Eur. J. Biochem. 150,85-88 (1985) 0 FEBS 1985

Diastereomers of adenosine 3’,5’-monothionophosphate (cAMP[S]) antagonize the activation of cGMP-dependent protein kinase Franz HOFMANN ’, Hans-Peter GENSHEIMER’, Wolfgang LANDGRAF ’, Roger HULLIN ’ and Bernd JASTORFF’

Pharmakologisches Institut der Universitat Heidelberg ‘ Fachbereich Biologic/ Chemie der Universitat Bremen

(Received March 8, 1985) - EJB 85 0245

cGMP-dependent protein kinase contains four cGMP-binding sites which are homologeous to the four CAMP-binding sites of CAMP-dependent protein kinase. The interaction of the diastereomers of adenosine 3’,5’- thionophosphate, (PS)-cAMP[S] and (PR)-cAMP[S], with cGMP-dependent protein kinase has been studied.

1. Autophosphorylation of cGMP-dependent protein kinase is stimulated by cAMP and (PS)-cAMP[S] with apparent KA values of 7 pM and 94 pM, respectively.

2. CAMP-stimulated autophosphorylation is inhibited competitively by (PR)-cAMP[S] with a Ki value of 15 pM.

3 . The phosphorylation of the peptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly) is stimulated by cGMP (approx. K A 1 pM) and cAMP (approx. K A 98 pM) but neither by the (PR) nor (PS) stereomer of cAMP[S].

4. (PR)-cAMP[S] and (PS)-cAMP[S] inhibit competitively CAMP- or cGMP-stimulated phosphorylation of the peptide substrate with Ki values of 52 pM and 73 pM, respectively.

5. (PS)-cAMP[S] stimulates the phosphorylation of the peptide substrate by an autophosphorylated enzyme. 6. Binding of [3H]cGMP to cGMP-dependent protein kinase is inhibited by (PS)-cAMP[S] and (PR)-cAMP[S]

with ICs0 values of 200 pM and 15 pM, respectively. 7. These results show that both diastereomers of cAMP[S] bind to cGMP-dependent protein kinase. (PR)-

cAMP[S] has properties of a pure antagonist whereas (PS)-cAMP[S] has properties of a partial agonist. The results provide further evidence that autophosphorylation of the enzyme affects the interaction between the cGMP-binding sites and the catalytic center of the enzyme by facilitating the activation of the phosphotransferase reaction.

cGMP has been implicated as an intracellular signal mole- cule which may regulate various cellular functions through the activation of a specific cGMP-dependent protein kinase. The nature and the function of the proteins phosphorylated by cGMP kinase have not been established in great detail. In contrast, the enzyme cGMP kinase has been purified and studied by several laboratories (for an overview see [l]). cGMP kinase is a dimer of apparently two identical polypeptide chains of 75 kDa which binds 2 mol cGMP/mol subunit [2-51. The two binding sites are distinct and selective for different analogues of cCMP and CAMP. The enzyme in- corporates up to 4 mol phosphate/mol subunit in an autocatalytic reaction [6 - 81 which is stimulated by the occupancy of only one of the two binding sites [3]. Autophosphorylation is stimulated to a greater extent by cAMP than cGMP and affects the properties of each binding site [5]. This suggested that the autophosphorylation reaction, observed so far only in in vitro experiments, may also be of some functional significance in vivo. To study this reaction in

Correspondence to F. Hofmann, Physiologische Chemie, Medizi- nische Fakultat, Universitat des Saarlandes, D-6650 Homburg/Saar, Federal Republic of Germany

Abbreviations. cGMP kinase, cGMP-dependent protein kinase; cAMP[S], adenosine 3’,5‘-monothionophosphate; ICs0, concentra- tion of compound which inhibits 50% of effect.

Enzymes. cGMP-dependent and CAMP-dependent protein kinases (EC 2.7.1.37).

more detail the availability of cyclic nucleotide derivatives which selectively inhibit the activation of the catalytic center of the enzyme would be of great value. Recently it has been shown [9 - 121 that the stereomers of cAMP[S] (Fig. 1) bind to the regulatory subunit of CAMP-dependent protein kinase. Whereas the (PS) stereomer of cAMP[S] activated the holoenzyme of CAMP-dependent protein kinase, no activa- tion of the holoenzyme was observed for the (PR) stereomer of cAMP[S]. Rather, (PR)-cAMP[S] inhibited the activation of the enzyme by CAMP. Consequently (PS)-cAMP[S] was classified as a partial agonist and (PR)-cAMP[S] as an antago- nist. It was expected that both stereomers of cAMP[S] bind to cGMP kinase, since the binding sites of cGMP kinase are homologeous to that of the cAMP kinase [13]. In this study we show that both stereomers of cAMP[S] bind to cGMP kinase, but that they affect differently the activation of the enzyme.

