Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.16.2 (tyrosine hydroxylase)
 14,760 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to study the role of protein kinase C in the regulation of tyrosine hydroxylase phosphorylation in PC12 cells, the effects of various agonists on diacylglycerol accumulation in PC12 cells were measured and the ability of these agonists to increase the phosphorylation tyrosine hydroxylase in protein kinase C-deficient cells was evaluated. Bradykinin (10 microM) and elevated extracellular K+ (55 mM) increased the accumulation of [3H]diacylglycerol in PC12 cells that had been prelabeled with [3H]arachidonic acid, and so might be expected to activate protein kinase C in these cells; in contrast, nerve growth factor did not increase diacylglycerol accumulation in PC12 cells. Protein kinase C-deficient PC12 cells were prepared by incubating the cells for 24 h with 1 microM phorbol dibutyrate. This treatment resulted in the loss of approximately 90% of the protein kinase C activity in the cells. Control and protein kinase C-deficient cells were incubated with 32Pi for 90 min and then stimulated with various agonists. 32P-labeled tyrosine hydroxylase was isolated from the cells by polyacrylamide gel electrophoresis and subjected to tryptic hydrolysis. 32P-containing phosphopeptides were separated by two-dimensional thin-layer electrophoresis and chromatography, visualized by autoradiography, and quantitated by scintillation counting Treatment of control cells with phorbol dibutyrate increased the incorporation of 32P into one tryptic phosphopeptide (referred to as T3) in tyrosine hydroxylase. Phorbol dibutyrate did not increase the phosphorylation of this peptide in protein kinase C-deficient cells. Bradykinin or 55 mM K+ increased the incorporation of 32P into four tyrosine hydroxylase phosphopeptides, including peptide T3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation of tyrosine hydroxylase in protein kinase C-deficient PC12 cells. 257 Mar 73

During investigations of the regulation of tyrosine hydroxylase (TH) by protein phosphorylation, a novel protein kinase activity has been discovered in rat pheochromocytoma. Originally detected as a trace contaminant in preparations of highly purified TH, this novel kinase activity phosphorylated TH at serine 8 in the proline-rich amino-terminal region of the enzyme. This particular site is not phosphorylated by, nor is the amino acid sequence surrounding this site selective for, any of the classical (i.e. well characterized) protein kinases. In this report, we describe the identification, characterization, and partial purification of this novel protein kinase. By utilizing a synthetic peptide corresponding to the amino-terminal region of TH, a selective assay for this protein kinase was developed. The kinase activity utilized ATP and magnesium, although GTP could also be utilized as a phosphate donor. The kinase activity was found to co-purify with TH activity through ammonium sulfate precipitation and DEAE-cellulose chromatography and could be only partially resolved from TH by heparin-agarose affinity chromatography. Substantial kinase activity could be resolved from TH by phosphocellulose chromatography. The novel kinase migrates as a protein with a molecular mass of approximately 45 kDa on gel permeation chromatography as well as sucrose density gradient centrifugation. Studies of site specificity indicate that this Ser/Thr kinase activity appears to be directed by an adjacent (carboxyl-terminal) proline residue, exhibiting a minimal recognition sequence of -X-Ser/Thr-Pro-X-. In addition to TH, this proline-directed protein kinase will also phosphorylate synapsin I, histone H1, and glycogen synthase, suggesting that this kinase may have multiple substrates in vivo. Additional findings indicate that the activity of proline-directed protein kinase is increased transiently in PC12 pheochromocytoma cells following treatment with nerve growth factor. Distinctions between this novel kinase and other well characterized protein kinases can be made on the basis of phosphorylation site specificity, chromatographic behavior, and physical characteristics.
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PMID:Identification of a novel proline-directed serine/threonine protein kinase in rat pheochromocytoma. 257 Jul 79

Peripherin is the main intermediate filament protein in sympathetic neurons. Immunoreactivity to peripherin was studied in mouse adrenal chromaffin cells after 6 days in culture, and compared to immunoreactivity to tyrosine hydroxylase used as a general marker of chromaffin cells in culture. Most of the cells immunoreactive to tyrosine hydroxylase were rounded, with a glandular phenotype and a few of them had processes. The cells reactive to peripherin only constituted a small proportion of the chromaffin cells (2%), and most of them sent out processes. However, not all the cells with processes were reactive for peripherin. These results did not change in the presence of nerve growth factor. The discussion focuses on the significance of the sub-population of cells reactive to peripherin. We suggest that these cells resemble the small granule chromaffin cells, regarded as an intermediate cell type between glandular cells and neurons. The cells that expressed peripherin here are compared to those selected to form the PC12 clone. The presence of peripherin in only a few of the cells sending out neurite-like processes is discussed in relation to the expression of other neurofilament proteins in developing cells and to the influence of non-chromaffin cells.
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PMID:Immunocytochemical localization of the intermediate filament protein peripherin in adult mouse adrenal chromaffin cells in culture. 257 56

