Protein Phosphorylation in Bovine Adrenal Medullary Chromaffin Cells: Histamine-Stimulated Phosphorylation of Tyrosine Hydroxylase

Histamine can cause the release of catecholamines from bovine adrenal medullary chromaffin cells by a mechanism distinct from that of the depolarizing agents nicotine or high K+ buffer. It was the aim of this study to determine the protein phosphorylation responses to histamine in these cells and to compare them with those induced by depolarization. A number of proteins showed increases in phosphorylation in response to histamine especially when analyzed on two-dimensional polyacrylamide gel electrophoresis or by phosphopeptide mapping; one protein of 20,000 daltons was markedly dephosphorylated. Emphasis was given to the effects of histamine on tyrosine hydroxylase (TOH) phosphorylation, because this protein showed the most prominent changes on one-dimensional gels. Histamine acted via H1 receptors to increase TOH phosphorylation; the response was blocked by the H1 antagonist mepyramine and could be mimicked by the H1 agonist thiazolylethylamine, but not by the H2 agonist dimaprit. The H3 agonist (R) alpha-methylhistamine increased TOH phosphorylation at high concentrations, but the response was blocked entirely by mepyramine. Histamine rapidly increased the phosphorylation of TOH, with a maximum reached within 5 s and maintained for at least 30 min. This was in marked contrast to nicotine-stimulated protein phosphorylation of TOH, which was rapidly desensitized. The initial phosphorylation response to histamine was independent of extracellular Ca2+ for at least 3 min, but the sustained response required extracellular Ca2+. This was in contrast to the situation with both nicotine and high K+ buffer, which under the conditions used here caused a response which was dependent on extracellular Ca2+ at all times investigated. In the presence of histamine, the phosphopeptide profiles for TOH were essentially the same with or without Ca2+, suggesting that the same protein kinases were involved, but at longer times there was evidence of new phosphorylation sites. The mechanism or mechanisms whereby histamine modulates TOH phosphorylation are discussed with emphasis on the differences from depolarizing agents.     

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.     

Insulin-Like Growth Factor-I Enhances Tyrosine Hydroxylase Activation in Bovine Chromaffin Cells

Previous studies have shown that insulin-like growth factor-I (IGF-I) enhances secretagogue-stimulated Ca2+ uptake and catecholamine release in bovine chromaffin cells. This report describes the effect of IGF-I on the activity of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2), the major regulatory enzyme in the pathway of catecholamine biosynthesis. Tyrosine hydroxylase activity was assayed by measuring 3,4-dihydroxyphenylalanine (Dopa) accumulation in the presence of brocresine, an inhibitor of Dopa decarboxylase. Chromaffin cells cultured in serum-free medium produced approximately 40% less Dopa when stimulated by 55 mM K+ than did cells that had been cultured in the presence of serum. Incubation of cells for 3 days in serum-free medium containing 10 nM IGF-I restored high K(+)-stimulated Dopa accumulation to a level comparable to that seen in cells cultured continuously in serum-containing medium. In eight experiments, IGF-I increased high K(+)-stimulated Dopa accumulation (expressed as picomoles per minute per milligram of protein) by 96 +/- 13%. IGF-I increased the protein content of chromaffin cells by approximately 30%; consequently, its effect on tyrosine hydroxylase activity was even greater when Dopa synthesis was expressed as picomoles per minute per 10(7) cells. IGF-I also enhanced the rate of Dopa accumulation in cells stimulated by dimethylphenylpiperazinium, 8-bromo-cyclic AMP, phorbol 12,13-dibutyrate, or Ba2+. The effect of IGF-I on high K(+)-stimulated tyrosine hydroxylase activity was measurable when enzyme activity was assayed in vitro, suggesting that this effect was due to a stable modification of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyrosine Hydroxylase Phosphorylation in Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells: The Effect of Protein Kinase and Phosphatase Inhibitors on Ser19 and Ser40 Phosphorylation

Abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [γ-³²P]ATP, in the presence or absence of 10 µ M Ca²⁺, 1 µ M cyclic AMP, 1 µ M phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca²⁺ increased the phosphorylation of Ser¹⁹ and Ser⁴⁰. Cyclic AMP, and phorbol dibutyrate in the presence of Ca²⁺, increased the phosphorylation of only Ser⁴⁰. Ser³¹ and Ser⁸ were not phosphorylated. The Ca²⁺-stimulated phosphorylation of Ser¹⁹ was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273–302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca²⁺-stimulated phosphorylation of Ser⁴⁰ was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5–22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19–31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser¹⁹ and Ser⁴⁰ were dephosphorylated by PP2A.     

Both Short- and Long-Term Effects of Nerve Growth Factor on Tyrosine Hydroxylase in Calf Adrenal Chromaffin Cells Are Blocked by S-Adenosylhomocysteine Hydrolase Inhibitors

We have previously shown that primary cultures of calf chromaffin cells respond to nerve growth factor (NGF) treatment with a selective induction of tyrosine hydroxylase (TH), which takes 48 h to be manifested. In the present study, we report that short exposure of calf chromaffin cells to NGF (5-60 min) results in TH activation, which involves a change in the Vmax of the enzyme with no change in the number of enzyme molecules, similar to an effect that has been previously reported in PC12 cells. This activation is markedly potentiated when the chromaffin cells are plated on a laminin substrate, such that after 5 min of NGF exposure, there is an approximately fourfold increase in the TH activity. Both short-term activation and long-term TH induction brought about by NGF treatment are blocked by 5'-deoxy-5'-methylthioadenosine and other drugs that act as S-adenosylhomocysteine (SAH) hydrolase inhibitors to block methylation by end-product inhibition. These drugs did not inhibit cyclic AMP-mediated TH activation or increases in the levels of TH. However, measurements of the degree of blockade of methylation in cells treated with these drugs, taken together with conceptual information regarding the nonregulatory nature of methylation in eukaryotic cells, were not consistent with inhibition of methylation as the crucial effect of the drugs to block the effects of NGF. Nonetheless, since SAH hydrolase inhibitors selectively inhibited NGF-mediated effects, and not comparable effects triggered by other stimuli, these compounds provide useful tools in future studies of the biochemical signalling mechanism of NGF.     

Bovine Tyrosine Hydroxylase Gene-Promoter Regions Involved in Basal and Angiotensin II-Stimulated Expression in Nontransformed Adrenal Medullary Cells

Abstract: The tyrosine hydroxylase gene is expressed specifically in catecholaminergic cells, and its activity is regulated by afferent stimuli. To characterize molecular mechanisms underlying those regulations, we have constructed chimeric genes consisting of bovine tyrosine hydroxylase gene promoters (wild-type or deletion mutants) and a luciferase reporter gene. The basal expression of these genes and their regulation by angiotensin II were examined in cultured bovine adrenal medullary cells. Luciferase activity was normalized to the amount of transfected plasmid DNA. A pTHgoodLUC plasmid containing the -428/+21-bp fragment of the tyrosine hydroxylase gene promoter expressed luciferase activity at severalfold higher levels than the promoterless pOLUC plasmid. Deletion of the -194/-54-bp promoter fragment containing POU/Oct, SP1, and other putative regulatory elements increased luciferase expression fivefold. An additional deletion further upstream (-269/-194 bp), including a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-like site, reduced promoter activity. These results indicate the presence of negatively and positively acting regions in the bovine tyrosine hydroxylase gene promoter controlling basal promoter activity in adrenal medullary cells. Angiotensin II stimulated the expression of endogenous tyrosine hydroxylase gene and pTHgood-LUC approximately threefold without affecting the expression of pOLUC. A comparable threefold stimulation was observed following the deletion of the -194/-54-bp promoter region, despite the increase in basal promoter activity. Additional deletion of the -269/-194-bp promoter fragment reduced stimulation by angiotensin II to 1.5-fold. These results indicate that the angiotensin II receptor-responsive element is located in the -269/-194-bp promoter region containing the TRE-like site. Additional angiotensin II-responsive site(s) may be present outside this region. Gel mobility shift assays demonstrated constitutive and angiotensin II-induced protein binding to the tyrosine hydroxylase gene promoter. Some DNA-protein complexes were displaced with c-Fos antibodies. The results suggest that c-Fos-related antigens support basal promoter activity and mediate activation of tyrosine hydroxylase by angiotensin II receptor.     

