title: Regulation of L-DOPA biosynthesis by site-specific phosphorylation of tyrosine hydroxylase in AtT-20 cells expressing wild-type and serine 40-substituted enzyme.
abstract: De novo L-DOPA biosynthesis was studied in stably transfected AtT-20 cells expressing wild-type- or [Leu40]-recombinant tyrosine hydroxylase (rTH). Basal rates of DOPA accumulation were much higher by cells expressing rTH in which Leu was substituted for Ser4O (S40L-rTH) than by those expressing wild-type rTH (WT-rTH). Treatment of WT-rTH cells with forskolin produced an increase in DOPA accumulation and a concomitant increase in WT-rTH phospho-Ser40 content, whereas DOPA production by cells expressing S40L-rTH was entirely unaffected by forskolin. After forskolin treatment of 32Pi-prelabeled cells, WT-rTH was phosphorylated at Ser8, Ser19, Ser31, and Ser40, whereas 32P incorporation into S40L-rTH was restricted to Ser8, Ser19, and Ser31. Relatively prolonged treatment of AtT-20 cells expressing WT-rTH with either a depolarizing agent (elevated potassium) or a phosphatase inhibitor (okadaic acid) increased DOPA production and increased the phosphorylation state of Ser40; but, unlike forskolin, these treatments also increased DOPA production by cells expressing S40L-rTH. Thus, the present studies demonstrate that Ser40 phosphorylation mediates forskolin-induced increases in DOPA biosynthesis directly but that mechanisms other than Ser40 phosphorylation can mediate the increases in DOPA biosynthesis produced either by depolarization or by protein phosphatase inhibition.
authors.affiliation: Neurochemistry Research Laboratories, New York University Medical Center, New York 10016, USA.
authors.lastname: Harada
authors.forename: J
authors.initials: J
authors.lastname: Wu
authors.forename: J W
authors.initials: JW
authors.lastname: Haycock
authors.forename: M
authors.initials: M
authors.lastname: Goldstein
mesh_terms: D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D007980:Levodopa / Q000096:biosynthesis*; D016297:Mutagenesis, Site-Directed; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D011994:Recombinant Proteins; D010768:Phosphoserine; D005576:Colforsin; D019319:Okadaic Acid; D007980:Levodopa; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D010749:Phosphoprotein Phosphatases; D011188:Potassium