Activation and Multiple‐Site Phosphorylation of Tyrosine Hydroxylase in Perfused Rat Adrenal Glands

Abstract

Tryptic digestion of tyrosine hydroxylase (TH) isolated from rat adrenal glands labeled with 32Pi produced five phosphopeptides. Based on the correspondence of these phosphopeptides with those identified in TH from rat pheochromocytoma cells, four phosphorylation sites (Ser8, Ser19, Ser31, and Ser40) were inferred. Field stimulation of the splanchnic nerves at either 1 or 10 Hz (300 pulses) increased 32P incorporation into TH. At 10 Hz, the phosphorylation of Ser19 and Ser40 was increased, whereas at 1 Hz, Ser19, Ser31, and Ser40 phosphorylation was increased. Stimulation at either 1 or 10 Hz also increased the catalytic activity of TH, as measured in vitro (pH 7.2) at either 30 or 300 microM tetrahydrobiopterin. Nicotine (3 microM, 3 min) increased Ser19 phosphorylation, vasoactive intestinal polypeptide (10 microM, 3 min) increased Ser40 phosphorylation, and muscarine (100 microM, 3 min) increased TH phosphorylation primarily at Ser19 and Ser31. Vasoactive intestinal polypeptide, but not nicotine or muscarine, mimicked the effects of field stimulation on TH activity. Thus, the regulation of rat adrenal medullary TH phosphorylation by nerve impulses is mediated by multiple first and second messenger systems, as previously shown for catecholamine secretion. However, different sets of second messengers are involved in the two processes. The action of vasoactive intestinal polypeptide as a secretagogue involves the mobilization of intracellular calcium, whereas its effects on TH phosphorylation are mediated by cyclic AMP. This latter effect of vasoactive intestinal polypeptide and the consequent increase in Ser40 phosphorylation appear to be responsible for the rapid activation of TH by splanchnic nerve stimulation.

Phosphorylation and activation of human tyrosine hydroxylase in vitro by mitogen‐activated protein (MAP) kinase and MAP‐kinase‐activated kinases 1 and 2

“…However, stimulation of rat PC12 cells for only 10-15 min with NGF [2] or phorbol esters [55] caused a relatively selective increase in the 32P-labelling of Ser3 1 (about threefold), suggesting that these agonists may only increase Ser4O phosphorylation after more prolonged stimulation. Stimulation of perfused rat adrenal glands for 3 min with nicotine increased the "P-labelling of Serl9 (5.8-fold), Ser31 (50%) and Ser4O (60%), while stimulation for 3 min with muscarine increased the 32P-labelling of these three sites by 3.7-fold, 4.3-fold and 40%, respectively [53]. Electrical stimulation of the medial forebrain bundle (containing the afferent dopaminergic fibres) for 20 niin increased the phosphorylation of Ser19 (2.4-fold), Ser31 (1 .%fold) and Ser40 (1.9-fold) [9], while stimulation of the splanchnic nerves (1 Hz for 5 min) increased the "P-labeling of Serl9 (3.2-fold), Ser31 (3.7-fold) and Ser40 (4.3-fold) 191.…”

“…Stimulation of perfused rat adrenal glands for 3 min with nicotine increased the "P-labelling of Serl9 (5.8-fold), Ser31 (50%) and Ser4O (60%), while stimulation for 3 min with muscarine increased the 32P-labelling of these three sites by 3.7-fold, 4.3-fold and 40%, respectively [53]. Electrical stimulation of the medial forebrain bundle (containing the afferent dopaminergic fibres) for 20 niin increased the phosphorylation of Ser19 (2.4-fold), Ser31 (1 .%fold) and Ser40 (1.9-fold) [9], while stimulation of the splanchnic nerves (1 Hz for 5 min) increased the "P-labeling of Serl9 (3.2-fold), Ser31 (3.7-fold) and Ser40 (4.3-fold) 191. MAP kinase is presumably responsible for the phosphorylation of Ser3 1 in vivo, and MAPKAP kinases-1 andlor 2 are candidates for the enzymes that phosphorylate Serl9 and Ser40 under these conditions.…”

“…Furthermore, the inclusion of 14.3.3. protein in our assays caused no further increase in the activity of TH phosphorylated by CaM kinase-11. Another laboratory also failed to observe an effect of the 14.3.3. protein on TH activity in extracts prepared from adrenal chromaffin cells stimulated by agonists which increase Serl9 phosphorylation [53]. It is possible that 14.3.3 may not activate TH, but stabilise the Serl9-phosphorylated enzyme under some conditions.…”

“…For example, stimulation of rat PC12 cells for 30min with phorbol dibutyrate or nerve growth factor (NGF) increased the 32P-labelling of both Ser31 (3.4-6-fold) and Ser40 (30-35%) [3]. Stimulation of rat PC 12 cells for 1-2 h with NGF increased the 3ZP-labelling of two tryptic peptides T1 and T3 by 4.4-fold and I5-fold, respectively [53]. T1 and T3 presumably correspond to the peptides containing Ser40 and Ser31, respectively, since the "P-labelling of T1 was also increased (4.5-fold) by CAMP-elevating agents.…”

Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation

“…Extensive work by many groups has shown that TH is phosphorylated by several multifunctional protein kinases [Z - 4,14,29]. The phosphorylation of Ser40 by CAMP-dependent protein kinase is by far the best characterized, and it is well established that this phosphorylation causes an activation of the bovine and rat enzymes [4,14,29,301. Kecently, it has also been found that phosphorylation of this amino acid in hTHl and hTH2 results in an activation of both isozymes and causes a 3 -4-fold increase in Ki for catecholamine inhibitors [12].…”