Abstract
The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused phosphorylation of phosphoproteins of 56-kDa which co-migrated with and had identical pI values to subunits of tyrosine hydroxylase. The phosphorylation was closely correlated with an increase of [3H]3,4-dihydroxyphenylalanine (DOPA) production which is a reflection of increased tyrosine hydroxylase activity. Only those phorbol esters which activate protein kinase C induced phosphorylation of the 56-kDa proteins and increased [3H]DOPA production. Neither TPA-induced phosphorylation of the 56-kDa proteins nor TPA-induced enhancement of [3H] DOPA production required extracellular Ca2+. TPA caused increases in phosphorylation of the 56-kDa proteins and increases in [3H]DOPA production over similar concentration ranges (10-1000 nM). TPA did not increase cellular cAMP. The data suggest that phorbol ester-induced phosphorylation of intracellular tyrosine hydroxylase, possibly by protein kinase C, results in increased tyrosine hydroxylase activity.
Highlights
Tyrosine hydroxylase is the rate-limiting enzyme in catecholaminebiosynthesis [1]
Cells which had been preincubated 30 min in [32P]phosphate con- and consistent the Ca2+/phospholipidacetate (TPA)-induced phosphorylatiwonas of proteins taining PSS were incubated in PSS solution with or withou3t 00 nM of 56 kDa which co-migrated with and had identiPcIalvalues of various phorbol estersfor 30 min and analyzed for phosphoproteins. (6.37, 6.27, and 6.15) to the nicotinic agonist-stimulated 56
Cells were incubated in hrocresine-containing PSS solution with or without 300 nM of various phorbol esters for 30 min
Summary
Cell Preparation-Primary dissociated cells from bovine adrenal medulla were preparedandmaintainedas monolayer culturesin Eagle’s minimal essential medium (GIBCO, Grand Island, NY) containing 10% heat-inactivated fetal calf serum as previously described [24].The culture medium contained 100 units/ml of penicillin, 100 Fg/rnl of streptomycin, 50 pg/ml of gentamycin, and 1.3 pg/ml of Fungizone (Squibb, Princeton, NJ) to prevent bacterial and fungal contamination. Experiments were usually terminated by replacing the medium with 0.10-0.25 ml of stop solution (3%sodium dodecyl sulfate, 2% mercaptoethanol, 5% glycerol, 62 mM Tris-C1 (pH 6.7), and 0.5 mg/ml of bromphenol blue). For two-dimensional electrophoresis, incubations in 6.4-mm diameter wellswere terminated by replacement of the incubation medium with 0.03 mlof a solution containing 9.5 M urea, 2% Nonidet P-40, 5% P-mercaptoethanol, 2% Ampholines (pH 3.5-10). Cells cultured in 16-mm diameter wells were incubated with 0.4 ml of 150 p~ brocresine (an inhibitor of DOPA decarboxylase) in PSS for 30 min a t 25 "C and replaced with 0.2mlof PSS containing 150 p~ brocresine, 10 or 50 p~ [3H]tyrosine (7 pCi/ml, side chain, 2,3[3H]tyrosine),and various drugs for indicated times. In some experiments 35-mm diameter dishes were used with 0.8 ml of brocresinecontaining PSS, 0.6 ml of [3H]tyrosine containing PSS, and 0.2ml of perchloric acid.
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