messenger systems in the control by pituitary cytotropic factor (CTF) of tyrosine hydroxylase (TH) expression in primary cultures of hypothalamic cells. Forskolin, an activator of adenylyl cyclase, as well as Sp-cAMP[S] [(Sp)-cyclic adenosine 3',5'-monophosphothioate], a cAMP agonist, and theophylline, an inhibitor of phosphodiesterase activity, stimulate the secretion of dihydroxyphenylalanine (DOPA) and dopamine (DA), suggesting a role for cAMP-dependent protein kinase in the secretion of catecholamines by hypothalamic dopaminergic cells. When cells were cultured with either CTF or forskolin for 14 days, a progressive increase in the secretion of DOPA and DA was observed throughout the period of incubation. At the end of the 2-week culture period, the amount of TH in the cells, determined by immunoblot analysis, was appreciably increased compared to controls. When the cells were analyzed immunocytochemically for TH, the TH-positive cells that had been incubated with CTF or forskolin for 2 weeks were found to have neurites that appeared larger than those of TH-positive cells in the controls. The diameters of the perikarya of TH-positive cells in cultures incubated with CTF also appeared larger than the controls. After incubation of hypothalamic cells with CTF for 96 h, the amount of TH mRNA in the cultures was significantly increased. When membranes isolated from PC12 cells were incubated for 10 min with 50 microM forskolin, the specific activity of adenylyl cyclase was increased 20-fold; CTF had no effect on adenylyl cyclase activity of PC12 cell membranes. Yet, CTF significantly (P less than 0.001) stimulated the secretion of DOPA and DA by PC12 cells. When hypothalamic cells were incubated with both forskolin and CTF, using doses of each that stimulated maximal secretion, the secretion of DOPA and DA was equal to sum of the secretions with each stimulant alone. These additive actions of forskolin and CTF and the failure of CTF to activate adenylyl cyclase in membranes of PC12 cells suggest that forskolin and CTF stimulate catecholamine secretion by hypothalamic dopaminergic cells through different mechanisms, perhaps through different protein kinases. When hypothalamic cells were incubated with CTF and W-7 [N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide], an inhibitor of calmodulin, the secretion of DOPA was significantly (P less than 0.001) less than that in cultures that were not incubated with W-7. The findings of this study suggest that TH expression in hypothalamic dopaminergic cells is controlled by redundant protein kinases, including cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase.
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PMID:Tyrosine hydroxylase expression in hypothalamic cells: analysis of the roles of adenosine 3',5'-monophosphate- and Ca2+/calmodulin-dependent protein kinases in the action of pituitary cytotropic factor. 168 36

Intact secretory granules isolated from bovine adrenal medulla express tyrosine hydroxylase (TH) activity. Granule-associated TH sediments on continuous sucrose gradients with dopamine beta-hydroxylase, a marker for granule membranes, indicating that TH is associated with chromaffin granules. Membranes prepared from lysed granules retain TH, whereas granule contents are free of the enzyme. TH immunoreactivity was detected in granule membranes by immunoblot analysis using a polyclonal antiserum against TH. TH immunoreactivity cannot be removed from membranes by washes in high ionic strength buffers and is only partially removed from membranes by treatment with either urea or Na2CO3. TH can be removed from granule membranes by the detergents Nonidet P-40, Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Treatment of membranes with a phosphatidylinositol-specific phospholipase C did not remove TH, ruling out the possibility of a glycosyl phosphatidyl anchor. Fractionation of granule membranes by temperature-induced phase separation in Triton X-114 revealed that TH is recovered in phases in which integral (detergent phase) and hydrophobic (phospholipid phase) membrane proteins are typically found. By contrast, TH from adrenal cytosol fractionated exclusively into the aqueous phase along with other soluble proteins. Digestion of granules with various protease enzymes revealed that TH is resistant to degradation, suggesting that the enzyme is embedded within membranes. TH becomes phosphorylated when intact granules are exposed to the catalytic subunit of the cAMP-dependent protein kinase, indicating that at least the N-terminal region of TH is exposed on the cytoplasmic surface of granules. These results establish that a fraction of TH is an integral component of bovine granule membranes. The association of TH with granule membranes may play a role in coordinating TH activity and catecholamine release.
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PMID:Tyrosine hydroxylase in secretory granules from bovine adrenal medulla. Evidence for an integral membrane form. 196 7

