Control of phenylalanine and tyrosine metabolism by phosphorylation mechanisms

CONTROL OF PHENYLALANINE AND TYROSINE METABOLISM BY PHOSPHORYLATION MECHANISMS CHRISTOPHER I. POGSON*, ALAN J. DICKSON, RICHARD G. KNOWLES*, MARK SALTER*, M. ANGELICA SANTANAt, JOHN C, STANLEY:~ and MICHAEL J. FISHER§ Department of Biochemistry, University of Manchester Medical School, Oxford Road, Manchester, MI3 9PL, U.K. INTRODUCTION Phenylalanine is an essential amino acid in mammals, and can serve as a source of tyrosine through the action of phenylalanine hydroxylase (EC. 1.14.16.1), so that the requirement for tyrosine can normally be met, at least in part, by a superfluity of phenylalanine in the diet. A distinctive feature is that both serve as precursors of neurotransmitters in the central nervous system. Because the concentrations of amino acid precursors may be factors in controlling the rate of neurotransmitter synthesis (I) and because both amino acids share, with tryptophan and possibly other amino acids, a common transport system into the brain (2), it is clear that the ratio, as well as the absolute value, of the concentrations of phenylalanine and tyrosine is important. The sequence of reactions involving the conversion of phenylalanine to tyrosine and its subsequent catabolism is shown in Figure 1. If the ratio of phenylalanine to tyrosine in the blood is to be maintained within certain limits, then the rates of the uptake of the amino acids into the liver, of phenylalanine hydroxylase and of tyrosine catabolism through tyrosine aminotransferase (EC, 2.6,1.5) must be coordinated. All these systems have been studied, and the interplay of the several regulatory factors is now clearer. Phenytalanine hydroxylase occurs almost exclusively in the liver, a very limited activity being found in kidney. It catalyzes the reaction L-phenylalanine + tetrahydrobiopterin + O2 L-tyrosine + dihydrobiopterin + H20. Present addresses: *Biochemistry Department, Wellcome Research Laboratories, Langley Court, Beckenham, Kent, BR3 3BS, U.K. tInstituto Nacional de la Nutricion, Salvador Zubiran, Calle Vasco de Quiroga 15, Delegacion Tlalpan, 14000 Mexico D.F., Mexico. ~Nestle Products Technical AssistanceCo. Ltd., Research Department, CH-1814 La Tour-DePeilz, Switzerland. §Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX, U.K. 309

310

CHRISTOPHER I. POGSON,

Phenylalanine

et aL

Tyrosine

OC

BLOOD CYTOPLASM

QLU

(5)//~ ) ZPHydroxyphenylpyrLivete

Phen, l a ~ o s l n e

0C6)~C02

(7)

(z)T " Phanylpyruvate Ph~ylllactate,Ph~y|~cetate

(z)

Homagmtisate

1 AcetoaceLate

and ( ~-I-lydroxybutyrate

t

Citric Acid

Cyclelnt~'modiat ~

FIG. 1. Pathways of metabolism of phenylalanine and tyrosine in mammalian liver. (1) Membrane transport: systems L and T (54); (2) Protein synthesis; (3) Proteolysis; (4) Phenylalanine hydroxylase; (5) Tyrosine aminotransferase; (6) 4-Hydroxyphenylpyruvate oxidase; (7) Phenylalanine transaminase.

The properties of the enzyme have been reviewed (3-6) and the mechanism of the reaction has been investigated in detail (7, 8). The full sequence of the human liver enzyme (9) and partial sequences of the rat liver enzyme (10) have been published. Earlier work indicated that two or more hydroxylase activities could be separated from extracts of rat liver. It is now generally accepted that the 'isoenzymes' reflect interconvertible phospho-forms of the enzyme, with a single amino acid sequence (11-13). In some strains of rats, however, two distinct hydroxylases have been shown to be the products of separate genes (14); Sprague-Dawley rat livers contain both hydroxylase activities. The rat liver enzyme is phosphorylated in vitro by the cyclic AMPdependent protein kinase (12, 15); this has also been found in vivo (16) and in liver cells exposed to glucagon (17). The human liver enzyme is also phosphorylated although without apparent change in kinetic properties (13). Tyrosine aminotransferase in rat liver is also a phosphoprotein (18-20), and it has been suggested (19) that phosphorylation may play a role in the control of the activity of this enzyme which has frequently been described as 'ratelimiting' for tyrosine catabolism. The activities of both the hydroxylase and the transaminase are sensitive to

PHENYLALANINE AND TYROSINE METABOLISM

311

changes in nutritional and hormonal conditions (see 21 for Refs), although not to the same extent or over the same time-scale. In this paper we present evidence to show the role ofphosphorylation in the control of phenylalanine and tyrosine metabolism, and discuss the importance of this phenomenon in relation to other factors of regulatory significance. MATERIALS

AND METHODS

Animals. Male Sprague-Dawley rats, 180-220 g, were used throughout and were fed ad libitum unless otherwise stated. Chemicals. The sources of chemicals were as given previously (22-25). Phenylalanine hydroxylase was purified by hydrophobic chromatography (26), and monospecific antibody was raised against this in sheep (24). Tyrosine aminotransferase was purified from rat liver, and monospecific antibody was raised in rabbits (27). Cell incubations. Liver cells were prepared by collagenase perfusion under standard conditions (25). On all occasions, cellular integrity was assessed by measurement of cellular ATP content (28). Cell suspensions (2 ml) were preincubated for 30 min with various substrates (lactate, pyruvate, glucose, glutamine) for flux measurements (3-5 mg dry wt per ml) and for 60 min with [3zP]Pi in a phosphate-depleted medium for immunoprecipitation experiments (10-15 mg dry wt per ml; 24). The intracellular specific activity of [32p]ATP reached a steady-state value by 60 min. Flux measurements. The principles and techniques of measurement of flux through phenylalanine hydroxylase have been detailed previously (24, 29, 30). Briefly, 3H from [4-3H]phenylalanine appears largely in the 3-position of tyrosine resulting from hydroxylase activity (only 8% being released directly as 3HzO). The ring-bound 3H is released either by further metabolism through homogentisate or by chemical reaction subsequently with N-iodosuccinimide. Thus noncharcoal-absorbable 3H gives a direct measurement of hydroxylase activity in cells. Flux through tyrosine aminotransferase is determined by release of 3H20 from L-[ring 2,3-3H]tyrosine (31). lmmunoprecipitation experiments. Phenylalanine hydroxylase antigenantibody precipitates were isolated as described in (24). Precipitates were washed by centrifugation through sucrose cushions, a step which decreased nonspecific association to minimal levels (19). Because the amount oftyrosine aminotransferase protein in cells is much lower than that of the hydroxylase, it was found necessary to include two extra procedures: (i) brief heating of cell