?JH7

OH OH

cAMP (PSI-cAMPlSI IPR)-cAMPISI

Fig. 1. Structures of the adenosine 3’,5‘-monothionophosphates

86

MATERIALS AND METHODS

Materials

Nucleotides were from Boehringer, Mannheim. The peptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly) was pur- chased from Sigma Chemicals. [3H]cGMP and 32P was from NEN Chemicals. All other chemicals were of the highest pu- rity available. Nitrocellulose filters (BA 85; 0.45 pm) were from Schleicher & Schull.

Assay

cGMP kinase activity was determined at 30°C in a total volume of 100 pl of 50 mM Mes, pH 6.9 containing 0.4 mM EGTA, 6 mM magnesium acetate, 10 mM NaC1, 0.7 mg/ml bovine serum albumin, 0.1 mg/ml peptide substrate, various concentrations of cyclic nucleotides and 50 ng pure enzyme. The reaction was started by the addition of 0.5 mM [Y-~~PIATP and was stopped after 3 min. The phosphorylated substrate was separated from [Y-~~P]ATP as described [14]. Autophosphorylation of the enzyme was carried out at 30 "C in a total volume of 100 pl of 40 mM Mes, pH 7.0, 0.3 mM EGTA, 150 mM NaC1, 0.1 mg/ml bovine serum albumin, 10 mM magnesium acetate, 0.8 pg cGMP kinase, and various concentrations of cyclic nucleotides. The reaction was in- itiated by the addition of 0.1 mM [Y-~~PIATP and was terminated after 14 min by spotting 80 pl onto a filter paper 181.

Miscellaneous methods

cGMP kinase was purified as described [5, 71, [Y-~~PIATP was prepared as described [15]. Binding of [3H]cGMP was carried out as in [5]. cAMP[S] stereomers were synthesized and purified as described [16, 171. The (PS) stereomer of cAMP[S] was also purchased from Boehringer, Mannheim. [3H]cGMP was always rechromatographed before use. The concentrations of nucleotides were calculated from their known absorption coefficients at 260 nm.

RESULTS

Autophosphorylation of cGMP-dependent protein kinase

cGMP kinase incorporates up to 4 mol phosphate/mol by an intramolecular reaction which is stimulated by cAMP and certain analogues of CAMP and cGMP. The autophos- phorylation proceeds at a high rate if one of the two cGMP- binding sites is occupied [3] and is inhibited if both binding sites are filled. (PS)-cAMP[S] and cAMP stimulated the autophosphorylation with apparent KA values of 94 pM and 7.3 pM, respectively (Fig. 2). The phosphorylation rate in the presence of (PS)-cAMP[S], however, was slower than that observed in the presence of CAMP. During a 17-min incuba- tion period, (PS)-cAMP[S] and cAMP stimulated the incor- poration of 1 and 2 mol phosphate/mol subunit, respectively. Prolongation of the incubation time to 180min led to the incorporation of 2 - 2.4 mol phosphate/mol subunit in the presence of each compound. This result suggests that (PS)- cAMP[S] acts similarly to cAMP but apparently activates cGMP kinase to a lesser degree than CAMP. The slower rate may be due to inhibition of the binding of ATP to the enzyme at the high concentration of (PS)-cAMP[S] used in these ex- periments (see below).

In contrast to (PS)-cAMP[S], (PR)-cAMP[S] did not stimulate the autophosphorylation reaction (Fig. 2) but in- hibited competitively CAMP-stimulated autophosphorylation with a Ki value of 15 pM (Fig. 2 and 3) suggesting that it binds to the same site(s) as cAMP but is not able to activate the autocatalytic phosphorylation reaction.

Binding of compounds to cGMP-dependent protein kinase

The conclusions given above are supported by following experiments. The binding of [3H]cGMP to cGMP kinase was inhibited by increasing concentrations of CAMP, (PS)- cAMP[S] (Fig. 4) and (PR)-cAMP[S] with apparent IC50 values of 10-20 pM, 200-300 pM and 10- 12 pM, re- spectively. The latter values were obtained in the presence of 10 mM magnesium acetate and 0.2 mM ATP and are close to the KA and Ki values obtained in the phosphorylation assays. The similarity of these values suggests that the IC50 values are similar or identical to the dissociation constants (Kd) for these compounds. Displacement of [3H]cGMP from site 1 and 2 under equilibrium conditions by (PS)- and (PR)- cAMP[S] suggested a similar affinity of these compounds for site 1 and 2. In addition, no striking selectivity for either binding site was observed when, after equilibration of cGMP kinase with [3H]cGMP in the absence and presence of (PR)- and (PS)-cAMP[S], the dissociation of [3H]cGMP from the enzyme was followed after the addition of 1 mM unlabelled cGMP (data not shown). Such experiments allow the determi- nation of a selective binding of a compound to one site, since [3H]cGMP dissociates with extremely different rates from binding sites 1 and 2 [2 - 51. Therefore these results support the previous conclusion [12] that the (PS) stereomer of cAMP[S] is an agonist and the (PR) stereomer is an antagonist of CAMP. However, these experiments do not prove that the stereomers of cAMP[S] affect in a similar manner a cGMP-stimulated function of the cGMP kinase, namely its phosphotransferase activity towards an exogeneous substrate.

