| Tyrosine hydroxylase (TH) is an enzyme and control point in the biosynthesis of catecholamine neurotransmitters. In addition to gene expression controls which regulate the level of TH holoenzyme, the activity of the enzyme is controlled through its phosphorylation. Four phosphorylation sites have been identified in the rat TH subunit, the most studied of all forms of TH. At least two of these sites, serine 19 and 40 of the rat TH subunit, are the target of protein kinases which themselves are the focal point of well-known signaling pathways. The cyclic AMP-dependent protein kinase (PK-A) phosphorylates serine 40, and calcium- and calmodulin-dependent protein kinase (CaMK) found in abundance in neuronal tissues targets serine 19. Serine 8 is reported to be phosphorylated by CDK2 (p34cdc2/p58cyclinA ), but the physiological significance of this modification is unknown. The identification of a (or the) protein kinase that phosphorylates the serine at position 31 was reported while this study was in progress. The microtubule-associated protein-2 kinase (MAP2K) is a serine/threonine protein kinase activated by tyrosine phosphorylation and threonine phosphorylation, first identified in insulin-stimulated cultured adipocytes, and found to be identical with an isoforms of the independently studied extracellularly regulated protein kinases (ERK). In these experiments, TH was purified as a substrate from bovine adrenal medulla and treated with a protein kinase obtained from bovine adrenal chromaffin cells which had been depolarized for an optimal period with potassium chloride, and which had been purified for enzymatic activity able to phosphorylate a synthetic peptide substrate forming the consensus sequence to rat TH serine-31. TH is activated when treated with this protein kinase preparation. Analysis of tryptic phosphopeptides obtained from the serine 31 kinase-treated TH substrate shows that serine 31 of TH was phosphorylated. Immunochemical testing of the purified enzyme indicates the presence of the MAP2K/ERK. These data indicate that MAP2K/ERK is the protein kinase activated in potassium-depolarized chromaffin cells and phosphorylates serine 31 of TH, with concomitant activation. |
