Jack C. Waymire

Assay of tyrosine hydroxylase by coupled decarboxylation of dopa formed from 1-14C-l-tyrosine

1. 1. A method has been presented for the measurement of tyrosine hydroxylase using a reaction coupled to aromatic l-amino acid decarboxylase. 2. 2. The specificity of the assay has been demonstrated in experiments in which various factors that influence either tyrosine hydroxylase or aromatic l-amino acid decarboxylase activity were omitted. 3. 3. The validity of the assay has been shown by the close agreement between the moles of CO2 produced and the moles of dopa formed, as well as the loss of all activity when the essential cofactors are omitted. 4. 4. The sensitivity of the method has been shown to be greater in our laboratory than tyrosine hydroxylase assays currently used.

Bovine adrenal chromaffin cells: high-yield purification and viability in suspension culture

A method for purifying chromaffin cells from adult, bovine, adrenal medullae and the techniques for maintaining the cells in suspension culture for at least 14 days are presented. Perfusion of medullae with a collagenase-containing medium produced a cell fraction that contained, in addition to chromaffin cells, a significant percentage of non-chromaffin cells. These cells were found to attach more rapidly than chromaffin cells to glass and tissue-culture plasticware. Using this property, we devised a selective plating procedure that yielded approximately 1-2 x 10(8) chromaffin cells per adrenal medulla at a purity of 95% or higher. On the basis of catecholamine levels and enzyme activities, suspension (as opposed to monolayer) cultures were chosen to further investigate their potential as a model system for the regulation of adrenergic function. In contrast to chromaffin cells cultured in monolayer, chromaffin cells in suspension had a more rounded appearance and formed multicellular aggregates with time in culture. Very few neurite-like structures, commonly observed in monolayer cultures, were present in the suspension cultures. Also, inhibitors of mitosis were not necessary to prevent overgrowth by non-chromaffin cells as there was little or no cell division in the suspension cultures. Catecholamine levels were relatively stable for at least 2 weeks, although a gradual decline in epinephrine occurred after day 5. Unlike other enzymes involved in catecholamine metabolism, phenylethanolamine N-methyl transferase activity declined significantly with time in culture in parallel to the gradual loss of epinephrine. In addition, both oxygen consumption and amino acid incorporation into proteins were relatively stable. Thus, the primary suspension cultures of adult, bovine chromaffin cells seem to offer several advantages for studying long-term regulation of chromaffin cell function and provide a stable source of adrenergic cells for examining short-term regulatory processes.

Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins.

We have identified the beta (beta) isoform of the 14‐3‐3 family of proteins as an activator of the Raf‐1 protein kinase. 14‐3‐3 was isolated in a yeast two‐hybrid screen for Raf‐1 kinase domain binding proteins. Purified bovine brain 14‐3‐3 interacted specifically with both c‐Raf‐1 and the isolated Raf‐1 kinase domain. Association was sensitive to the activation status of Raf‐1; 14‐3‐3 bound to unactivated Raf‐1, but not Raf‐1 activated by protein kinase C alpha or Ras and Lck. The significance of these interactions under physiological conditions was demonstrated by co‐immunoprecipitation of Raf‐1 and 14‐3‐3 from extracts of quiescent, but not mitogen‐stimulated, NIH 3T3 cells. 14‐3‐3 was not a preferred Raf‐1 substrate in vitro and did not significantly affect Raf‐1 kinase activity in a purified system. However, in cell‐free extracts 14‐3‐3 acted as a Ras‐independent activator of both c‐Raf‐1 and the Raf‐1 kinase domain. The same results were obtained in vivo using transfection assays; 14‐3‐3 enhanced both c‐Raf‐1‐ and Raf‐1 kinase domain‐stimulated expression of AP‐1‐ and NF‐kappa B‐dependent reporter genes and accelerated Raf‐1 kinase domain‐triggered differentiation of PC12 cells. We conclude that 14‐3‐3 is a latent co‐activator bound to unactivated Raf‐1 in quiescent cells and mediates mitogen‐triggered but Ras‐independent regulatory effects aimed directly at the kinase domain.

