in Fibroblasts by Intracellular Concentrations of Cyclic Adenosine ...

12 downloads 85 Views 669KB Size Report
Commun. 44, 1192-1198. 4. Peery, C. V., Johnson, G. S. & Pastan, I. (1971) J. Biol. Chem. 246, 5785-5790. 5. Pastan, I. & Perlman, R. L. (1971) NatureNew Biol.
Proc. Nat. Acad. Sci. USA

Vol. 69, No. 2, pp. 459-462, February 1972

Regulation of Adenosine 3':5'-Cyclic Monophosphate Phosphodiesterase Activity in Fibroblasts by Intracellular Concentrations of Cyclic Adenosine Monophosphate (3T3/dibutyryl cyclic AMP/SV40-transformed cells/Michaelis constants/L cells/prostaglandin El)

MASSIMO D'ARMIENTO, GEORGE S. JOHNSON, AND IRA PASTAN Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014

Communicated by Earl W. Sutherland, December 10, 1971

ABSTRACT Cyclic AMP-phosphodiesterase is present in various mouse fibroblasts. Contact-inhibited 3T3 cells contain two forms of the enzyme, one with a Km of 2.5 uIM and the second with a Km of 71 AM. As 3T3 cells grow to confluency and cAMP concentrations rise, the activity of the first enzyme increases, whereas that of the second is unchanged. A line of SV40-transformed 3T3 cells with low cAMP concentrations also has low levels of the cAMPphosphodiesterase with a K,,, of 2.5 1AM. Treatment of 3T3 and SV40-transformed 3T3 cells with dibutyryl cAMP and theophylline increases cAMP-phosphodiesterase accumulation. This accumulation is blocked by cycloheximide and actinomycin D. The newly formed enzyme resembles the higher affinity enzyme present in unstimulated cells, since it has a Km of 1.2-2.0 MM, and is stimulated by snake venom. In L cells in which cAMP concentrations are elevated by treatment with prostaglandin El, cAMP phosphodiesterase also accumulates. We conclude that intracellular concentrations of cAMP regulate the synthesis of cAMP-phosphodiesterase, and that cAMP functions as an inducer of the enzyme.

Previous studies indicated that cAMP regulates the growth, morphology, and adhesiveness of normal and transformed fibroblasts (1-4). Thus, an understanding of cAMP metabolism is important to elucidate the regulation of these properties in normal and cancer cells. cAMP concentrations are determined by the activity of the enzyme adenylate cyclase, which catalyzes the formation of cAMP from ATP, and cAMP phosphodiesterase, which degrades cAMP into 5'-AMP (for review, see ref. 5). cAMP can also leak from cells into the extracellular fluid. We have previously reported on the properties of adenylate cyclase in normal and transformed fibroblasts (4). Here, we report on the properties of the cAMP phosphodiesterase. We find that mouse 3T3 cells have high levels of cAMP phosphodiesterase activity; kinetic analysis, suggests the activity is due to two enzymes. However, one line of SV4Otransformed cells has very low activities of cAMP phosphodiesterase. The total activity of the enzyme is dependent on the intracellular concentration of cAMP, and this activity is increased by treatment of cells with But2cAMP (N6-2'-Odibutyryl cAMP) and theophylline. These findings suggest that cAMP functions as an inducer of the enzyme. MATERIALS AND METHODS

[3H]cAMP (14.3 Ci/mmole) was obtained from SchwarzMann and purified on Dowex AG 50W-X8 resin. cAMP was Abbreviations: But2cAMP,

