Cholinergic receptor-mediated phosphorylation and activation of tyrosine hydroxylase in cultured bovine adrenal chromaffin cells.
We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phosphorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one- and two-dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Two-dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+-dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP-dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+-dependent protein kinase. After 2 min phosphorylation by a cyclic AMP-dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.
Pocotte
S L
SL
Holz
R W
RW
Ueda
T
T
D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D002837:Chromaffin Granules / Q000187:drug effects / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D000242:Cyclic AMP / Q000494:pharmacology; D004295:Dihydroxyphenylalanine / Q000096:biosynthesis; D004246:Dimethylphenylpiperazinium Iodide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008970:Molecular Weight; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011950:Receptors, Cholinergic / Q000378:metabolism*; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D010750:Phosphoproteins; D011950:Receptors, Cholinergic; D004246:Dimethylphenylpiperazinium Iodide; D004295:Dihydroxyphenylalanine; D003994:Bucladesine; D002217:Carbachol; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D002118:Calcium