Calcium/phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase.

Abstract

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.

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

“…The phosphorylation of Ser40 by PKA increases TH activity in vitro, the extent of activation observed depending on the pH of the assay, the ptel-in cofactor concentration, whether catecholamines are present [4,7,40,49,501 and perhaps on the source and method of isolation of the enzyme. The use of recombinant TH in the present study could also affect the extent of activation resulting from phosphorylation.…”

Differences in tyrosine hydroxylase-like immunoreactivity characterize the mesostriatal innervation of striosomes and extrastriosomal matrix at maturity.

“…TyrOHase can exist in catalytically active and inactive forms (29,30). Differences in the rates of dopamine turnover of striosomal and matrix nigrostriatal innervations could reflect corresponding differences in the activity states of TyrOHase in the two systems.…”

Protein kinase C phosphorylates the inhibitory guanine‐nucleotide‐binding regulatory component and apparently suppresses its function in hormonal inhibition of adenylate cyclase

“…Only a limited number of proteins have been identified or visualized: a 40000-MI (47000-MI) protein in platelets that is phosphorylated in response to thrombin or plateletactivating factor [15,24,251, unidentified proteins in cardiac tissue (M, = 38000, 42000, 49000) [26-281, myelin basic protein [27], vinculin [29] and tyrosine hydroxylase [30]. It is very unlikely that the 40000-Mr substrate from platelets is the same as Cia, since the former is soluble and migrates differently during sodium dodecylsulfate/polyacrylamide gel electrophoresis (data not shown).…”