Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline, and adrenaline. In response to short term stimuli TH activity is primarily controlled by phosphorylation of serine 40. We have previously shown that phosphorylation of serine 19 in TH can indirectly activate TH via a hierarchical mechanism by increasing the rate of phosphorylation of serine 40. Here we show that phosphorylation of serine 31 in rat TH increases the rate of serine 40 phosphorylation 9-fold in vitro. Phosphorylation of serine 31 in intact bovine chromaffin cells potentiated the forskolin-induced increase in serine 40 phosphorylation and TH activity more than 2-fold. Humans are unique in that they contain four TH isoforms but to date no significant differences have been shown in the regulation of these isoforms. Phosphorylation of the human TH isoform 1 at serine 31 by extracellular signal-regulated protein kinase (ERK) also produced a 9-fold increase in the rate of phosphorylation of serine 40, whereas little effect was seen in the TH isoforms 3 and 4. ERK did not phosphorylate human TH isoform 2. The effect of serine 19 phosphorylation on serine 40 (44 in TH2) phosphorylation is stronger in TH2 than in TH1. Thus hierarchical phosphorylation provides a mechanism whereby the two major human TH isoforms (1 and 2) can be differentially regulated with only isoform 1 responding to the ERK pathway, whereas isoform 2 is more sensitive to calcium-mediated events.Tyrosine hydroxylase (TH) 2 [EC1.14.16.2] is the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline, and adrenaline (1). Short term regulation of TH is accomplished by dynamic changes in the phosphorylation state of the enzyme (2, 3). Although four serine residues have been shown to be phosphorylated in TH, only three of these serine residues (Ser 19 , Ser 31 , and Ser 40 ) are regulated in vivo (4). The most important mechanism of TH activation is phosphorylation of Ser 40 , which decreases the feedback inhibition by the catecholamines (5-7). Phosphorylation of dopamine bound TH at Ser 40 by protein kinase A (PKA) can activate TH by up to 20-fold (5). The direct effect of Ser 19 and Ser 31 phosphorylation on TH activation is much more modest. Phosphorylation of Ser 19 by calcium calmodulindependent protein kinase (CaMKII) will only increase TH activity in the presence of the 14-3-3 protein (8 -10), and this results only in a 2-fold increase in the activity. The phosphorylation of Ser 31 by extracellular signal-regulated protein kinase (ERK) produces less than a 2-fold increase in TH activity, primarily by decreasing the affinity of the cofactor tetrahydrobiopterin (BH 4 ) (11-13). Phosphorylation of Ser 31 in TH has also been shown to increase the stability of TH (14).Humans have four TH protein isoforms, whereas anthropoids have two, and other mammalian species only have one (15). TH is encoded by a single gene, and the multiple isoforms are because of multiple mRNAs generated by alternativ...

Autophosphorylation of Ca 2+-calmodulin stimulated protein kinase II (CaMKII) at two sites (Thr286 and Thr305/306) is known to regulate the subcellular location and activity of this enzyme in vivo. CaMKII is also known to be autophosphorylated at Thr253 in vitro but the functional effect of phosphorylation at this site and whether it occurs in vivo, is not known. Using antibodies that specifically recognize CaMKII phosphorylated at Thr253 together with FLAG-tagged wild type and phospho-and dephospho-mimic mutants of a-CaMKII, we have shown that Thr253 phosphorylation has no effect on either the Ca 2+ -calmodulin dependent or autonomous kinase activity of recombinant a-CaMKII in vitro. However, the Thr253Asp phosphomimic mutation increased a-CaMKII binding to subcellular fractions enriched in postsynaptic densities (PSDs). The increase in binding was similar in extent, and additive, to that produced by phosphorylation of Thr286. Thr253 phosphorylation was dynamically regulated in intact hippocampal slices. KCl induced depolarisation increased Thr253 phosphorylation and the phospho-Thr253-CaMKII was specifically recovered in the subcellular fraction enriched in PSDs. These results identify Thr253 as an additional site at which CaMKII is phosphorylated in vivo and suggest that this dynamic phosphorylation may regulate CaMKII function by altering its distribution within the cell.

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