312

CHRISTOPHER I. POGSON, et al.

extracts at 60°C to remove a proportion of other proteins (32), and (ii) addition of 234 milliunits of semi-purified antigen to achieve pellets of appreciable size. The precipitate was allowed to form by incubation for 2 hr at 37°C and overnight at 4°C. Otherwise, procedures were as described for the hydroxylase (24). 32p-Containing immunoprecipitates were characterized by polyacrylamide gel electrophoresis under denaturing conditions. Contamination with 32p not associated with antigen was very low, so that immunoprecipitates were routinely counted directly. 3H-Containing immunoprecipitates derived from 3H-labelled phenylalanine hydroxylase contained somewhat greater levels of contamination with other 3H-labelled material, but this did not change during the course of the experiment (33).

RESULTS

AND DISCUSSION

We have been concerned in studying the relationship between posttranslational modifications in vitro and their impact in vivo where other regulatory factors may be present. Our investigations with phenylalanine hydroxylase derive from a longer-term concern with aromatic amino acid metabolism and its control and from the strictly practical point that the hydroxylase is present in relatively large quantities in liver. Tyrosine aminotransferase is of course also involved in aromatic amino acid metabolism. Despite many publications devoted to the isolated enzyme or crude extracts, it has, however, been little studied under physiologicallyrelevant conditions. Measurements of metabolic flux through specific steps in vivo are difficult and time-consuming. We have therefore used the isolated liver cell as our model of the animal in vivo, and have derived simple procedures which enable us to make large numbers of measurements under various conditions (21, 24, 25, 29, 30, 31, 33, 34).

Phenylalanine Hydroxylase: Activity and Phosphorylation Effect of glucagon and cyclic AMP. Phosphorylation of purified phenylalanine hydroxylase with cyclic AMP-dependent protein kinase is associated with an activation of up to 4-fold (35). In liver cells increased activity is found in the presence of glucagon but only at low concentrations of phenylalanine (29). Although these concentrations are in the range found in vivo, the maximal stimulation of flux is rarely more than 100%, and certainly never reaches that seen in vitro. The reasons for this are complex and will be discussed below. Analysis of the degree of phosphorylation stimulated by glucagon shows that this phosphorylation closely parallels metabolic flux with physiological

PHENYLALANINE AND TYROSINE METABOLISM

313

220

180 m

._¢ ~e 140

S

Phosphorylation

~----~"~ ' I I I ~i 11 10 9 8 nohormone -Ioglo[glucagon](M)

I

7

FIG. 2. The relationship between the metabolic flux through, and phosphorylation of, phenylalanine hydroxylase in rat liver cells incubated with glucagon. The final concentration of phenylalaninewas 50 ,aM. Other details were as described in (24) and (29). This figure is corrected from that given in (36). Error bars have been omitted for clarity.

substrate concentrations (Fig. 2; Refs. 24, 36). As with the activity, the phosphorylation never approaches 1 mole phosphate per mole of subunit in cells, even with maximally-effective concentrations ofglucagon. Cyclic AMP, added as the dibutyryl derivative, produces effects almost identical to those seen with 10"7M glucagon (Fig. 3).

Effects of adrenergic agents. B-Adrenergic effects are seen in rats, most readily in young (37) and/or female (38) animals. In experiments with liver cells from 80 g rats, we found that B-agonists increased flux through the hydroxylase, and to the same extent as did cyclic AMP (34). As anticipated, cells from older, male rats were insensitive to B-stimuli (Fig. 3). They were, nevertheless, responsive to epinephrine and norepinephrine themselves (Fig. 3) acting as ot-agonists (Figs. 3, 4). The calcium ionophore, A23187, mimicked these c~-effeets, evidence supportive of a role for intracellular calcium in increasing both metabolic flux and phosphorylation under these conditions (Fig. 3). These results are in agreement with studies in vitro which showed that purified enzyme was readily phosphorylated by the calmodulin-dependent protein kinase (39). Although other calcium-dependent activities cannot be ruled out, it appears unlikely that phosphorylase kinase plays a role (40). Effect ofvasopressin. We anticipated that vasopressin might have effects on phenylalanine hydroxylase which were similar to those of the c~-adrenergic

314

C H R I S T O P H E R I. POGSON, et al. 200

og

300

8~

'1,

1

F==

100

200

O

o

EL

"I" .L

100

A

B

C

D

A

B

C

.D

E

F

G

H

Y

Experiment

I

II

FIG. 3. The effects of various agents on the metabolic flux through, and phosphorylation of, phenylalanine hydroxylase in rat liver cells. All experiments were performed at a final phenylalanine concentration of 50 ~zM. All results are the means + S.E.M. from at least 3 independent cell preparations. The significance of differences between means (all vs control) was assessed by the paired t test: *P < 0.05. Experiment 1: A, control; B, 10-4 M dibutyryl cyclic AMP; C, 10-7 M glucagon; D, 10-7 M insulin. Experiment H: A, control; B, 10-7 M glucagon; C, 10-5M epinephrine; D, 10-5 ~¢ norepinephrine; E, 10-6M isoprenaline; F, 2 × 10-s M phenylephrine; G, 2.5 )< 10-6M A23187; H, 10-7M vasopressin. Further details are given in (24) and (34).

agents. However, despite being able clearly to demonstrate an effect of the hormone on glycogenolysis, we were unable to detect any change in hydroxylase activity in cells or in enzyme phosphorylation (Fig. 3). This is not inconsistent with other recent studies with vasopressin (41-44) and may indicate greater differentiation between control systems than hitherto recognized. We have, in separate experiments, found no effect of phorbol ester on the hydroxylase and been unable, in vitro, to phosphorylate the purified enzyme with protein kinase C.