N'-2'-O-dibutyryl cAMP. 459

also obtained from Schwarz-Mann, snake venom (Ophiophagus hannah) from Sigma Chemical Co., and Dowex 1-X2, 200-400 mesh from Bio-Rad. The resin was washed with 0.5 N NaOH, 0.5 N HCl, and deionized water, to a final pH of 5.0, before use. Separatipn and Assay of phosphodiesterase. The cells were grown under standard conditions (4). The cAMP phosphodiesterase was prepared by washing the monolayer four times with ice-cold phosphate-buffered saline (pH 7.4) and four times with ice-cold homogenizing medium containing 0.04 M Tris- HCl (pH 8.0)-10.9% sucrose (w/v). The cells were scraped from the culture dish with a rubber stopper, and homogenized in 0.5-1.0 ml of homogenizing medium, at a final concentration of protein of 2-5 mg/ml, in a glass Dounce homogenizer (tight pestle; 20 strokes). All steps were at 04°C. The resulting homogenate was used directly as the enzyme in the assay. Protein measurements were made by the method of Lowry et al. (6), with bovine serum albumin as the standard. The assay for cAMP phosphodiesterase was essentially the two-step assay of Butcher and Sutherland (7), with snake venom used in the second step as a source of 5'-nucleotidase to convert the product of cAMP phosphodiesterase (i.e., 5'-AMP) to adenosine and inorganic phosphate. In the first stage, the 0.18-ml reaction mixture contained 40 mM Tris HCl (pH 8), 10 mM MgCl2, 4 mM 2-mercaptoethanol, and an appropriate concentration of enzyme. After an equilibration period of 10 min at 300C, during which no loss of enzyme activity occurred, the reaction was initiated by the addition of 20 ul of a solution containing [3H]cAMP (200,000 cpm) and unlabeled cAMP. After 10 min at 300C, the reaction was stopped by heating at 1000C for 1 min. 50 ug of snake venom was added and the components were incubated for a further 10 min at 30°C to convert 5'-AMP to adenosine. This reaction was stopped by the addition of 1.0 ml of a 1:3 slurry of Bio-Rad AG 1X-2 resin; after they were mixed, the tubes were centrifuged 10 min at 4000 X g and the radioactivity in the supernatant was measured. When snake venom was used to stimulate the cAMP phosphodiesterase (8), 50 ,g was present in the first stage of the reaction. The blank for each experiment was prepared with heat-inactivated enzyme, or by omission of the enzyme from the incubation mixture. The blank value was subtracted from each experimental value. In all assays the rate of [3H]cAMP hydrolysis was linear for at least 15 min and directly proportional to protein concentration

Biochemistry: d'Armiento et al.

460

Proc. Nat. Acad. Sci. USA 69

(1972)

E.E E c

E

8 -

B -..0

C

.E

S.4

A

0O-,

4

~

~

~

~

~

E

-0~ ~~~~0

I-

2

.026 .040

.064 .OeO

.106

.160

I/cAMPxI M

FIG. 1A and B. Kinetic analysis by double-reciprocal plot of cAMP phosphodiesterase activity in 3T3-4 cells: --- -0, cAMP phosphodiesterase activity in the presence of snake venom: 0-*, cAMP phosphodiesterase activity in the absence of venom.

over a range of 5-250 j&g/ml. In each assay not more than 1520% of the substrate was consumed. The relative velocity was expressed in nmol of cAMP hydrolyzed per mg of protein per min at 30'C. All of the experiments were repeated from 3 to 5 times, and each experimental value represents the mean of two or three closely agreeing numbers. RESULTS Kinetic analysis of the cAMP phosphodiesterase activity in 3T34 cells by double-reciprocal plots is shown in Fig. 1A and B. We find that these cells, like many tissues of the rat (9),

TABLE 1. cAMP phosphodiesterase actinty during the growth of 3TS cells Log-phase growing cells

Cyclic AMP (pM) (ref. 3) Venom Vma.. (nmol per mg of protein per min)* V.., (nmol per mg of protein per mini)t

Contact-inhibited cells

1 1.6

32 4 53 II /cAWMPxIO

phosphodiesterase activity in 3T3-SV40 cells. 0-- 0, cAMP phosphodiesterase activity in the presence of venom: 0-0, cAMP phosphodiesterase activity in the absence of venom. appear to contain two enzymatic activities. One has an apparent Km of 2.5,uM and a Vm.x of 200 pmol per mg of protein per min (Fig. 1A). The increase of Vm.. due to the presence of snake venom in the first step of the reaction is from 200 to 300 pmol per mg protein per min. The second enzymatic activity has an apparent Km of 71 ,uM and a Vma. of 2500 pmol per mg protein per min (Fig. 1B). The presence of snake venom seems to slightly decrease the activity of this second enzyme. Table 1 shows the increase in the cAMP phosphodiesterase activity during growth of 3T34 cells. When these cells become contact-inhibited, the cAMP phosphodiesterase activity rises from 3- to 5-fold. This rise is present in both the low- and the high-Km enzymes, and occurs whether the enzyme is measured in the presence or in the absence of venom. The apparent Km

TABLE 2. cAMP phosphodiesterase activity in STS-4 cells treated with But2cAMP and theophylline

cAMP phosphodiesterase activity (pmol/mg of protein per min) Venom

11

3.3 -

+

-

0.1

0.2

0.2

+

_+

0.3

Control Butc2AMP and theophylline 1.4

0.6

* K., was 2,uM for all four conditions. t Kin was 71 uM for all four conditions.

2.2

8

"M FIG. 2. Kinetic analysis by double-reciprocal plot of cAMP

26 67

48 103

2.9

But2cAMP (1.2 mM) and theophylline (1 mM) were added for 40 hr to a 4-day-old culture. Enzyme activity was measured at 0.625 uM cAMP.

