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Tyrosine Hydroxylase Is Activated and Phosphorylated on Different Sites in Rat Pheochromocytoma PC12 Cells Treated with Phorbol Ester and Forskolin
Author(s) -
Tachikawa Eiichi,
Tank A. William,
Weiner David H.,
Mosimann Werner F.,
Yanagihara Nobuyuki,
Weiner Norman
Publication year - 1987
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1987.tb05673.x
Subject(s) - forskolin , phosphorylation , tyrosine hydroxylase , adenylate kinase , tyrosine phosphorylation , activator (genetics) , protein kinase c , ionomycin , phorbol , protein kinase a , chemistry , cyclase , medicine , protein phosphorylation , endocrinology , biochemistry , biology , enzyme , microbiology and biotechnology , receptor , intracellular
Incubation of rat pheochromocytoma PC12 cells with 4β‐phorbol‐12β‐myristate‐13α‐acetate (PMA), an activator of Ca 2+ /phospholipid‐dependent protein kinase (protein kinase C), or forskolin, an activator of adenylate cyclase, is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase. Neither the activation nor increased phosphorylation of tyrosine hydroxylase produced by PMA is dependent on extracellular Ca 2+ . Both activation and phosphorylation of the enzyme by PMA are inhibited by pretreatment of the cells with trifluo‐perazine (TFP). Treatment of PC 12 cells with l‐oleoyl‐2‐acetylglycerol also leads to increases in the phosphorylation and enzymatic activity of tyrosine hydroxylase; 1, 2‐diolein and 1, 3‐diolein are ineffective. The effects of forskolin on the activation and phosphorylation of the enzyme are independent of Ca 2+ and are not inhibited by TIT 5 . Forskolin elicits an increase in cyclic AMP levels in PC 12 cells. The increases in both cyclic AMP content and the enzymatic activity and phosphorylation of tyrosine hydroxylase following exposure of PC 12 cells to different concentrations of forskolin are closely correlated. In contrast, cyclic AMP levels do not increase in cells treated with PMA. Tryptic digestion of the phosphorylated enzyme isolated from untreated cells yields four phosphopeptides separable by HPLC. Incubation of the cells in the presence of the Ca 2+ ionophore ionomycin increases the phosphorylation of three of these tryptic peptides. However, in cells treated with either PMA or forskolin, there is an increase in the phosphorylation of only one of these peptides derived from tyrosine hydroxylase. The peptide phosphorylated in PMA‐treated cells is different from that phosphorylated in forskolin‐treated cells. The latter peptide is identical to the peptide phosphorylated in dibutyryl cyclic AMP‐treated cells. These results indicate that tyrosine hydroxylase is activated and phosphorylated on different sites in PC 12 cells exposed to PMA and forskolin and that phosphorylation of either of these sites is associated with activation of tyrosine hydroxylase. The results further suggest that cyclic AMP‐dependent and Ca 2+ / phospholipid‐dependent protein kinases may play a role in the regulation of tyrosine hydroxylase in PC 12 cells.