PHENYLALANINE A N D TYROSINE METABOLISM

315

40

II

30

8

! 10 U-

L A

B

C

D

E

F

FIG. 4. Antagonism of adrenergic effects of metabolic flux through phenylalanine hydroxylase in rat liver cells. All experiments were performed at a final phenylalanine concentration of 50/aM. All results are means __. S.E.M. from 4 separate cell preparations. The significance of differences (i.e. between C and A and F and D) between means was assessed by the paired t test: *P < 0.05. Neither antagonist on its own had any effect on the system. A, 10-6M epinephrine; B, 10-6M epinephrine plus 10-5 M propranolol; C, 10-6 M epinephrine plus 10-5 M phentolamine; D, 10-6 M norepinephrine; E, 10-6 M norepinephrine plus 10-5 M propranolol; F, 10-6 M norepinephrine plus 10-5 M phentolamine. Further details are given in (34),

Phosphorylation in liver cells and in vitro. In vitro, phosphorylation of the purified hydroxylase with either cyclic AMP-dependent (35, 45) or Ca 2÷dependent (39) protein kinase can lead to the incorporation of 1 mole of phosphate per mole of subunit. This incorporation shows a linear relationship with enzyme activity assayed with natural cofactor (35, 39). In our experiments, control incubations contained hydroxylase with 0.2-0.3 mole of phosphate per mole of subunit. With maximally-effective concentrations of glucagon, the values obtained approached 0.6-0.7 mole per mole, while aagonist-induced phosphorylation was maximally little more than 0.4-0.5 mole per mole. When both types of agonist were added together at high concentrations, the extent of phosphorylation resembled that seen with glucagon alone (Fig. 5). As with the experiments with purified proteins, however, a linear relationship could be demonstrated between expressed activity in whole cells and the extent ofphosphorylation, however caused (Fig.

6). A major difference between the two experimental systems, purified enzymes and isolated cells, is that reaction in the purified system will proceed to maximal phosphorylation, whereas, in cells, it will reach a new steady-state representing the balance between increased kinase activity and protein phosphatase activity. Unless phosphatase activity becomes zero or is very low, stoichiometric incorporation of phosphate is unlikely to occur. One specific

316

C H R I S T O P H E R I, POGSON, et al. Flux

Phosphorylation

300

"

8

t

200 !l

,00 Glucagon (10"7M)

--

+

Norepinephrine (10"5M)

--

+

--

+

+

+

--

--

+ +

+

FIG. 5. The effect of glucagon and norepinephrine on metabolic flux through, and phosphorylation of, phenylalanine hydroxylation in rat liver cells. The final concentration of phenylalanine was 50 pM with additions as shown. The control values were, for flux, 4.55 --- 0.16 nmol/hr per mg dry wt, and for phosphorylation, 0.21 + 0.03 tool P/tool subunit. Results are means -2_ S.E.M. for at least 4 separate cell preparations. Further details are given in (33).

240

./

220 e0 >. x o "o >. .I-

200

180

160

i

u 0l ¢

140

~=

Q.

120

100

,

,

0.1

0.2

~&,

,

0,3

0.4

0.5

0.6

Phosphate content (mol P/rnol subunit)

FIG. 6. The relationship between the metabolic flux through, and phosphorylation of, phenylalanine hydroxylase in rat liver cells under various conditions. Data are taken from experiments with glucagon (control, 10-]] M- 10-7 M) (e) and those with calcium-mobilizing agents (&). Each point is the mean from at least 2 separate cell preparations. Reproduced from (59)by permission of the Biochemical Society, London.

P H E N Y L A L A N I N E A N D TYROSINE METABOLISM

317

question arises from the cell studies: why do glucagon or other agents acting through cyclic AMP induce higher levels ofphosphorylation than a-agonists? There is as yet no clear answer but the experiment in Table 1 is perhaps indicative. When glucagon is added together with [np]p~, the rate of incorporation of label into the enzyme is less than observed for controls without the hormone. Under these conditions, one factor of importance in determining the rate of labelling is the rate at which previously existing unlabelled phosphate is removed by the appropriate protein phosphatase. The results in this experiment are consistent with a mechanism whereby glucagon both increases phosphorylation and decreases phosphatase activity. Any agent, such as oL-agonists, which did not affect phosphatase activity, would then induce phosphorylation only to a lower new steady-state level. Experiments are in progress to test this hypothesis. TABLE 1. E F F E C T O F G L U C A G O N (10-7 M) ON THE R A T E O F PHOSPHORYLATION OF PHENYLALANINE HYDROXYLASE U N D E R PRE-STEADY STATE CONDITIONS Time (min) 15 30 60 90

Control 3.0 8.5 17.8 18.8

+ 0.2 + 2.4 + I. 1 --. 0.3

Plus glucagon 2.6 5.5 10.4 13.5

[nP]Pi (100 ~Ci/ml, 0.4 raM-E) and glucagon were added simultaneously after the preincubation period. Values are counts/min per pmol of hydroxylase subunit. Results are means + S.E.M. from 3 separate cell preparations (control) or means from 2 such preparations (plus glucagon).

Effect of insulin, Two kinds of insulin effect on the hydroxylase can be demonstrated. Chronic diabetes (10 day) is associated with an increase in phenylalanine hydroxylase activity (29, 46; see also Table 3) which is attributable to an increase in enzyme protein (33). This activity is further increased in cells exposed to glucagon (presumably as a consequence of phosphorylation) but not in cells incubated with noradrenaline (Fig. 7). When normal cells are incubated with insulin (10-7 M) alone, no effect can be discerned on flux or phosphorylation (Fig. 3). When activity is increased by low concentrations of glucagon, however, insulin significantly opposes both the changes in activity (Fig. 8) and in phosphorylation (Table 2; 47). A similar effect is seen when norepinephrine is the primary stimulus.