Long-term neural crest cultures grown in the continuous absence of exogenous nerve growth factor (NGF) contain a subpopulation of cells with NGF receptors exclusively of the low affinity subtype (Kd of approximately 3.2 nM). The current studies combined immunocytochemistry, using GIN1 (a support cell marker) or tyrosine hydroxylase antibodies, with radioautography following exposure to iodinated nerve growth factor (125I-NGF). The majority of cells specifically binding 125I-NGF were found to be immunoreactive for GIN1, indicating that the primary cell phenotype expressing receptors for NGF appear to be support cell precursors, at least under these conditions. These cells are likely to be responsive to and/or dependent upon NGF; the nature of this response or dependency remains to be determined. Some cells exhibiting silver grains were not immunoreactive for GIN1, suggesting that other cell phenotypes in neural crest cultures also have NGF receptors. In addition, some neural crest cells were found that stained with GIN1 and lacked 125I-NGF binding. Tyrosine hydroxylase-like immunoreactive cells apparently did not bind 125I-NGF under these culture conditions. Catecholaminergic sympathetic and sensory neurons from embryonic ganglia, derived from the neural crest, express both the high and low affinity forms of the NGF receptor. In order to determine whether the microenvironment played a role in the type of catecholaminergic cells appearing in culture, neural crest cells were grown in the continuous presence of exogenous NGF. Under these conditions, many tyrosine hydroxylase-like immunoreactive cells were found that specifically bound 125I-NGF. In addition, silver grains were still detected on these cells following a chase with nonradioactive NGF, designed to eliminate 125I-NGF bound to low affinity sites. Therefore, the catecholaminergic cells possess both the low and high affinity forms of the receptor. NGF's ability to modulate tyrosine hydroxylase activity, as it does in mature catecholaminergic neurons, was tested in this system. Surprisingly, there was no statistically significant difference in tyrosine hydroxylase activity in cultures grown in the absence or presence of exogenous NGF. This raises the possibility that embryonic catecholaminergic cells are unable to respond to NGF in this specific way, even though the receptors for the factor are present.
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PMID:Catecholaminergic cells and support cell precursors in neural crest cultures differentially express nerve growth factor receptors. 257 44

The effects of retinoic acid (RA), a naturally occurring metabolite of vitamin A, on the growth, morphology and neurochemical differentiation of the PC12 clone of rat pheochromocytoma cells were investigated. RA added to the medium inhibited the growth of PC12 cells in a dose-dependent manner up to 10 microM without affecting their morphology. In PC12 cells cultured in the presence of 10 microM RA for 8 days, the specific activity of choline acetyltransferase (ChAT) was increased 2-fold, while the specific activity of tyrosine hydroxylase (TH) was decreased 0.5-fold compared with cells cultured in the absence of RA. Specific activities of acetylcholinesterase (AChE), glutamate decarboxylase (GAD) and lactate dehydrogenase (LDH) were not affected by RA. Both the increase of ChAT and the decrease of TH induced by RA exhibited similar time and dose dependencies. RA inhibited the increase of TH activity induced by nerve growth factor (NGF), an adrenergic neuronotrophic factor on PC12 cells. From these observations it was concluded that RA induces a cholinergic neurochemical differentiation of PC12 cells independent of a morphological differentiation.
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PMID:Cholinergic differentiation of clonal rat pheochromocytoma cells (PC12) induced by retinoic acid: increase of choline acetyltransferase activity and decrease of tyrosine hydroxylase activity. 257 10