Tyrosine Kinases Are Required for Catecholamine Secretion and Mitogen-Activated Protein Kinase Activation in Bovine Adrenal Chromaffin Cells

Abstract: Nicotine-induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen-activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by >90% in cells stimulated by nicotine, 55 m M KCI, or the Ca²⁺ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie down-stream of Ca²⁺ influx and upstream of MEK activation. This Ca²⁺-activated tyrosine kinase activity could not be accounted for entirely by c-Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca²⁺-activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.     

LA－90细胞在温度转化过程中蛋白质酪氨酸磷酸化作用研究

ＬＡ－９０细胞在温度转化过程中蛋白质酪氨酸磷酸化作用研究夏英,高漫,颜卉君,吴国利（北京师范大学生物系生物化学及分子生物学研究室,１００８７５）关键词酪氨酸蛋白激酶;磷酸酪氨酸蛋白磷酸酶;细胞转化ｉｓ－ＲＳＶＬＡ－９０细胞是ＲＳＶ转染的小鼠３Ｔ３细胞...     

Long-Term Activation of Protein Kinase C by Nicotine in Bovine Adrenal Chromaffin Cells

Previous results from our laboratory suggest that long-term treatment of primary cultured bovine adrenal medullary (BAM) chromaffin cells with nicotine or phorbol 12-myristate 13-acetate, either of which directly activates protein kinase C (PKC), increases the mRNA levels encoding catecholamine-synthesizing enzymes and proenkephalin. In the present study, we have examined the effects of nicotine on BAM cell PKC activity with special emphasis on long-term effects. Nicotine increased particulate PKC activity in a concentration-dependent manner when measured using in vitro enzyme assay with histone as the substrate. This effect is mediated through nicotinic cholinergic receptors, because 1,1-dimethylphenylpiperazinium, a nicotinic agonist, had a similar effect. In addition, chlorisondamine, a specific nicotine-receptor blocking drug, antagonized the effect of nicotine. Nicotine also increased specific [3H]phorbol 12,13-dibutyrate ([3H]PdBu) binding within 1 min, the effect of which was maximal between 3 and 12 min. This effect was reversed by chlorisondamine similarly after 12 min and after 18 h of nicotine treatment, indicating that continual nicotinic-receptor occupancy is required for persistent PKC activation. Compared to PKC activation, the onset of nicotine-stimulated diacylglycerol production was slow, and it was observed after 12 min of incubation with nicotine. The diacylglycerol levels, specific [3H]PdBu binding, and PKC activity remained significantly elevated for at least 18 h with continuous nicotine incubation. Furthermore, nicotine increased the PKC immunoreactivity of a particulate protein with a molecular mass of 82 kDa in the western blot. These results suggest that nicotinic-receptor activation increases PKC activity and immunoreactivity in BAM cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Roles for Protein Kinase C and Mitogen-Activated Protein Kinase in Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Abstract: Both the Ca²⁺/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca²⁺ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca²⁺ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC₅₀ 1–5 µM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC₅₀～10 µM) than that induced by nicotine (IC₅₀～30 µM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca²⁺ ionophore and the participation of both PKC and MEK/MAPK in optimal secretion induced by nicotine.     