318

C H R I S T O P H E R I. POGSON, Normal

200

e t al.

Diabetic

4 :

÷

-t-

x ii

A

B

C

D

A

B

C

D

FIG. 7. The effects of glueagon and norepinephrine on metabolic flux through phenylalanine hydroxylase in liver cells from normal and chronically (10 day)-diabetic rats. All experiments were performed at a final phenylalanine concentration of 50 #M. The control rates (A) for ceils from normal and diabetic rats were 4.55 + 0.16 and 10.13 + 0.82 nmol/hr per mg dry wt. All results are means + S.E.M. from 6 independent cell preparations. The significance of differences between means (all v s control) was assessed by Student's t test: *P < 0.05. A, control; B, 10"7 M glucagon; C, I0-SM norepinephrine; D, 10-TM glucagon plus 10-SM norepinephrine. Further details are given in (33).

÷

too

8

!

I

50

E 0

Glucagon (M)

10 "10

Insulin (10"7M) -

÷

5xi0-10 --

+

+

10-7

10-8

10"9 --

+

--

+

FIG. 8. Antagonism of the action of glucagon on flux through phenylalanine hydroxylase by insulin. The final concentration of phenylalanine was 50#M, with additions of insulin and glucagon as shown. Results are means + S.E.M. for 3 separate cell populations. The differences between means (insulin plus glucagon vs insulin alone) were assessed by Student's t test: *P < 0.05. Further details are given in (47).

319

P H E N Y L A L A N I N E A N D TYROSINE M E T A B O L I S M TABLE 2. E F F E C T O F INSULIN ON G L U C A G O N - 1 N D U C E D PHOSPHORYLATION OF PHENYLALANINE HYDROXYLASE Conditions

Mole phosphate incorporated/mole subunit

Control

0.22 __. 0.04 0.37 + 0.04' 0.26 + 0.02t

Glucagon (10-9M) Glucagon (10"gM), Insulin (10-TM)

Results are means +__S.E.M. from 3 separate celt preparations. *P < 0.05 vs control; i P < 0.05 v s glucagon alone (Student's t test).

Effect of polyamines. Phenylalanine hydroxylase phosphorylated by the cyclic AMP-dependent protein kinase is readily dephosphorylated by protein phosphatase-2A (EC. 3.1.3.-) (48). The physiological control of this phosphatase remains largely unknown, but recent work (49) has shown that the isolated enzyme is strongly activated by spermine, less so by spermidine and not at all by putrescine. This activation is expressed towards a number of substrates including phosphorylated phenylalanine hydroxylase (49). It would be anticipated, therefore, that, if this effect was indeed physiologically relevant, spermine would counteract any stimulation of the hydroxylase by increased phosphorylation in vivo. Figure 9 shows that spermine antagonizes the effect of glucagon both on flux and phosphorylation in liver cells, and that this effect can be seen at concentrations below 10-4 M. It is at present difficult to translate these observations to the liver in vivo because there is limited

100

200

T/I

,,Q

?so

±

I

ik

spermine |mM)

....... 0

glucagon (nM) ............

+

0.01 0.1 1.0 +

+

+

0

1.0

0

1.0

+

+

"O 7 o

..¢

E 10o

o 8

a_

FIG. 9. The effect of spermine on the glucagon activation of phenytalanine hydroxylase activity and phosphorylation in rat liver cells, The final concentration of phenylalanine was 50 #M throughout. Results are means + S.E.M. from 3 separate cell preparations. Flux data are shown on the left, phosphorylation data on the right,

320

CHRISTOPHER I. POGSON, et aL

information on the 'normal' concentration, distribution and chemical availability of polyamines. It is of more than passing interest, however, that polyamines have been recently shown to be potential regulators of diverse metabolic processes (50-53).

Identity of phosphorylation sites. Using purified enzymes, D~skeland et al. (39) have clearly shown that the same site is phosphorylated by both cyclic AMP- and Ca2÷-dependent protein kinases. This is consistent with our results with liver cells (Fig. 5). We have confirmed the observation in cells by analysis of phosphopeptides generated from immunoprecipitated enzyme by various proteases; the patterns of phosphopeptides on polyacrylamide gel electrophoresis were similar whatever the nature of the original stimulus (59). Tyrosine Aminotransferase: Activity and Phosphorylation Effects of various potential agonists. We have investigated the effects of, inter alia, glucagon, o~-and/3-agonists, and vasopressin on the expressed activity of tyrosine aminotransferase in cells metabolizing tyrosine at physiological (and other) concentrations. Under no conditions have we noted any change in metabolic flux from 5 min up to 2 hr. Immunoprecipitation of aminotransferase, necessarily in the presence of cold carrier, showed that the enzyme did incorporate 32p, in agreement with previous observations (18, 19). This incorporation was, in contrast with that of the hydroxylase, very slow, proceeding linearly for 90 min before levelling off at 120 rain. At this time, the stoichiometry of incorporation was 2.1 mole phosphate per mole of aminotransferase subunit. This phosphorylation was unaffected by any of the potential agonists tried, and seems to be more consistent with the formation of the constitutively-bound phosphate noted by Hargrove et al. (20).

Control of Phenylalanine and Tyrosine Metabolism Long-term control mechanisms. Although changes in the pattern of phosphorylation of phenylalanine hydroxylase may occur during longer-term nutritional or other changes (46), much of the response to such changes may be attributed to changes in total enzyme activity reflecting parallel changes in enzyme protein (33). Both the hydroxylase and tyrosine aminotransferase activities are readily increased by glucocorticoid but to different extents and with different time-courses of induction (21). Table 3 shows the effect of various conditions on metabolic fluxes through the reactions catalyzed by the two enzymes in liver cells.