Depolarization-stimulated catecholamine biosynthesis : involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ

Depolarizing stimuli increase catecholamine (CA) biosynthesis, tyrosine hydroxylase (TH) activity, and TH phosphorylation at Ser19, Ser31, and Ser40 in a Ca2+‐dependent manner. However, the identities of the protein kinases that phosphorylate TH under depolarizing conditions are not known. Furthermore, although increases in Ser31 or Ser40 phosphorylation increase TH activity in vitro, the relative influence of phosphorylation at these sites on CA biosynthesis under depolarizing conditions is not known. We investigated the participation of extracellular signal‐regulated protein kinase (ERK) and cAMP‐dependent protein kinase (PKA) in elevated K+‐stimulated TH phosphorylation in PC12 cells using an ERK pathway inhibitor, PD98059, and PKA‐deficient PC12 cells (A126‐B1). In the same paradigm, we measured CA biosynthesis. TH phosphorylation stoichiometry (PS) was determined by quantitative blot‐immunolabeling using site‐ and phosphorylation state‐specific antibodies. Treatment with elevated K+ (+ 58 mm) for 5 min increased TH PS at each site in a Ca2+‐dependent manner. Pretreatment with PD98059 prevented elevated K+‐stimulated increases in ERK phosphorylation and Ser31 PS. In A126‐B1 cells, Ser40 PS was not significantly increased by forskolin, and elevated K+‐stimulated Ser40 PS was three‐ to five‐fold less than that in PC12 cells. In both cell lines, CA biosynthesis was increased 1.5‐fold after treatment with elevated K+ and was prevented by pretreatment with PD98059. These results suggest that ERK phosphorylates TH at Ser31 and that PKA phosphorylates TH at Ser40 under depolarizing conditions. They also suggest that the increases in CA biosynthesis under depolarizing conditions are associated with the ERK‐mediated increases in Ser31 PS.

Vasoactive intestinal peptide stimulates catecholamine biosynthesis in isolated adrenal chromaffin cells: evidence for a cyclic AMP-dependent phosphorylation and activation of tyrosine hydroxylase.

Vasoactive intestinal peptide (VIP) increased catecholamine biosynthesis in bovine adrenal chromaffin cells by 50–200%. Six related peptides produced no effects. In addition, VIP increased tyrosine hydroxylase (TH) activity measured in gel‐filtered supernatants prepared from homogenates of treated cells. The hypothesis that cyclic AMP is the second messenger involved in these effects of VIP was also evaluated. VIP led to an elevation of cyclic AMP levels, and this increase occurred over a similar concentration range and time course as the activation of TH and the increase in catecholamine biosynthesis. Each measure reached maximal levels at 10–20 γM VIP within 1 min and remained elevated for at least 16 min. These changes produced by VIP were paralleled by enhanced phosphorylation of TH, and this phosphorylation occurred on a single tryptic peptide that was the same peptide whose phosphorylation has been previously shown to be stimulated by forskolin. In contrast to VIP and forskolin, 12‐O‐tetradecanoylphorbol 13‐acetate, a phorbol ester known to activate protein kinase C, increased the phosphorylation on a total of three tryptic peptides of TH. Our results indicate that VIP stimulates catecholamine biosynthesis in chromaffin cells through the phosphorylation and activation of TH and support the conclusion that a cyclic AMP‐dependent phosphorylation of TH is responsible for these effects.

Activation of Protein Kinase C by Purified Bovine Brain 14-3-3: Comparison with Tyrosine Hydroxylase Activation

Abstract: In the course of the purification of 14‐3‐3 protein (14‐3‐3) we found that 14‐3‐3 isolated from bovine forebrain activates protein kinase C (PKC), rather than the previously reported protein kinase C inhibitory activity (KCIP). We have characterized the 14‐3‐3 activation of PKC. The physical properties of purified PKC activator are the same as those previously reported for 14‐3‐3 and KCIP; i.e., (1) it is composed of subunits of molecular weight 32,000, 30,000, and 29,000; (2) it is homogeneous with respect to molecular weight, as judged by native gradient‐gel electrophoresis, with a molecular weight of 53,000; and (3) it is composed of at least six isoforms when analyzed by reverse‐phase HPLC. The concentration dependence of PKC activation by 14‐3‐3 is in the same range as that shown previously for KCIP inhibition of PKC, and as that required for 14‐3‐3 activation of tyrosine hydroxylase; a maximal stimulation of two‐ to three‐fold occurs at 40–100 µg/ml. 14‐3‐3s activation of PKC is sensitive to α‐chymotrypsin digestion but is not heat labile. Activation is specific to PKC; at least two other protein kinases, cyclic AMP‐ and calcium/calmodulin‐dependent protein kinases, are not activated. The activation of PKC by 14‐3‐3 is independent of phosphatidylserine and calcium and, as such, is an alternative mechanism for the activation of PKC that obviates its translocation to membranes.