Tyrosine Hydroxylase Activation and Inactivation by Protein Phosphorylation Conditions

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, catalyzes the conversion of tyrosine to DOPA, Cyclic AMP-dependent protein phosphorylation conditions alter tyrosine hydroxylase activity in rat striatal homogenates. In agreement with other laboratories, we find that short-term pre-incubation (3 min) of extracts under phosphorylating conditions (Mg . ATP, cAMP) increases enzyme activity two- to tenfold over control as measured during a subsequent 15-min assay. We now report that preincubation under phosphorylating conditions for longer periods (30 min) results in a loss of activity to levels equal to or below that of the control enzyme. Addition of purified bovine brain protein kinase catalytic subunit and Mg . ATP enhances activation and increases the rate of inactivation. To demonstrate that inactivation is not associated with proteolytic degradation or irreversible denaturation, the inactivated form of the enzyme can be reactivated. The protein kinase inhibitor protein decreases the activation process and prevents inactivation of the enzyme to below control values. The sedimentation coefficient is not changed by phosphorylation conditions (S = 8.8 +/- 0.1). Although the apparent Km of the enzyme for the 6-methyltetrahydropterine (6-MPH4) cofactor is reduced (0.86 mM, control; 0.32 mM, activated), it is also reduced in the inactivated form (0.38 mM). The Ki for dopamine is increased from 4.5 microM for the control to 28 microM for the activated enzyme, whereas the inactivated form of the enzyme exhibits a Ki of 10 microM. Removal of catecholamines by gel filtration fails to alter activity and the apparent cofactor Km. Moreover, both the activated and the inactivated states persist following gel filtration. It therefore appears that the activation-inactivation process is not mediated solely by the modulation of enzyme feedback inhibition or changes in the Km for 6-MPH4. We also describe a coupled decarboxylase assay in which labeled dopamine is resolved from the precursors tyrosine and DOPA by low-voltage paper electrophoresis.     

In Vitro Phosphorylation of Bovine Adrenal Chromaffin Cell Tyrosine Hydroxylase by Endogenous Protein Kinases

Under phosphorylating conditions, addition of Ca2+ or cyclic AMP to the 100,000 g supernatant of purified bovine adrenal chromaffin cells increases both the incorporation of 32P into tyrosine hydroxylase and the activity of the enzyme. Combining maximally effective concentrations of each of these stimulating agents produces an additive increase in both the level of 32P incorporation into tyrosine hydroxylase and the degree of activation of the enzyme. The increased phosphorylation by Ca2+ is due to stimulation of endogenous Ca2+-dependent protein kinase activity and not inhibition of phosphoprotein phosphatases. When the chromaffin cell supernatant is subjected to diethylaminoethyl (DEAE) chromatography to remove calmodulin and phospholipids, tyrosine hydroxylase is no longer phosphorylated or activated by Ca2+; on the other hand, phosphorylation and activation of tyrosine hydroxylase by cyclic AMP are not affected. Subsequent replacement of either Ca2+ plus calmodulin or Ca2+ plus phosphatidylserine to the DEAE-fractionated cell supernatant restores the phosphorylation, but not activation of the enzyme. Reverse-phase HPLC peptide mapping of tryptic digests of tyrosine hydroxylase from the 100,000 g supernatant shows that the Ca2+-dependent phosphorylation occurs on three phosphopeptides, whereas the cyclic AMP-dependent phosphorylation occurs on one of these peptides. In the DEAE preparation, either cyclic AMP alone or Ca2+ in the presence of phosphatidylserine stimulates the phosphorylation of only a single phosphopeptide peak, the same peptide phosphorylated by cyclic AMP in the crude supernatant. In contrast, Ca2+ in the presence of calmodulin stimulates the phosphorylation of three peptides having reverse-phase HPLC retention times that are identical to peptides phosphorylated by Ca2+ addition to the crude unfractionated 100,000 g supernatant. Rechromatography of the peaks from each of the in vitro phosphorylations, either in combination with each other or in combination with each of the seven peaks generated from phosphorylation of tyrosine hydroxylase in situ, established that cyclic AMP, Ca2+/phosphatidylserine, and Ca2+/calmodulin all stimulate the phosphorylation of the same reverse-phase HPLC peptide: in situ peptide 6. Ca2+/calmodulin stimulates the phosphorylation of in situ peptides 3 and 5 as well. Thus, tyrosine hydroxylase can be phosphorylated in vitro by protein kinases endogenous to the chromaffin cell. Phosphorylation occurs on a maximum of three of the seven in situ phosphorylated sites, and all three of these sites can be phosphorylated by a Ca2+/calmodulin-dependent protein kinase.     