321

PHENYLALANINE AND TYROSINE METABOLISM TABLE 3. EFFECT OF NUTRITIONAL AND HORMONAL STRESS ON METABOLIC FLUX THROUGH PHENYLALANINE HYDROXYLASE AND TYROSlNE AMINOTRANSFERASE IN RAT LIVER CELLS

Conditions Control Starved (48 hr) Tryptophan-treated (750 mg/kg) Adrenalectomy Adrenalectomy plus dexamethasone (2.5 mg/kg per day) Diabetes (3d) Diabetes (14d)

Flux (nmol/hr per mg dry wt) Through pbenylalanine Through tyrosine hydroxylase aminotransferase 5.07 + 0.27 7.62 + 0.29 4.60 _+0.23 3.73 + 0.56 5.93 + 0.33§ 8.53 + 0.74* 14.93 + 1.57t§

3.32 + 4.42 + 5.00 + 4.50 +

0.20 0. I0 0.5I* 0.33*

9.70+ 0.4lit 8.42 + 1.22" 11.80 _ 0.12:~§

Results are means _ S.E.M. from 3 separate preparations. Flux through the hydroxylase was measured with 50 ~ phenylalanine, that through the aminotransferase with 100 tyrosine. Details of animal treatments are given in (21) and (25). Differences between means were assessed by Student's t test: P (vs control)* < 0.05; "1"< 0.01; :~ < 0.001: (vs adrenalectomized or 3d diabetic groups) §< 0.05; II < 0.01. Changes in starvation were not significant when assessed on a DNA basis.

T h e lack o f parallelism in the b e h a v i o r o f the two enzymes highlights a p o t e n t i a l p r o b l e m . I f the h y d r o x y l a s e activity exceeds that o f the a m i n o transferase, there is a 'risk' that there will be a greater flow o f c a r b o n f r o m p h e n y l a l a n i n e to tyrosine t h a n f r o m tyrosine o n w a r d s with a c o n s e q u e n t i m b a l a n c e in the ratio o f the two a m i n o acids in the circulation. Alternatively, increased a m i n o t r a n s f e r a s e activity alone could deplete the tyrosine concentration. W e have recently s h o w n t h a t liver cells c o n t a i n a t r a n s p o r t system which is specific for a r o m a t i c a m i n o acids, and t h a t this has the characteristics o f a Tsystem (54). P h e n y l a l a n i n e a n d tyrosine are substrates for this T-system and for the previously r e p o r t e d L-system. A p a r t f r o m its substrate specificity, the T-system differs f r o m the L-system in having a higher affinity but lower m a x i m a l activity. At physiological c o n c e n t r a t i o n s o f the a m i n o acids, b o t h systems are involved. M e a s u r e m e n t s o f the rates at which p h e n y l a l a n i n e and tyrosine enter liver cells (54) indicate that t r a n s p o r t is by n o m e a n s as rapid a process as f r e q u e n t l y assumed, F u r t h e r e x p e r i m e n t a l analysis (60) has p e r m i t t e d the calculation o f c o n t r o l coefficients (see 55) for t r a n s p o r t a n d for the steps catalyzed by the h y d r o x y l a s e a n d a m i n o t r a n s f e r a s e (Table 4). U n d e r basal conditions, the c o n t r o l o f p h e n y l a l a n i n e m e t a b o l i s m (neglecting t r a n s a m i n a t i o n (21)) is s h a r e d between t r a n s p o r t a n d the hydroxylase. This m e a n s that inhibition (or activation) o f either step will have a m a j o r effect o n flux. T r a n s p o r t o f p h e n y l a l a n i n e is inhibited competitively by tyrosine and

C H R I S T O P H E R I. POGSON, et al.

322

TABLE 4. CONTROL C O E F F I C I E N T S FOR THE CATABOLISM OF P H E N Y L A L A N I N E A N D TYROSINE BY RAT LIVER CELLS

Conditions

Control coefficients on flux (C J) Transport into the cell Enzyme

Phenylalanine hydroxylase Basal Plus glucagon (10-TM)

0.49 + 0.01 0.88 +-- 0.01"

0.51 + 0.01 0.12 + 0.01"

0.22 _ 0.01 0.58 + 0.02*

0.71 + 0.02 0.29 + 0.01"

Tyrosine aminotransferase Basal Dexamethasone-stimulated

Basal conditions refer to incubations of cells from normal fed animals with either 50 #M phenylalanine or 100 #M tyrosine. Results are means _+S.E.M. for 3 separate preparations. The significance of differences between means was assessed by Student's t test. P (vs basal): * < 0.005.

tryptophan (T-system) and by the range of L-system amino acid substrates (54), so that potential 'imbalances' in circulating amino acid concentrations may be self-regulating; that is, low concentrations of, for example, phenylalanine will be transported less effectively at constant concentrations of competing amino acids, and will thereby be protected from metabolism through hydroxylation. Activation of the hydroxylase by phosphorylation due to glucagon (or, presumably, other appropriate stimuli) will increase flux, but the increase seen will (because C J < 1.0) be less than observed with the isolated, purified, enzyme. As the enzyme is phosphorytated, so the distribution of control coefficients changes, so that the system becomes increasingly more sensitive to control at the level of transport and less so at the hydroxylation step (assuming that other factors remain constant). A similar argument applies in the case of tyrosine metabolism. As enzyme activity is increased by dexamethasone (or other stimuli; Table 3), so the system becomes more responsive to control at the plasma membrane. It is becoming increasingly apparent that the basic phosphorylationdephosphorylation cycle is but a part, albeit an important one, of the system for the regulation of phenylalanine disposal. Figure 10 illustrates the actions of various factors on the components of the phenylalanine hydroxylase system. The question marks on the Figure are a reminder of the work still to be done. Although the phosphorylation of the aminotransferase seems to be 'silent' (at least in terms of readily observable kinetic parameters), problems still remain regarding the factors responsible for the absolute rate of the flux from tyrosine to 4-hydroxyphenylpyruvate and onwards. The ratio of 2oxoglutarate to glutamate, sensitive to many metabolic effectors and stresses may well be a major influence on aminotransferase in vivo (21). Observations

PHENYLALANINE AND TYROSINE METABOLISM

Gluoagon

cA-PrK

323

~-agonists

or

2÷

Ca-PrK Phe

PAHA ~ '

PAHI

Pi

PAHPi

?

