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 [Leu(40)]-recombinant tyrosine hydroxylase (rTH). Basal rates of DOPA accumulation were much higher by cells expressing rTH in which Leu was substituted for Ser(40) (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-Ser(40) content, whereas DOPA production by cells expressing S40L-rTH was entirely unaffected by forskolin. After forskolin treatment of P-32(i)-prelabeled cells, WT-rTH was phosphorylated at Sers, Ser(19), Ser(31), and Ser(40), whereas P-32 incorporation into S40L-rTH was restricted to Ser(8), Ser(19), and Ser(31), 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 Ser(40); but, unlike forskolin, these treatments also increased DOPA production by cells expressing S40L-rTH, Thus, the present studies demonstrate that Ser(40) phosphorylation mediates forskolin-induced increases in DOPA biosynthesis directly but that mechanisms other than Ser(40) phosphorylation can mediate the increases in DOPA biosynthesis produced either by depolarization or by protein phosphatase inhibition.