Proc. Nat. Acad. Sci. USA 69

Cyclic AMP-Phosphodiesterase in Fibroblasts

(1972)

values, however, seem to be unchanged. We found that when 3T3-4 cells become contact-inhibited, the intracellular concentration of cAMP rises by about 3 to 4-fold (3). Therefore, it seems likely that cAMP itself is the inducer of the cAMP phosphodiesterase. To test this possibility, we treated 3T3-4 cells with But2cAMP and theophylline. The results of these experiments are shown in Table 2. After 40 hr of treatment with 1.2 mM But2cAMP and 1mM theophylline, the cAMP phosphodiesterase activity is more than doubled. We have measured cAMP phosphodiesterase activity in various transformed cell lines. One of these lines, transformed by SV40 virus, has quite low concentrations of cAMP. A kinetic analysis of cAMP phosphodiesterase in a crude homogenate of this line (3T3-SV40) is shown in Fig. 2. The apparent Km is 2.5 ,.M and the Vm.. is quite low, about 20 pmol per mg of protein per min. The presence of snake venom increases the Vm.s to 160 pmol per mg per min, but does not change the apparent Km. The rate of accumulation of phosphodiesterase when 3T3SV40 cells are treated with But2cAMP and theophylline is shown in Fig. 3. After 46 hr of treatment, there is a 5-fold increase in cAMP phosphodiesterase activity assayed in the presence of venom and an 18-fold increase in activity assayed in its absence. But2cAMP alone is active, but much-less effective in raising cAMP phosphodiesterase activity. Cycloheximide and actinomycin D completely prevent the induction of cAMP phosphodiesterase (Table 3). This result indicates that the effect is dependent on RNA and protein synthesis, and is not simply due to an activation of cAMP phosphodiesterase by But2cAMP and/or theophylline. Kinetic analysis of the enzyme accumulated after 30 hr of treatment with But2cAMP and theophylline show that it has a Km of 1.5 AM both in the presence and absence of venom. The Vm.s is 70 pmol per mg per min without venom and 160 in its presence. Thus, the enzyme produced appears to resemble the low-Km enzyme present in 3T3 cells containing high concentrations of cAMP. As shown in Fig. IA and B, it is the low-Km enzyme whose activity is stimulated by the addition of venom; the activity of the high-Km enzyme is slightly inhibited. TABLE 3. Effect of actinomycin D and cycloheximide on the synthesis of cAMP phosphodiesterase of SV40 ceUs

Addition to the culture medium None Theophylline But2cAMP But2cAMP and theophylline ButscAMP and theophylline and cycloheximide But2cAMP and theophylline and actinomycin D

cAMP phosphodiesterase activity (pmol/mg of ptotein per min) Venom + 19 1 22 3 27 9 35 16 2

18

1

15

But2cAMP (1.2 mM), theophylline (1 mM), cycloheximide (10 mg/ml), and actinomycin D (0.5 jg/ml) were added for 10 hr to a 2-day-old culture. Enzyme activity was measured at 0.625 AM cAMP.

Beut2cAMP , ._

461

Control

10

20

5

40

30

HOURS OF INCUBATION

FIG. 3. Rate of cAMP phosphodiesterase accumulation in 3T3-SV40 cells treated with But2cAMP (1.2 mM) or But2cAMP (1.2 mM) and theophylline (1 mM). Enzyme activity was measured at 0.625 gM cAMP. Open circles, with venom; dosed circles, no venom added.

Bovine-brain cAMP phosphodiesterase is resolved into two components during purification by DEAE-chromatography (8). One component ("catalytic fraction") has low catalytic activity, whereas the other ("activator"), a heat-stable fraction, has no activity. The two components yield fully active enzyme when recombined. To examine whether we were stimulating the synthesis of an excess of activator, we mixed the enzyme prepared from control and treated cells. The activity obtained is the sum of the two separate activities, indicating that the increase in cAMP phosphodiesterase activity in cells treated with But2cAMP and theophylline is not due to synthesis of an activator (Table 4). This experiment also indicates the low cAMP phosphodiesterase activity in the extract of 3T3-SV40 cells does not result from the presence of an inhibitor, but from the presence of smaller amounts of cAMP phosphodiesterase. Treatment of the purified catalytic fraction from DEAEcellulose with snake venom or trypsin also increases its activity. -Although this activation is not observed with the unpurified brain enzyme (8-10), we found that either venom TABLE 4. cAMP phosphodiesterase activity in mixed extracts from But*cAMP and theophylline-treated, and from nontreated, SV40 cells cAMP phosphodiesterase activity (pmol/ mg-of protein per min) Venom A. SV40 control B. SV40 treated with But2cAMP and theophylline 1/ A + 1/2B Theoretical

l/2A~l/2Foud

1

19

16 8.5

27

8

25

35

The samples shown in Table 3 were mixed as indicated and assayed for activity at 0.625 uM cAMP.