, PAHPA

Active protein ~osphatasa- 2A . Polyarninell

)

cA-PrK ~

Glucagon

/:ac'l:::li:ro~e: - ~ / / / pho~ohatBm-

2A

FIG. 10. Mechanisms for the control of the phosphorylation of phenylalanine hydroxylase. Abbreviations: BH4, tetrahydrobiopterin; cA-PrK, cyclicAMP-dependent protein kinase; Ca~'PrK, calcium-dependentprotein kinase; PAH, phenylalanine hydroxylase; PAHP, phenylalanine hydroxylase (phosphorylated); Phe, phenylalanine. The subscripts A and I indicate respectively the substrate-activated and inactive states of the enzyme. It is probable that the hydroxylase is phosphorylated in both A and I forms. Phenylalanine is reported to increase, and tetrahydrobiopterin to inhibit, the rate of phosphorylation by the cyclicAMP-dependent kinase in vitro (58); the former may, however, act as an inhibitor when the calcium-dependent kinase is involved (39). that tyrosine derived from phenylalanine in vivo (56) and in liver cells (A. R. Nicholas, R. G. Knowles, A. J. Dickson and C. I. Pogson, unpublished data) does not readily equilibrate with the cellular pool derived from exogenous tyrosine suggest that further complexities may also exist in what seemed initially a simple system! SUMMARY A system for the parallel determination of enzyme phosphorylation and expressed activity in rat liver ceils, and its application to studies of phenylalanine hydroxylase and tyrosine aminotransferase, is described. Phenylatanine hydroxylase is phosphorylated by agents which stimulate cyclic A M P - and Ca2÷-dependent protein kinase activity. The phosphorylation site(s) appear to be the same for both kinases. Phosphorylation is accompanied by increased metabolic flux at low, physiologically relevant,

324

CHRISTOPHER I. POGSON, et al.

substrate c o n c e n t r a t i o n s . Insulin a n d spermine b o t h inhibit the p h o s p h o r y lation o f the e n z y m e , possibly b y increasing d e p h o s p h o r y l a t i o n . T y r o s i n e a m i n o t r a n s f e r a s e is p h o s p h o r y l a t e d in liver cell i n c u b a t i o n s but the rate is slow a n d insensitive to additions to the m e d i u m . N o parallel changes in flux c o u l d be detected. Both e n z y m e s are subject to c o m p l e x r e g u l a t o r y m e c h a n i s m s , short- and l o n g - t e r m . Their activities m a y be c o o r d i n a t e d in vivo by c o n t r o l exerted at the level o f the p l a s m a m e m b r a n e where b o t h a m i n o acids share the same t r a n s p o r t processes. D e t e r m i n a t i o n o f the c o n t r o l coefficients for the several c o m p o n e n t s indicates that m e m b r a n e t r a n s p o r t m a y be a m a j o r limitation on flux.

AC KNO W L E D G E M E N T S W e are grateful to the Medical Research C o u n c i l a n d the W e U c o m e T r u s t for financial s u p p o r t .

REFERENCES 1. R.I. PETERS and B. R. BUHR, Tryptophan and serotonin metabolism after sustained tryptophan infusion, Neurochem. Int. 6, 685-691 (1984). 2. J.D. FERNSTROM, Effects of precursors on brain neurotransmitter synthesis and brain functions, Diabetologia 20 (Suppl.), 281-289 (1981). 3. S. KAUFMAN, Phenylketonuria: biochemical mechanisms, Adv. Neurochem. 2, 1-132 (1977). 4. S. KAUFMAN, The phenylalanine hydroxylating system from mammalian liver, Adv. Enzymol. Relat. Areas Mol. Biol. 35, 249-319 (1971). 5. S. KAUFMAN, Regulatory properties of phenylalanine, tyrosine and tryptophan hydroxylases, Biochem. Soc. Trans. 13, 433-436 (1985). 6. C. I. POGSON, M. A. SANTANA and M. J. FISHER, Phenylalanine hydroxylase: metabolic aspects, Biochem. Soc. Trans. 13, 439-441. 7. T.A. DIX, G. E, BOLLAG, P. L. DOMANICO and S. J. BENKOVIC, Phenylalanine hydroxylase: Absolute configuration and source of oxygen of the 4a-hydroxytetrahydropterin species, Biochemistry 24, 2955-2958 (1985). 8. S. BENKOVIC, D. WALLICK, L. BLOOM, B. J. GAFFNEY, P. DOMANICO, T. A. DIX and S. PEMBER, On the mechanism of action of phenylalanine hydroxylase, Biochem. Soc. Trans, 13, 436-438 (1985). 9. S.C.M. KWOK, F. D. LEDLEY, A. G. DILELLA, K. J. H. ROBSON and S. L. C. WOO, Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence of human phenylalanine hydroxylase, Biochemistry 24, 556--561 (1985). 10. K.J.H. ROBSON, W. BEATTIE, R. 3. JAMES, R. G. H. COTTON, F. J. MORGAN and S. L. C. WOO, Sequence comparison of rat liver phenylalanine hydroxylase and its cDNA clones, Biochemistry 23, 5671-5675 (1984). 11. J. DONLON and S. KAUFMAN, Modification of the multiple forms of rat hepatic phenylalanine hydroxylase by in vitro phosphoryfation,Biochem. Biophys. Res. Commun. 78, 1011-1017 (1977). 12. J. DONLON and S. KAUFMAN, Relationship between the multiple forms of rat hepatic phenylalanine hydroxylase and degree of phosphorylation, J. Biol. Chem. 255, 2146-2152 (1980). 13. S.C. SMITH, B. E, KEMP, W. J. McADAM, J. F. B. MERCER and R. G. H. COTTON,

PHENYLALANINE AND TYROSINE METABOLISM

14.

15.

16. 17. 18. 19. 20.

21. 22.

23.

24. 25.

26. 27. 28, 29. 30. 31. 32. 33.

