Activation of rat striatal tyrosine hydroxylase [TyrOHase; tyrosine monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] by ATP/Mg2+ and endogenous protein kinase can be produced without the addition of cAMP. This activation is not due to endogenous free catalytic subunit derived from cAMP-dependent protein kinase. In the presence of amounts of protein kinase inhibitor sufficient for complete inhibition of striatal cAMP-dependent protein kinase and the cAMP-mediated activation of TyrOHase, addition of ATP/Mg2+ results in an enhancement of TyrOHase activity. Enzyme activation does not occur when the nonhydrolyzable form of ATP, adenylyl imidodiphosphate, is substituted for ATP. When TyrOHase is assayed in the presence of ATP/Mg2+ and different concentrations of either tyrosine or 6-methyltetrahydropterin cofactor, a 2-fold increase in enzyme Vma, is demonstrable, with no change in the Km for either substrate or cofactor. In contrast, in the presence of cAMP and ATP/Mg2+, both an increase in Vma.and an enhanced affinity for pterin cofactor are demonstrable. In the latter circumstance, the 2-fold increase in Vmax can be attributed entirely to the action of cAMP-independent protein kinase.The addition of either EGTA or CaCl2 does not modify the effect seen in the presence of ATP, suggesting that the effect of ATP/ Mg2+ is not mediated by a Ca2+-dependent protein kinase. These data support the existence of a cAMP-independent striatal protein kinase that can catalyze the activation of TyrOHase.In striatum, as in peripheral adrenergic tissues, the enzyme tyrosine hydroxylase [TyrOHase; tyrosine monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1. 14.16.2] catalyzes the first and rate-limiting step in the biosynthesis of the neurotransmitter dopamine (1-4). cAMP and analogs of this cyclic nucleotide are able to increase the activity of the enzyme in crude tissue preparations in the presence of added ATP and Mg2+ (ATP/Mg2+) (5)(6)(7)(8)(9)(10)(11), and this activation is associated with either an increased affinitv of enzyme for cofactor (5,7,11,12) or a reduction of feedback inhibition by dopamine or norepinephrine (7, 13). Presumably, cAMP activates cAMP-dependent protein kinase, releasing free catalytic subunit which in turn activates TyrOHase. In recent studies (14-18) it has been shown that the free catalytic subunit of cAMP-dependent protein kinase activates TyrOHase by directly phosphorylating the enzyme.In many of the earlier studies on the activation of TyrOHase by protein kinase, crude tissue extracts were used, and the constituents of the system responsible for the activation of the enzyme in the presence of cAMP and ATP/Mg2+ were not rigorously defined. In a number of studies (7-10), activation of TyrOHase by ATP/Mg2+ alone has been reported, and it had been assumed that this activation was attributable to the presence of free catalytic subunit of cAMP-dependent protein kinase, thus accounting for the lack of an...