462

Biochemistry: d'Armiento et al.

or trypsin markedly increased the activity of the unpurified fibroblast enzyme. To further examine whether we were preferentially stimulating the synthesis of one or both components of the fully active enzyme, we assayed all our samples in the presence or absence of snake venom. If we were stimulating synthesis of the catalytic subunit, we could expect there to be a preferential stimulation of the activity of the induced enzyme by snake venom, whereas if we were stimulating the synthesis of the activator, we might see less increase in activity by venom treatment. In general, the induced enzyme is somewhat less dependent on stimulation by venom, suggesting some excess of activator subunit.

L-929 cells contain an adenylate cyclase that is activated by prostaglandin E1 (PGE,) (4); there is a 6- to 10-fold increase in the cAMP content of the cells when they are incubated with this compound (Otten, Johnson, and Pastan, unpublished results). We find a 20% increase in cAMP phosphodiesterase activity as assayed in the presence of venom and a 30% increase in the enzyme assayed its absence after 24 hr of incubation of L-929 cells with 50 Mg/ml of PGEI. DISCUSSION

The results presented in this paper show that cAMP phosphodiesterase activity increases when contact-inhibited 3T3-4 cells become confluent, when 3T3-4 and 3T3-SV40 cells are treated with But2cAMP and theophylline, and when L-929 cells are treated with PGE,. These data suggest that the level of activity of cAMP phosphodiesterase is regulated by the intracellular concentration of the substrate, cAMP. Most of our studies on the synthesis of cAMP phosphodiesterase have been performed with a line of SV40-transformed 3T3 cells, which we previously observed contained low concentrations of cAMP (3). These cells also contain low levels of cAMP phosphodisterase. However, shortly after the addition of But2cAMP and theophylline, the enzyme begins to accumulate; this accumulation continues for about 48 hr, until a plateau value is achieved. Apparently, accumulation requires new RNA and protein synthesis, since treatment with either actinomycin D or cycloheximide prevents enzyme accumulation.

Proc. Nat. Acad. Sci. USA 69

(1972)

This increase in enzymatic activity could be due to an increase in synthesis of enzyme, a decrease in degradation of enzyme, or conversion from an inactive to an active form. The inhibition by actinomycin D and cycloheximide suggests that accumulation is due to an increase in the rate of synthesis. However, it is possible that the requirement for RNA and protein synthesis reflects a need to synthesize a component that converts inactive cAMP phosphodiesterase to an active form, or that prevents degradation of the enzyme. Most tissues of rat have at least two cyclic AMP phosphodiesterases, which can be identified kinetically and can be separated by gel filtration (9). We find in mouse 3T3 cells kinetic evidence for two phosphodiesterases. In the SV40transformed cells, there is kinetic evidence for only one cAMP phosphodiesterase, with a Km of about 2.5 MM, but the presence of a low level of a second enzyme cannot be excluded. After treatment of the SV40 cells with But2cAMP and theophylline, the enzyme activity approaches that of 3T3 cells, but there is still kinetic evidence for only one cAMP phosphodiesterase. NOTE ADDED IN PROOF

Manganiello and Vaughn have recently found that L cells treated with prostaglandin E1 also have an elevation of cAMP phosphodiesterase activity (Proc. Nat. Acad. Sci. USA 69, 269-273, 1972). 1. Johnson, G. S., Friedman, R. M. & Pastan, I. (1971) Proc. Nat. Acad. Sci. USA 68, 425-429. 2. Johnson, G. S. & Pastan, I. (1971) J. Nat. Cancer Inst. in press. 3. Otten, J., Johnson, G. S. & Pastan, I. (1971) Biochem. Biophys. Res. Commun. 44, 1192-1198. 4. Peery, C. V., Johnson, G. S. & Pastan, I. (1971) J. Biol. Chem. 246, 5785-5790. 5. Pastan, I. & Perlman, R. L. (1971) Nature New Biol. 229, 5-7. 6. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 7. Butcher, R. W. & Sutherland, E. W. (1962) J. Biol. Chem. 237, 1244-1250. 8. Cheung, W. Y. (1971) J. Biol. Chem. 246, 2859-2869. 9. Thompson, W. J. & Appleman, M. M. (1971) Biochemistry 10, 311-316. 10. Cheung, W. Y. (1970) Biochem. Biophys. Res. Commun. 38, 533-538.