325

Two apparent molecular weight forms of human and monkey phenylalanine hydroxylase are due to phosphorylation, J. Biol. Chem. 259, 11284-11289 (1984). J . F . B . MERCER, A. GRIMES, I. JENNINGS and R. G. H. COTTON, Identification of two molecular-mass forms of phenylalanine hydroxylase that segregate independently in rats: Specific association of each form with certain rat strains, Biochem. Z 219, 891-898 (1984). J.-P. ABITA, S. MILSTIEN, N. CHANG and S. KAUFMAN, In vitro activation of rat liver phenylalanine hydroxylase by phosphorylation, J. Biol. Chem. 251, 5310-5314 (1976). J. DONLON and S. KAUFMAN, Glucagon stimulation of rat hepatic phenylalanine hydroxylase through phosphorylation in vivo, J. Biol, Chem. 253, 6657-6659 (1978). J.-P. ABITA, H. CHAMRAS, G. ROSSELIN and F. REY, Hormonal control of phenylalanine hydroxylase activity in isolated rat hepatocytes, Biochem. Biophys. Res. Commun. 92, 912-918 (1980). K.L. LEE and J. M. NICKOL, Phosphorylation of tyrosine aminotransferase in vivo, J. Biol. Chem. 249, 6024-6026 (1974). W. D. WICKS and J.-L. SU, Relationship between phosphorylation of tyrosine aminotransferase and regulation of its synthesis by cyclic AMP and hormones, J. Cycl. Nucleotide Res. 4, 113-122 (1978). J.L. HARGROVE and D. K. GRANNER, Physical properties, limited proteolysis, and acetylation of tyrosine aminotransferase from rat liver, J. Biol. Chem. 256, 8012-8017 (1981). J.C. STANLEY, M. J. FISHER and C. I. POGSON, The metabolism of L-phenylalanine and L-tyrosine by liver cells isolated from adrenalectomized rats and from streptozotocindiabetic rats, Biochem. £ 225, 249-255 (1985). S.A. SMITH and C. I. POGSON, The metabolism of L-tryptophan by isolated rat liver cells: Effect of albumin binding and amino acid competition on oxidation of tryptophan by tryptophan 2,3-dioxygenase, Biochem. J. 186, 977-986 (1980). S.A. SMITH, F. P. A. CARR and C. I. POGSON, The metabolism of L-tlyptophan by isolated rat liver cells: Quantification of the relative importance of, and the effect of nutritional status on, the individual pathways of tryptophan metabolism, Biochem. J. 192, 673-686 (1980). M.J. FISHER and C. I. POGSON, Phenylalaninehydroxylase in liver cells: Correlation of glucagon-stimulated enzyme phosphorylation with expressed activity, Biochem. J. 219, 79-85 (1984). M. SALTER, J. C. STANLEY, M. J. FISHER and C. I. POGSON, The influence of starvation and tryptophan administration on the metabolism ofphenylalanine,tyrosine and tryptophan in isolated rat liver cells, Biochem. J. 221,431-438 (1984). R. SHIMAN, D. W. GRAY and A. PATER, A simple purification of phenylalanine hydroxylase by substrate-induced hydrophobic chromatography, J. Biol. Chem. 254, 1130-1306 (1979). J . C . STANLEY, A. R. NICHOLAS, A. J. DICKSON, I. M. THOMPSON and C. I. POGSON, Tryptophan aminotransferase activity in rat liver, Biochem. J. 220, 341-344 (1984). A.J. DICKSON and C, I. POGSON, The metabolic integrity of hepatocytes in sustained incubations, FEBS Lett. 83, 27-32 (1977). F . P . A . CARR and C. I. POGSON, Phenylalaninemetabolism in isolated rat cells: Effects of glucagon and diabetes, Biochem. J, 198, 655-660 (1981). M . J . FISHER and C. I. POGSON, The determination of flux through phenylafanine hydroxylase and homogentisate oxidase in isolated hepatocytes, Bioscience Rep. 4, 835-841 0984). A.J. DICKSON, F. A. O. MARSTON and C. I. POGSON, Tyrosine aminotransferase as the rate-limiting step for tyrosine catabolism in isolated rat liver cells, FEBSLett. 127, 28-32 (1981). E. J. LEWIS, P. CALIE and W. D. WICKS, Differences in rates of tyrosine aminotransferase deinduction with cyclic AMP and glucocorticoids, Proc. Natl. Acad. Sci. U.S.A. 79, 5778-5782 (1982). M. A. SANTANA, M. J. FISHER, A. J. BATE and C. I. POGSON, The effect of

326

34. 35.

36. 37.

38. 39.

40.

41. 42.

43.

44. 45. 46. 47. 48. 49. 50. 51.

