Tyrosine Hydroxylase Activation and Inactivation by Protein Phosphorylation Conditions

Abstract

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, catalyzes the conversion of tyrosine to DOPA, Cyclic AMP-dependent protein phosphorylation conditions alter tyrosine hydroxylase activity in rat striatal homogenates. In agreement with other laboratories, we find that short-term pre-incubation (3 min) of extracts under phosphorylating conditions (Mg . ATP, cAMP) increases enzyme activity two- to tenfold over control as measured during a subsequent 15-min assay. We now report that preincubation under phosphorylating conditions for longer periods (30 min) results in a loss of activity to levels equal to or below that of the control enzyme. Addition of purified bovine brain protein kinase catalytic subunit and Mg . ATP enhances activation and increases the rate of inactivation. To demonstrate that inactivation is not associated with proteolytic degradation or irreversible denaturation, the inactivated form of the enzyme can be reactivated. The protein kinase inhibitor protein decreases the activation process and prevents inactivation of the enzyme to below control values. The sedimentation coefficient is not changed by phosphorylation conditions (S = 8.8 +/- 0.1). Although the apparent Km of the enzyme for the 6-methyltetrahydropterine (6-MPH4) cofactor is reduced (0.86 mM, control; 0.32 mM, activated), it is also reduced in the inactivated form (0.38 mM). The Ki for dopamine is increased from 4.5 microM for the control to 28 microM for the activated enzyme, whereas the inactivated form of the enzyme exhibits a Ki of 10 microM. Removal of catecholamines by gel filtration fails to alter activity and the apparent cofactor Km. Moreover, both the activated and the inactivated states persist following gel filtration. It therefore appears that the activation-inactivation process is not mediated solely by the modulation of enzyme feedback inhibition or changes in the Km for 6-MPH4. We also describe a coupled decarboxylase assay in which labeled dopamine is resolved from the precursors tyrosine and DOPA by low-voltage paper electrophoresis.

Phosphorylation and Activation of Brain Aromatic l‐Amino Acid Decarboxylase by Cyclic AMP‐Dependent Protein Kinase

“…Tyrosine hydroxylase (TH), the rate‐limiting enzyme for the synthesis of catecholamines, undergoes PKA‐dependent phosphorylation, resulting in enzyme activation (Kumer and Vrana, 1996). Multiple‐site phosphorylation and activation of striatal TH occur in vivo and in vitro (Joh et al, 1978 ; Vrana et al, 1981 ; Haycock, 1987 ; Salah et al, 1989 ; Haycock and Haycock, 1991). We have previously shown that the activities of AAAD and TH of striatum are modulated in parallel by cyclic AMP (presumably PKA)‐dependent pathways in vivo (Young et al, 1998).…”

Calcium/phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase.

“…A decrease in activity with increasing time of phosphorylation has also been reported in studies with cAMP-dependent phosphorylation of tyrosine hydroxylase (e.g., see ref. 31) and has been attributed to an increased susceptibility to denaturation of the phospho form of the enzyme relative to the dephospho form (32). There was little or no change in tyrosine hydroxylase activity as a function of time of preincubation in the absence of protein kinase C (Fig.…”

Alteration of tyrosine hydroxylase activity in PC12 cells infected with herpes simplex virus type 1

“…An altered synthetic rate equips the cell to deal with longterm neural demands. More rapid alteration in enzyme activity can be effected by activation or inactivation of existing TH molecules (38). Because of the rapidity of the fall and rise in TH activity noted within the first 9 hours after HSV-1 infection, we anticipated that, inactivation and activation of TH likely accounted for our experimental findings.…”

“…either from activation of existing enzyme molecules or synthesis of new enzyme (25,38,40,41). In order to distinguish which of these processes predominated in determining the alterations in TIt activity accompanying HSV-1 infection, we carried out an immunotitration assay using antibody against TH (3, i4, 4t).…”