Localization of Basic Fibroblast Growth Factor in Bovine Adrenal Chromaffin Cells

Abstract: We have investigated basic fibroblast growth factor (FGF-2) localization in and release from isolated bovine adrenal chromaffin cells. In contrast to previous reports, we found no evidence of fibroblast growth factor (FGF) storage in catecholamine-containing chromaffin granules. Subcellular fractionation studies did not show enrichment of FGF-2 immunoreactivity in granules, and cholinergic stimulation failed to release FGF-2 into the medium. Our results suggest that adrenal chromaffin cells resemble other FGF-2-synthesizing cell types with respect to FGF storage and secretion.     

Arachidonic Acid Activates Cation Channels in Bovine Adrenal Chromaffin Cells

Abstract— Microscopic fluorescence analysis of fura-2-loaded bovine adrenal chromaffin cells demonstrates that ～70% of the cells responded to arachidonic acid in increasing the intracellular Ca²⁺ concentration. Because this increase was markedly less in the absence of external Ca²⁺, we examined the effect of arachidonic acid on Ca²⁺ influx electrophysiologically. Bath application of 10 μM arachidonic acid induced a long-lasting inward current when the cell was clamped at -50 mV. Other fatty acids, such as oleic acid, linoleic acid, eicosatrienoic acid, and eicosa-pentaenoic acid, were all ineffective. The current-voltage relationships suggest that arachidonic acid may activate voltage-insensitive channels. Arachidonic acid (2μM) activated a single-channel current in the inside-out patch, even in the presence of inhibitors of cyclooxygenase and lipoxygenase, possibly suggesting that arachidonic acid could activate channels directly. The onset delay of the inward channel current in the outside-out patch configuration (54.02 ± 63.5 s; mean SD) was significantly shorter than that in the inside-out patch one (197.3 ± 177.7 s). Washout of arachidonic acid decreased the probability of channel openings in the outside-out patch but not in the inside-out one. These results suggest that arachidonic acid activates channels reversibly from outside of the plasma membrane. The unitary conductarce for Ca²⁺ of arachidonic acid-activated channel was ～17 pS. The arachidonic acid-activated channel was permeable to Ba²⁺, Ca²⁺, and Na⁺ but not to Cl^?. The opening probability of the arachidonic acid-activated channel did not depend on membrane potential. These results demonstrate that arachidonic acid activates cation-selective, Ca²⁺-permeable channels in bovine adrenal chromaffin cells.     

Phosphorylation of Tyrosine Hydroxylase by Cyclic GMP-Dependent Protein Kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated and activated by purified cyclic GMP-dependent protein kinase as well as by cyclic AMP-dependent protein kinase catalytic subunit. The extent of activation was correlated with the degree of phosphate incorporated into the enzyme. Comparable stoichiometric ratios (0.6 mol phosphate/mol tyrosine hydroxylase subunit) were obtained at maximal concentrations of either cyclic AMP-dependent or cyclic GMP-dependent protein kinases. The enzymes appeared to mediate the phosphorylation of the same residue based on the observation that incorporation was not increased when both enzymes were present. The major tryptic phosphopeptide obtained from tyrosine hydroxylase phosphorylated by each protein kinase exhibited an identical retention time following HPLC. The purified phosphopeptides also exhibited identical isoelectric points. These data provide support for the notion that the protein kinases are phosphorylating the same residue of tyrosine hydroxylase.     