CHRISTOPHER I. POGSON, et al. experimental diabetes on phenylalanine metabolism in isolated liver ceils, Biochem. J. 227, 169-175 (1985). M . J . FISHER, M. A. SANTANA and C. I. POGSON, Effects of adrenergic agents, vasopressin and ionophore A23187, on the phosphorylation of, and flux through, phenylalanine hydroxylase in rat liver cells, Biochem. J. 219, 87-90 (1984). R. SHIMAN, G. E. MORTIMORE, C. M. SCHWORER and D. W. GRAY, Regulation of phenylalanine hydroxylase activity by phenylalanine in vivo, in vitro, and in perfused rat liver, J. Biol. Chem. 257, 11213-11216 (1982). C.I. POGSON, W. R. CARPENTER, J. S. COOK, M. J. FISHER, M. A. LOMAX, M. SALTER and J. C. STANLEY, A critical approach to the use of isolated liver cells for the study of metabolic events, Proc. Nutr. Soc. 43, 119-132 (1984). J.B. BLAIR, M. E. JAMES and J. L. FOSTER, Adrenergic control of glucose output and adenosine Y:5'-monophosphate levels in hepatocytes from juvenile and adult rats, J. Biol. Chem. 254, 7579-7584 (1979). R.K. STUDER and A. B. BORLE, Differences between male and female rats in the regulation of hepatic glycogenolysis: The relative role of calcium and cAMP in phosphorylase activation by catecholamines, J. BioL Chem. 257, 7987-7993 (1982). A. P. DOSKELAND, C. M. SCHWORER, S. O. DOSKELAND, T. D. CHRISMAN, T. R. SODERLING, J. D. CORBIN and T. FLATMARK, Some aspects of the phosphorylation of phenylalanine 4-monooxygenase by a calcium-dependent and calmodulin-dependent protein kinase, Eur. J, Biochem. 145, 31-37 (1984). J . C . GARRISON, D. E. JOHNSEN and C. P. CAMPANILE, Evidence for the role of phosphorylase kinase, protein kinase C, and the other Ca2+-sensitive protein kinases in the response of hepatocytes to angiotensin II and vasopressin, Z Biol. Chem, 259, 3283-3292 (1984). F. ASSIMACOPOULOS-JEANNET, R. M. DENTON and B. JEANRENAUD, Stimulation of hepatic lipogenesis and acetyl-coenzyme A carboxylase by vasopressin, Biochem. J. 198, 485-490 (1981). J.A. CREBA, C. P, DOWNES, P. T. HAWKINS, G. BREWSTER, R. H. MICHELL and C. J. KIRK, Breakdown of phosphatidylinositol 4-phosphate andphosphatidylinositol4,5bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+-mobilizing hormones, Biochem. Z 212, 733-747 (1983). N . M . KNEER and H. A. LARDY, Regulation of gluconeogenesis by norepinephrine, vasopressin and angiotensin II: A comparative study in the absence and presence of extracellular Ca2+, Arch. Biochem, Biophys. 225, 187-195 (1983). J.A. GARCIA-SAINZ, F. MENDLOVIC and M. A. MARTINEZ-OLMEDO, Effects of phorbol esters on t~-adrenergic-mediated and glucagon-mediated actions in isolated rat hepatocytes, Biochem. J. 228, 277-280 (1985). M. PARNIAK, H. HASEGAWA, J. WlLGUS and S. KAUFMAN, On the phosphate content of rat liver phenylalanine hydroxylase purified by hydrophobic chromatography Biochem. Biophys. Res. Commun. 99, 707-714 (1981). 3. DONLON and E. BEIRNE, Modulations of rat hepatic phenylalaninehydroxylase due to induced diabetes on high-protein diet,Biochem. Biophys. Res. Commun. 108, 747-751 (1982). M.J. FISHER, A. J. DICKSON and C. I. POGSON, Insulin antagonizes the glucagonmediated stimulation of phenylalanine hydroxylase activity in isolated liver cells, Biochem. Soc. Trans. (1985), in press. S. PELECH, P. COHEN, M. J. FISHER, C. I. POGSON, M, R. EL-MAGHRABI and S. J. PILKIS, The protein phosphatases involved in cellular regulation: Glycolysis, gluconeogenesis and aromatic amino acid breakdown in rat liver, Eur. J. Biochem. 145, 39-49 (1984). H.Y.L. TUNG, S. PELECH, M. J. FISHER, C. I. POGSON and P. COHEN,The protein phosphatases involved in cellular regulation: Influences of polyamines on the activities of protein phosphatase-1 and protein phosphatase-2A, Eur. J. Biochem. 149, 305-313 (1985). Z. DAMUNI, J. S. HUMPHREYS and L. J, REED, Stimulation of pyruvate dehydrogenase phosphatase activity by polyamines, Biochem. Biophys. Res. Commun. 124, 95-99 (1984). A. ELGAVISH, R. W. WALLACE, D. J. PILLION and E. MEEZAN, Polyamines

PHENYLALANINE AND TYROSINE METABOLISM

52. 53.

54. 55.

56.

57. 58.

59.

60.

327

stimulate D-glucose transport in isolated renal brush-border membrane vesicles, Biochim. Biophys. Acta 777, 1-8 (1984). P. AUBERGER, M. SAMSON, G. LE CAM and A. LE CAM, Regulation of protein phosphorylation by polyamines in hepatocytes, Biochim. Biophys. A cta 81)1,461-469 (1984). P. MARTIN-SANZ, R. HOPEWELL and D. N. BRINDLEY, Spermine promotes the translocation of phosphatidate phosphohydrolase from the cytosol to the microsomal fraction of rat liver and it enhances the effects of oleate in this respect, FEBS Left. 179, 262-266 (1985).. M. SALTER, R. G. KNOWLES and C. I. POGSON, Transport of the aromatic amino acids into isolated rat liver cells: properties of uptake by T- and L-systems, Biochem. J. 233, 499-506 (1986). A.K. GROEN, R. VAN DER MEER, H. V. WESTERHOFF, R. J. A. WANDERS, T. P. M. AKERBOOM and J. M. TAGER, Control of metabolic fluxes, pp. 9-37 in Metabolic Compartmentation (H. SIES, ed.), Academic Press, New York (1982). L.L. MOLDAWER, I. KAWAMURA, B. R. BISTRIAN and G. L. BLACKBURN, The contributions of phenylalanineto tyrosine metabolism in vivo. Studies in the post-absorptive and phenylalanine-loaded rat, Biochem. Z 210, 811-817 (1983). M . J . FISHER and C. I. POGSON, Phosphorylation of phenylalanine hydroxylase: its hormonal control and metabolic consequences, Biochem. Soc. Trans. 13, 466-467 (1985). A. P. DOSKELAND, S. O. DOSKELAND, D. I~GREID and T. FLATMARK, The effect of ligands of phenylalanine 4-monooxygenase on the cAMP-dependent phosphorylation of the enzyme, J. Biol. Chem. 259, 11242-11248 (1984). M. J. FISHER, A. J. DICKSON and C. I. POGSON, Phosphopeptide analysis of phenylalaninehydroxylase isolated from liver cells exposed to hormonal stimuli, Biochem. J. (1986), in press. M. SALTER, R. G. KNOWLES and C. I. POGSON, Quantification of the importance of individual steps in the control of aromatic amino acid metabolism, Biochem. J. (t986), in press.