Protein kinase C, purified to homogeneity, was found to phosphorylate and activate tyrosine hydroxylase that had been partially purified from pheochromocytoma PC 12 cells. These actions of protein kinase C required the presence of calcium and phospholipid. This phosphorylation of tyrosine hydroxylase reduced the K. for the cofactor 6-methyltetrahydropterine from 0.45 mM to 0.11 mM, increased the K; for dopamine from 4.2 jIM to 47.5 pjM, and produced no change in the Km for tyrosine. Little or no change in apparent V. was observed. These kinetic changes are similar to those seen upon activation of tyrosine hydroxylase by cAMP-dependent protein kinase. Two-dimensional phosphopeptide maps of tyrosine hydroxylase were identical whether the phosphorylation was catalyzed by protein kinase C or by the catalytic subunit of cAMP-dependent protein kinase. Both protein kinases phosphorylated serine residues. The results suggest that protein kinase C and cAMP-dependent protein kinase phosphorylate the same site(s) on tyrosine hydroxylase and activate tyrosine hydroxylase by the same mechanism.Nerve stimulation and neurotransmitters have been demonstrated to accelerate catecholamine biosynthesis in central and peripheral nervous tissue, adrenal medulla, and pheochromocytoma cells (1-4). This acceleration is thought to result from an increase in the catalytic activity of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) (cf. ref. 5), the rate-limiting enzyme in the biosynthesis of catecholamines (6). Several lines of evidence have suggested that cAMP-dependent phosphorylation may be one means of activation of tyrosine hydroxylase in situ (cf. ref. 7 and references therein). Analogues of cAMP have been reported to accelerate catecholamine biosynthesis in intact tissue preparations (8-11). In broken cell preparations, tyrosine hydroxylase has been shown to be activated, in the presence of ATP and Mg2', by cAMP or cAMP-dependent protein kinase (e.g., see refs. 8 and 12). Finally, purified tyrosine hydroxylase has been shown to be phosphorylated and activated by purified cAMP-dependent protein kinase (13)(14)(15).Recent evidence has suggested a role for calcium as well as cAMP in the activation of tyrosine hydroxylase in situ (16,17 Other reagents and enzymes were obtained from the following commercial sources: H1 histone (III-S), catalase, bovine serum albumin, bovine brain L-a-phosphatidyl-L-serine, diolein, dithioerythritol, EGTA, Tris, and ATP (Sigma); leupeptin (Chemicon, El Segundo, CA); 6,7, Purification of Tyrosine Hydroxylase. Tyrosine hydroxylase was partially purified as described (13) Abbreviation: 6-MePH4, 6-methyltetrahydropterine. 7713The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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The activities of the catecholamine synthetic enzymes tyrosine hydroxylase and phenylethanolamine N-methyltransferase, and the concentrations of the catecholamines and their respective metabolites, have been measured in the dorsal and ventral halves of the brainstem at various ages in the embryonic and adult rat. The activity of phenylethanolamine N-methyltransferase in both parts of the brainstem at day 14 of gestation is at or greater than adult levels and thereafter displays relatively small variations during ontogeny. Tyrosine hydroxylase activity, in contrast, is undetectable at day 14 and increases slowly, achieving only 20-25% of adult values by day 18 of gestation. Adrenaline concentrations correlate well with the activity of phenylethanolamine N-methyltransferase, showing a precocious development, whereas noradrenaline and 3,4-dihydroxyphenylethylamine (dopamine) concentrations are more closely related to the enhancement of tyrosine hydroxylase activity; at day 18 of gestation, for example, they are only 5 and 10%, respectively, of the adult values. The concentrations of the metabolites of noradrenaline and dopamine are suggestive of a high rate of turnover. These results confirm previous immunocytochemical evidence of a tardy appearance of tyrosine hydroxylase-like immunoreactivity in the phenylethanolamine N-methyltransferase-positive perikarya of the embryonic medulla oblongata. In addition, the abundance of adrenaline in this area at early gestational stages strongly suggests that, despite the paucity of tyrosine hydroxylase, phenylethanolamine N-methyltransferase is active in vivo and is utilizing a substrate other than noradrenaline. It is likely, however, that at later stages of gestation, when tyrosine hydroxylase is present at sufficient activity to supply noradrenaline, the conventional synthetic pathway for adrenaline formation comes into being.

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