The structure of the cofactor binding domain of tyrosine hydroxylase (TH) was examined at physiological pH by determining kinetic parameters of (R)-tetrahydrobiopterin [(R)-BH4] and a series of tetrahydropterin (PH4) derivatives (6-R1-6-R2-PH4: R1 = H and R2 = methyl, hydroxymethyl, ethyl, methoxymethyl, phenyl, and cyclohexyl; R1 = methyl and R2 = methyl, ethyl, propyl, phenyl, and benzyl). A minimally purified TH preparation that was not specifically phosphorylated (designated as "unphosphorylated") was compared with enzyme phosphorylated with cAMP-dependent protein kinase. The Km for tyrosine with most tetrahydropterin analogues ranged between 20 and 60 microM with little decrease upon phosphorylation. Two exceptions were an unusually low Km of 7 microM with 6-ethyl-PH4 and a high Km of 120 microM with 6-phenyl-6-methyl-PH4, both with phosphorylated TH. Tyrosine substrate inhibition was elicited only with (R)-BH4 and 6-hydroxymethyl-PH4. With unphosphorylated TH (with the exception of 6-benzyl-6-methyl-PH4, Km = 4 mM) an inverse correlation between cofactor Km and side-chain hydrophobicity was observed ranging from a high with (R)-BH4 (5 mM) to a low with 6-cyclohexyl-PH4 (0.3 mM). An 8-fold span of Vmax was seen overall. Phosphorylation caused a 0.6-4-fold increase in Vmax and a 35-2000-fold decrease in Km for cofactor, ranging from a high of 60 microM with 6-methyl-PH4 to a low of 0.6 microM with 6-cyclohexyl-PH4. A correlation of the size of the hydrocarbon component of the side chain with affinity is strongly evident with phosphorylated TH, but in contrast to unphosphorylated enzyme, the hydroxyl groups in hydroxymethyl-PH4 (20 microM) and (R)-BH4 (3 microM) decrease Km in comparison to that of 6-methyl-PH4. Although 6,6-disubstituted analogues were found with affinities near that of (R)-BH4 (e.g., 6-propyl-6-methyl-PH4, 4 microM), they were frequently more loosely associated with phosphorylated TH than their monosubstituted counterparts (6-phenyl-PH4, 0.8 microM; cf. 6-phenyl-6-methyl-PH4, 8 microM). A model of the cofactor side-chain binding domain is proposed in which a limited region of nonpolar protein residue(s) capable of van der Waals contact with the hydrocarbon backbone of the (R)-BH4 dihydroxypropyl group is opposite to a recognition site for hydroxyl(s). Although interaction with either the hydrophilic or hydrophobic regions of unphosphorylated tyrosine hydroxylase is possible, phosphorylation by cAMP-dependent protein kinase appears to optimize the simultaneous operation of both forces.
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PMID:Changes in the cofactor binding domain of bovine striatal tyrosine hydroxylase at physiological pH upon cAMP-dependent phosphorylation mapped with tetrahydrobiopterin analogues. 256 33

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by the cAMP as well as the calcium and cGMP second messenger systems. Treatment of intact rat PC12 cells with neuropeptides including secretin and vasoactive intestinal polypeptide (VIP) stimulated tyrosine hydroxylase activity 2 to 3-fold in vitro. Secretin (EC50 = 10 nM) was about 3 orders of magnitude more potent than VIP (EC50 = 3 microM). A combination of several protease inhibitors failed to enhance the potency of either peptide. Other members of the secretin family including glucagon and peptide histidine isoleucine (PHI) stimulated tyrosine hydroxylase activity to a lesser extent. Somatostatin, which is not homologous to secretin, was ineffective. The maximal response of tyrosine hydroxylase activation to 1 microM secretin occurred within 6-15 sec. Secretin, VIP, and forskolin also enhanced tyrosine hydroxylase activity (3,4-dihydroxyphenylalanine production) in intact cells, as determined by high performance liquid chromatography and electrochemical detection. Secretin, VIP, PHI, and glucagon increased the levels of cAMP in PC12 cells more than 10-fold, as determined by radioimmunoassay. We also demonstrated that cAMP is released from the cells into the incubation medium following secretin treatment. Secretin and VIP treatment also enhanced the activity of cAMP-dependent protein kinase in a concentration-dependent fashion, as measured subsequently in vitro. Based on the greater potency of secretin in comparison with VIP, PHI, and glucagon, we suggest that the PC12 cells contain a secretin-preferring receptor that increases cAMP levels and brings about an activation of tyrosine hydroxylase activity through the stimulation of cAMP-dependent protein kinase.
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PMID:Regulation of tyrosine hydroxylase activity in rat PC12 cells by neuropeptides of the secretin family. 257 21


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