Proc. Nati. Acad. Sci. USA Vol. 80, pp. 2097-2101, April 1983 Biochemistry Evidence for the involvement of a cyclic AMP-independent protein kinase in the activation of soluble tyrosine hydroxylase from rat striatum* (tyrosine monooxygenase/enzyme phosphorylation/kinetics) DAVID W. ANDREWS, THOMAS A. LANGAN, AND NORMAN WEINER Department of Pharmacology, University of Colorado School of Medicine, 4200 East 9th Avenue, Denver, Colorado 80262 Communicated by Edwin G. Krebs, November 29, 1982 ABSTRACT Activation of rat striatal tyrosine hydroxylase been assumed that this activation was attributable to the pres- [TyrOHase; tyrosine monooxygenase; L-tyrosine, tetrahydropter- ence of free catalytic subunit of cAMP-dependent protein ki- idine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] by nase, thus accounting for the lack of an absolute requirement ATP/Mg2+ and endogenous protein kinase can be produced with- for cAMP to produce this effect. out the addition ofcAMP. This activation is not due to endogenous In the present study, using purified heat-stable protein ki- free catalytic subunit derived from cAMP-dependent protein ki- nase. In the presence of amounts of protein kinase inhibitor suf- nase inhibitor, we have attempted to demonstrate TyrOHase ficient for complete inhibition of striatal cAMP-dependent protein activation in crude striatal extracts when the cAMP-dependent kinase and the cAMP-mediated activation of TyrOHase, addition protein kinase is completely inhibited. Under these conditions, of ATP/Mg2+ results in an enhancement of TyrOHase activity. TyrOHase is activated significantly and this activation is de- Enzyme activation does not occur when the nonhydrolyzable form pendent on the presence of ATP/Mg2 . Furthermore, the ki- of ATP, adenylyl imidodiphosphate, is substituted for ATP. When netics of this activation differ from those seen with cAMP-me- TyrOHase is assayed in the presence of ATP/Mg2+ and different diated activation of the enzyme. These results support the concentrations of either tyrosine or 6-methyltetrahydropterin co- existence of a cAMP-independent protein kinase in striatal tis- factor, a 2-fold increase in enzyme Vma, is demonstrable, with no sue which is capable of activating TyrOHase. change in the Km for either substrate or cofactor. In contrast, in the presence of cAMP and ATP/Mg2+, both an increase in Vma. MATERIALS and an enhanced affinity for pterin cofactor are demonstrable. In the latter circumstance, the 2-fold increase in Vmax can be attrib- Materials were obtained as follows: L-tyrosine, 2-(N-morpho- uted entirely to the action of cAMP-independent protein kinase. lino)ethanesulfonic acid (Mes), N-[tris (hydroxymethvl)methyl]- The addition of either EGTA or CaCl2 does not modify the effect aminoethanesulfonic acid (Tes), ATP, cAMP, NADPH, histone seen in the presence of ATP, suggesting that the effect of ATP/ 2-a, dithiothreitol, and adenylyl imidodiphosphate (p[NH]ppA) Mg2+ is not mediated by a Ca2+-dependent protein kinase. These from Sigma; 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahvdro- data support the existence of a cAMP-independent striatal protein pteridine (6-MeH4Pter) from Calbiochem; [ y-32P]ATP tetra- kinase that can catalyze the activation of TyrOHase. (triethylammonium) salt (10-40 Ci/mmol; 1 Ci = 3.7 x 1010 Bq), and L-[3,5-3H]tyrosine (40-60 Ci/mmol) from New En- In striatum, as in peripheral adrenergic tissues, the enzyme ty- gland Nuclear. cAMP-dependent protein kinase (HK1), and the rosine hydroxylase [TyrOHase; tyrosine monooxygenase; L-ty- two cAMP-independent kinases, growth-associated histone ki- rosine, tetrahydropteridine:oxygen oxidoreductase (3-hydrox- nase and histone kinase 2 (HK2), and calf thymus histone H1 ylating), EC 1. 14.16.2] catalyzes the first and rate-limiting step were prepared according to Langan (19). in the biosynthesis of the neurotransmitter dopamine (1-4). cAMP and analogs of this cyclic nucleotide are able to increase METHODS the activity of the enzyme in crude tissue preparations in the presence of added ATP and Mg2+ (ATP/Mg2+) (5-11), and this Tissue Preparation. Male Sprague-Dawley rats (Charles River activation is associated with either an increased affinitv of en- Laboratories, Wilmington, MA) weighing 150-250 g were zyme for cofactor (5, 7, 11, 12) or a reduction of feedback in- stunned by a blow to the head and decapitated, and the brains hibition by dopamine or norepinephrine (7, 13). Presumably, were removed. Striata were dissected according to the pro- cAMP activates cAMP-dependent protein kinase, releasing free cedure of Glowinski and Iversen (20) and frozen at -70'C until catalytic subunit which in turn activates TyrOHase. In recent assayed. Tissue was homogenized in 5 vol of 0.32 M sucrose/ studies (14-18) it has been shown that the free catalytic subunit 20 mM Tris/0.2% Triton X-100, pH 6.2. The homogenate was of cAMP-dependent