Tyrosine Hydroxylase Phosphorylation in Bovine Adrenal Chromaffin Cells: The Role of Intracellular Ca2+ in the Histamine H1 Receptor-Stimulated Phosphorylation of Ser8, Ser19, Ser31, and Ser40

Abstract: Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H₁ receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca²⁺. In contrast, the H₁-mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 µ M ) and significantly reduced by prior exposure to caffeine (10 m M for 10 min) to deplete intracellular Ca²⁺. Trypticphosphopeptide analysis by HPLC revealed that the H₁ response in the presence or absence of extracellular Ca²⁺ resulted in a major increase in the phosphorylation of Ser¹⁹ with smaller increases in that of Ser⁴⁰ and Ser³¹. In contrast, although a brief stimulation with nicotine (30 µ M for 60 s) also resulted in a major increase in Ser¹⁹ phosphorylation, this response was abolished in the absence of extracellular Ca²⁺. These data indicate that the mobilization of intracellular Ca²⁺ plays a crucial role in supporting H₁-mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis.     

The Use of Permeabilized Cells to Assay Protein Phosphorylation and Catecholamine Release

A number of approaches can be used to determine the protein kinases and protein phosphatases acting on particular phosphoproteins in vivo. Cell permeabilization represents one such approach. In this overview we discuss the different permeabilization procedures used in bovine adrenal chromaffin cells and in particular the use of digitonin. The effect of various factors on the extent of digitonin-permeabilization, protein phosphorylation and catecholamine release are also discussed. The factors include the permeabilization medium, the ions such as calcium, and the second messengers, such as cAMP, IP₃, cADPR and calmodulin. The effect of specific peptide inhibitors of protein kinases on tyrosine hydroxylase phosphorylation is illustrated. Advantages and disadvantages of cell permeabilization procedures are discussed throughout the text.     

Bovine Adrenal Tyrosine Hydroxylase: Comparative Study of Native and Proteolyzed Enzyme, and Their Interaction with Anions

Abstract: The pH optimum of native adrenal medulla tyrosine hydroxylase activity is shifted from 5.8 to 6.4 by polyanions (heparin, dextran sulphate), salts (NaCl, Na₂SO₄) and the anionic buffer 2-( N -morpholino)ethanesulphonic acid (MES). Simultaneously, the activity at the optimal pH is increased. Kinetic studies have shown that this activation is associated with a decrease of the apparent K _m of the enzyme for the cofactor 6,7-dimethyltetrahydropterin (DMPH₄) and an increase in the V _max for tyrosine and DMPH₄. The K _m for the tyrosine remained unchanged. These data have been interpreted in terms of the polyelectrolyte theory. The adsorption of tyrosine hydroxylase on various affinity gels containing heparin, dextran sulphate or unsulphated polymer dextran as ligands indicate that the activation of the enzyme is mediated by electrostatic interactions with the anionic species. The site of electrostatic interaction possesses some specificity since the binding constants are higher for heparin or dextran sulphate than for NaCl or MES buffer. Moreover, 3-( N -morpholino)propanesulphonic acid (MOPS) a slightly structurally different buffer inhibits the enzyme activity whereas N -(2-acetamido)-2-amino-ethanesulphonic acid (ACES) has no effect. A limited proteolytic digestion which preserves the enzymatic activity, destroys the effects of the anions. The isoelectric point and the molecular parameters of tyrosine hydroxylase are markedly altered after limited digestion. It is therefore suggested that the interaction between the hydroxylase and anionic compounds occurs on a part of the protein which is different from the active site and which is lost by proteolysis. This portion of the protein might be involved in regulation of native tyrosine hydroxylase.