The effects of chronic nerve growth factor administration on the development of neuropeptides in the embryonic chick peripheral nervous system were quantitated by radioimmunoassays. Starting at embryonic Day 3.5, daily doses of 20 micrograms of nerve growth factor (NGF) increased the substance P content of lumbosacral spinal sensory ganglia at all ages studied (Days 10-14), while having no effect on substance P levels of thoracic sensory ganglia. In contrast, the contents of somatostatin were increased in both thoracic and lumbosacral ganglia, but only at comparatively late time points (Day 14). Nerve growth factor administration was also found to decrease the somatostatin contents of lumbosacral paravertebral sympathetic ganglia at early time points (Day 8) while increasing levels at later stages (Day 14), thus acting to accelerate the normally occurring developmental changes in level of this peptide. These changes were shown to be specific for somatostatin by demonstrating that NGF increased tyrosine hydroxylase levels in sympathetic neurons at Day 8, and had no effect on sympathetic vasoactive intestinal polypeptide levels at Day 14. It has been concluded that exogenous NGF does not simply act to increase or prolong the expression of neuron-specific phenotypes in the chick, but rather its action is time and location dependent to accelerate development.
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PMID:Nerve growth factor changes the relative levels of neuropeptides in developing sensory and sympathetic ganglia of the chick embryo. 257 2

It is known that nerve growth factor (NGF) induces neurite outgrowth and elevation of the activity of adrenergic marker enzyme, tyrosine hydroxylase (TH) in clonal rat pheochromocytoma cells (PC12), whereas glioma-conditioned medium (GCM) induces neurite outgrowth and elevation of the activity of cholinergic marker enzyme, choline acetyltransferase (ChAT) in PC12 cells. In the previous study we have shown that retinoic acid (RA) induces specific elevation of ChAT activity and depression of TH activity without morphological differentiation (Matsuoka, I. et al., Brain Res., 502 (1989]. In the present study, we compared the effects of NGF, GCM and RA on the intracellular signalings in PC12 cells in relation to the mechanism of cholinergic differentiation. Addition of NGF, GCM or RA to the culture medium of PC12 cells caused a rapid rise in intracellular Ca2+ concentration [( Ca2+]i) reaching the level of almost 2.5-fold the resting condition within 3-18 h. Thereafter, [Ca2+]i of NGF-treated cells were decreased to the resting level within 12 h. On the other hand, [Ca2+]i of GCM-and RA-treated cells decreased to a level which was 1.8- to 2-fold the resting condition within 24-48 h and stayed at this level for up to 4-7 days. When homogenates of GCM- and RA-treated PC12 cells were incubated with [gamma-32P]ATP, phosphorylation of a protein with molecular mass of 27 kDa (27 K-protein) was specifically enhanced. The phosphorylation of the 27 K-protein was not seen in the homogenate of the NGF-treated cells. The phosphorylation of the 27 K-protein was dependent on Ca2+ and inhibited by inhibitors of Ca2+-dependent protein kinase, H-7 and W-7. Addition of H-7 and W-7 to the culture medium of PC12 cells abolished the elevation of ChAT activity specifically induced by GCM and RA. These observations suggested that the sustained increase of [Ca2+]i and Ca2+-dependent protein phosphorylation are involved in the intracellular signaling mechanism required for the cholinergic differentiation of PC12 cells induced by GCM and RA.
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PMID:Possible involvements of intracellular Ca2+ and Ca2+ -dependent protein phosphorylation in cholinergic differentiation of clonal rat pheochromocytoma cells (PC12) induced by glioma-conditioned medium and retinoic acid. 258

We describe features of the regulation of a neural-specific gene, SCG10, which is induced by nerve growth factor (NGF) during the neuronal differentiation of the rat pheochromocytoma cell line PC12. Induction of SCG10 mRNA occurs within 12-24 hr of exposure to NGF, is sustained in the continued presence of the neurotrophic factor, and involves a mechanism that is, at least in part, transcriptional. Unlike the rapid, transient transcriptional activations of genes such as c-fos, SCG10 induction requires ongoing protein synthesis, suggesting the participation of a de novo synthesized regulatory protein in mediating the effects of NGF on this gene. Although c-fos itself may play this role, its induction is clearly insufficient to cause an induction of SCG10. NGF, FGF, and, to a lesser extent, phorbol esters induced SCG10, whereas EGF and dibutyryl cAMP did not. In these characteristics, SCG10 induction appears to constitute a reliable molecular index of the transcription-dependent neuronal differentiation induced by NGF. Glucocorticoids, which inhibit NGF-induced neurite outgrowth from normal primary chromaffin cells, partially blocked SCG10 induction in PC12 cells. A reciprocal pattern of regulation by NGF and glucocorticoids was observed for tyrosine hydroxylase mRNA. These data suggest that environmental signals such as NGF may act on specific genes, both positively and negatively, to control the choice of alternative fates by developing neural crest cells.
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PMID:The induction of a neural-specific gene, SCG10, by nerve growth factor in PC12 cells is transcriptional, protein synthesis dependent, and glucocorticoid inhibitable. 283 17