protein kinase activates TyrOHase by di- centrifuged at 20,000 X g for 20 min at 40C, and the super- rectly phosphorylating the enzyme. natant served as the enzvme source. In many of the earlier studies on the activation of TyrOHase Purification of Protein Kinase Inhibitor. Protein kinase in- by protein kinase, crude tissue extracts were used, and the con- hibitor was prepared from whole bovine brain through the stituents of the system responsible for the activation of the en- zyme in the presence of cAMP and ATP/Mg2+ were not rig- Abbreviations: TyrOHase, tyrosine hydroxylase; 6-MeH4Pter, 2-amino- defined. In a number of studies activation of 4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine; p[NH]ppA, 5'-ade- orously (7-10), nylyl imidodiphosphate. TyrOHase by ATP/Mg2+ alone has been reported, and it had * A preliminary report of a portion of these findings was presented at meetings of the Western Pharmacology Society, Colorado Springs, CO, The publication costs of this article were defrayed in part by page charge Jan. 21-26, 1979, and the Western Clinical Research Forum, Carmel, payment. This article must therefore be hereby marked "advertisement` CA, Feb. 15-19, 1982, and at the National Student Research Forum, in accordance with 18 U. S. C. §1734 solely to indicate this fact. Galveston, TX, Apr. 26-30, 1982. 2097 Downloaded by guest on September 24, 2021 2098 Biochemistry: Andrews et al. Proc. Natl. Acad. Sci. USA 80 (1983) phosphocellulose step according to the procedure of Demaille toluene scintillation cocktail. Counts per minute were cor- et al. (21). The peak fractions were pooled, dialyzed against dis- rected for column recovery (consistently 60%) and counting ef- tilled water overnight, Iyophilized, and stored at -20TC. ficiency (20%). Protein Kinase Assay. Supernatant enzyme (25 1.l) was added Assay II. During the course of these studies, it was found to the incubation system which contained, in a final volume of that 1 mM ascorbate served as a more effective reducing agent 100 pl (pH 6.2): 75 mM Mes, 12 mM potassium phosphate, 154 than the NADPH/pteridine reductase reducing system, allow- ,ug of dithiothreitol, 5 mM NaF, 60 AM EGTA, 800 AM the- ing for a more sensitive assay and obviating the need for a rel- ophylline, 5 mM magnesium acetate, 100 Ag of histone, and atively crude preparation of pteridine reductase, in agreement 800 ,M [y-32P]ATP (0.25 ACi per tube). Test agents included with the earlier results of Lerner et al. (24). With the use of 1 125 ,uM cAMP and various concentrations of protein kinase in- mM ascorbate, the incubation time was decreased to 15 min. hibitor. Values for protein kinase blanks were obtained by using Where designated, 25 mM NaF was used instead of 5 mM as tubes lacking histone substrate and containing amounts of pro- in assay I. All other details of the assay were carried out as in tein kinase inhibitor sufficient for full inhibition of cAMP-de- assay I. pendent histone kinase. The reaction was initiated by the ad- Protein Assay. Protein was assayed by the method of Lowry dition of enzyme to the mixture and was allowed to proceed for et al. (25) with bovine serum albumin as standard. 5 min at 37TC, after which it was terminated by the addition of 2.25 ml 28% (wt/vol) trichloroacetic acid. Samples were al- lowed to stand for 15 min and then mixed on a Vortex shaker, RESULTS and the contents of each tube were filtered separately through Specificity of Protein Kinase Inhibitor. Assay of histone ki- type HAWP Millipore filters to trap phosphorylated histone. nases in the presence of different concentrations of protein ki- Filters were dried under a heat lamp, and assayed for radio- nase inhibitor revealed complete inhibition of histone phos- activity in 4 ml of an Omnifluor scintillation cocktail. Counting phorylation by cAMP-dependent protein kinase at 3 ug ofprotein efficiency was 85-90%. kinase inhibitor, whereas 100 ug of the inhibitor had no effect Protein Phosphorylation Reaction. The activation of Tyr- on histone phosphorylation catalyzed by either of two cAMP- OHase and the subsequent assay of the enzyme was carried out independent histone kinases. by a two-step incubation procedure. A 5-min incubation was Titration of Protein Kinase Inhibitor Against Protein Kinase carried out under essentially the same conditions as the protein and TyrOHase. Additions of up to 100 4g of protein kinase kinase assay; histone was omitted because it interfered with the inhibitor failed to modify either basal protein kinase or basal activation of TyrOHase, presumably by competition of sub- TyrOHase activity (Fig. 1). These results indicate that there is strates for the kinase. This phase of the assay was terminated no free endogenous catalytic subunit of cAMP-dependent pro- by adding 10 ,ul of 1 M potassium phosphate, pH 7.2/25 mM tein kinase in our preparations ofcrude rat striatal supernatant.