A further study on the morphology and biochemistry of X-ray and dibutyryl cyclic AMP-induced differentiated neuroblastoma cells in culture

Abstract Dibutyryl cyclic AMP-induced morphological differentiation of mouse neuroblastoma cells in culture required the synthesis of new protein but not of new RNA. An appreciable increase in RNA synthesis, a slight increase in protein synthesis and a marked decrease in DNA synthesis occurred in the dibutyryl cyclic AMP-induced differentiated cells. Tyrosine hydroxylase activity which is barely detectable in the control neuroblastoma culture markedly increased after treatment with dibutyryl cyclic AMP. Sodium butyrate, which inhibited cell division without causing morphological differentiation also increased tyrosine hydroxylase activity, but to a lesser degree. 3′5′ Cyclic AMP and 5′AMP which inhibited cell division without causing morphological differentiation did not increase the enzyme activity. X-irradiation produced inhibition of cell division and morphological differentiation similar to that observed with dibutyryl cyclic AMP, but the tyrosine hydroxylase activity did not increase. The irradiated cells, nevertheless, retained the potential to manifest increased tyrosine hydroxylase levels upon subsequent exposure to either dibutyryl cyclic AMP or sodium butyrate. The present data suggest that increased tyrosine hydroxylase levels in neuroblastoma cells is not inextricably linked to morphological changes which are presumed to reflect cellular differentiation.

Enhancement of Tyrosine Hydroxylase Phosphorylation and Activity by Glial Cell Line-derived Neurotrophic Factor

Although glial cell-line derived neurotrophic factor (GDNF) acts as a potent survival factor for dopaminergic neurons, it is not known whether GDNF can directly alter dopamine synthesis. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for dopamine biosynthesis, and its activity is regulated by phosphorylation on three seryl residues: Ser-19, Ser-31, and Ser-40. Using a TH-expressing human neuroblastoma cell line and rat primary mesencephalic neuron cultures, the present study examined whether GDNF alters the phosphorylation of TH and whether these changes are accompanied by increased enzymatic activity. Exposure to GDNF did not alter the TH protein level in either neuroblastoma cells or in primary neurons. However, significant increases in the phosphorylation of Ser-31 and Ser-40 were detected within minutes of GDNF application in both cell types. Enhanced Ser-31 and Ser-40 phosphorylation was associated with increased TH activity but not dopamine synthesis in neuroblastoma cells, possibly because of the absence of l-aromatic amino acid decarboxylase activity in these cells. In contrast, increased phosphorylation of Ser-31 and Ser-40 was found to enhance dopamine synthesis in primary neurons. Pharmacological experiments show that Erk and protein kinase A phosphorylate Ser-31 and Ser-40, respectively, and that their inhibition blocked both TH phosphorylation and activity. Our results indicate that, in addition to its role as a survival factor for dopaminergic neurons, GDNF can directly increase dopamine synthesis.

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

Abstract: 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.

ADRENERGIC ENZYMES IN CULTURED MOUSE NEUROBLASTOMA: ABSENCE OF DETECTABLE AROMATIC‐l‐AMINO‐ACID DECARBOXYLASE

The relative activities of tyrosine hydroxylase, aromatic‐l‐amino‐acid decarboxylase and dopamine beta‐hydroxylase were established in a number of clones of neuroblastoma cells isolated from the uncloned mouse C‐1300 tumor. One clone, NBD‐2, was chosen for further analysis on the basis of its relatively high activities of tyrosine hydroxylase and dopamine beta‐hydroxylase. The levels of these enzymes, and monoamine oxidase and catechol O‐methyltransferase, were at least 20‐80 fold lower in the neuroblastoma culture than in mouse superior cervical ganglion. More importantly, aromatic‐l‐amino‐acid decarboxylase activity was not even detectable in any neuroblastoma clone examined. Based on the relative sensitivities of the tyrosine hydroxylase and aromatic‐l‐amino‐acid decarboxylase assays and on the ratio of these two enzymes in the mouse ganglion, decarboxylase activity is more than 10 fold lower in the cultured cells than would be predicted on the basis of tyrosine hydroxylase activity.