A rapid phosphorylation of tyrosine hydroxylase occurs in the PC12 nerve-like clonal cell line in response to nerve growth factor (NGF), epidermal growth factor (EGF), dibutyryl-cAMP, cholera toxin, phorbol- 12-myristate-13-acetate (PMA), or potassium depolarization in the presence of calcium ions. Complete tryptic digestion and two-dimensional peptide mapping reveals four available sites of phosphorylation in the enzyme. Phosphoamino acid analysis demonstrates that serine is the amino acid residue phosphorylated in each peptide. Specific phosphorylation of each of the four sites is achieved by different subsets of the above agents. One peptide site is phosphorylated in response to EGF alone. A second site is phosphorylated only in response to NGF, cholera toxin or dibutyryl-cAMP. A third site is phosphorylated only in response to potassium depolarization and requires the presence of extracellular Ca2+. The fourth site is the only site phosphorylated in response to PMA. These data indicate that at least 4 distinct kinase systems can act to phosphorylate tyrosine hydroxylase in PC12 cells. The PMA-stimulated peptide site is also phosphorylated in response to every one of the other agents. Further proteolytic digestions and phosphopeptide mapping of this common peptide, using Staphylococcus V8 protease and thermolysin, did not generate different phosphopeptides resulting from the different agents. These data suggest that the phosphorylation of this common peptide in response to all of the agents may be mediated by a common kinase, and, hence, that tyrosine hydroxylase phosphorylation by some agents may be mediated by two kinases. Although phosphopeptide maps of tyrosine hydroxylase resulting from cAMP elevation or NGF are qualitatively similar, quantitative differences exist, suggesting differential regulation of the same kinases by these agents. Tyrosine hydroxylase was found to be activated 2--4-fold in response to each phosphorylating agent. Thus, NGF and EGF present novel, natural means of regulating the activation state of tyrosine hydroxylase in responsive neurons.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Nerve growth factor and other agents mediate phosphorylation and activation of tyrosine hydroxylase. A convergence of multiple kinase activities. 286 43

A specific antiserum was used to compare phosphorylation of tyrosine hydroxylase (TH) (EC 1.14.16.2, tyrosine 3-monooxygenase) as regulated by elevated K+ and nerve growth factor (NGF) in cultured PC12 pheochromocytoma cells. Exposure of cultures to either elevated K+ or to NGF significantly enhanced the incorporation of [32P]orthophosphate into TH. The effect of elevated K+ was evident at 10 mM and was maximal by 40-80 mM. Increased phosphorylation of TH was detected at 0.1 nM (3 ng/ml) NGF and reached a maximal level by 0.3-1 nM (10-30 ng/ml) NGF. Elevated K+ showed a biphasic time course of action with one maximum of phosphorylation at about 30 sec of exposure and a second after about 10 min of exposure. In contrast, the NGF effect showed an initial lag of several minutes followed by a monophasic increase in phosphorylation to reach a plateau. Both treatments enhanced TH activity, but in each case the time courses of this did not strictly correlate with that of phosphorylation. The effect of elevated K+ on TH phosphorylation required the presence of extracellular Ca2+ and was suppressed by trifluoperazine (100 microM). N-(6-Aminohexyl)-5-(chloronaphthalene)-1-sulfonamide (W-7) (100 microM), a potent inhibitor of calmodulin activity, also blocked the enhancement of phosphorylation by elevated K+, whereas N-(6-aminohexyl)-1-(naphthalene)sulfonamide (W-5) (100 microM), a less potent analogue of W-7, did not. In contrast to these findings, the increase in TH phosphorylation brought about by NGF did not require extracellular Ca2+, and was only slightly affected by trifluoperazine or W-7. When TH phosphorylated under various conditions (control medium, elevated K+, NGF) was subjected to peptide mapping after exposure to Staphylococcus aureus protease V8, multiple phosphorylated peptides were observed. Elevated K+ and NGF each produced increases in labeling of each of the peptides. However, the relative degree of labeling of different peptides was distinct for each condition. These data suggest that elevated K+ and NGF bring about rapid enhancement of the phosphorylation of TH by means of different mechanisms.
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PMID:Regulation of tyrosine hydroxylase phosphorylation in PC12 pheochromocytoma cells by elevated K+ and nerve growth factor. Evidence for different mechanisms of action. 286 75


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