Regulation of L-DOPA biosynthesis by site-specific phosphorylation of tyrosine hydroxylase in AtT-20 cells expressing wild-type and serine 40-substituted enzyme.
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.
Harada
Neurochemistry Research Laboratories, New York University Medical Center, New York 10016, USA.
Wu
J
J
Haycock
J W
JW
Goldstein
M
M
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*
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