Phosphorylation of the peptide substrate

The phosphorylation of the peptide substrate was stimulated by cGMP and cAMP but neither by (PR)-cAMP[S] nor (PS)-cAMP[S] even at millimolar concentrations (Fig. 2). Double-reciprocal replots of these data yielded K, values of 1.2 pM (cGMP) and 100 pM (CAMP) and Vvalues of 11 pmol and 6.3 pmol phosphate transferred min- ' (mg enzyme)-'. In contrast to the data on autophosphorylation, both stereomers of cAMP[S] antagonize the cGMP- or CAMP- stimulated peptide phosphorylation. (PR)-cAMP[S] competi- tively inhibited cGMP-stimulated peptide phosphorylation with a Ki of 52.5 pM (Fig. 3). (PS)-cAMP[S] competitively inhibited CAMP-stimulated peptide phosphorylation (range of Ki values obtained in several experiments 73 - 250 pM) and in an apparently mixed type or uncompetitive manner cGMP- stimulated enzyme activity (Fig. 5). This difference in the kinetics of the inhibition of CAMP- and cGMP-stimulated activities is surprising, since all available evidence suggests that cGMP and cAMP activate cGMP kinase by binding to identical sites. The discrepancy is overcome if one takes into account that at high concentrations cAMP but not cGMP binds to the ATP site (Ki 1 mM) and thereby prevents the phosphotransferase activity of the enzyme [3]. It is therefore possible that, at the high concentration necessary to inhibit the cGMP-stimulated phosphotransferase activity, (PS)- cAMP[S] binds not only to the two cGMP-binding sites but

1.5 - c C 3 n : 1.0

E - \

0.5 E

a -

0

0 20 5c

3 0 75 150 (PR)-cAMPISI (PM)

87

I

Fig. 2. Stimulation of the autophosphorylation and the peptide phosphorylation by the stereomers of cAMP[S]. Autophosphorylation ( A ) and phosphorylation of peptide substrate by native (B) and autophosphorylated (C) cGMP kinase was determined as described under Methods. The autophosphorylated enzyme contained a total of 3.9 mol phosphate/mol subunit. S and R refer to (PS)-cAMP[S] and (PR)-cAMP[S] respectively

Fig. 3. Dixon plots of the kinetics of the inhibitory effect of (PR-cAMP[S]. Inhibition o f CAMP-stimulated autophosphorylation (A) and of cGMP-stimulated peptide phosphorylation (B) was measured as described in Methods and in the legend to Fig. 2

1 0

0 s 0 5

0 0 -6 -L -2

log IcNMPl IM)

Fig. 4. Displacement of [ 3H]cGMP by CAMP and (PSJ-CAMPIS]. Native cGMP kinase (0.2 pg) was incubated for 60 min at 2°C in the presence of 0.1 FM [’HIcGMP and the concentration of compounds indicated. Thereafter Mg . ATP in a final concentration of 10 mM and 0.2 mM was added and the reaction was quenched after 2 min by the addition of stopping solution [3]. For further details see Methods and [ 5 ]

also to the ATP site. As shown in Fig. 5, (PS)-cAMP[S] in- hibited competitively ATP binding with a Ki of 1 mM. This finding suggests therefore that the unclear inhibition kinetics of the cGMP-stimulated enzyme activity by (PS)-cAMP[S] is caused by the simultaneous binding of the compound to the

cGMP-binding sites and the ATP-binding site. From different experiments with cAMP and ATP it can be calculated that the affinity of (PS)-cAMP[S] for the cGMP-binding sites was only 5 - 10-fold higher than that for the ATP site. A similar kinetic phenomenon as observed in the presence of cGMP is not evident from the experiments carried out with CAMP. This is due to the fact that cAMP itself interferes with ATP binding at higher concentrations and masks therefore the interaction of (PS)-cAMP[S] with this site. In summary both stereomers of cAMP[S] are competitive antagonists of cGMP and CAMP, if an exogeneous substrate is present.

This conclusion is not necessarily in contradiction to the interpretation of the data obtained by measurement of the autophosphorylation reaction. It is known that binding of a protein substrate affects the activity and the extent of stimula- tion of cGMP kinase [4, 181. Binding of the peptide substrate may therefore affect the interaction of the cGMP-binding sites with the catalytic center in such a way that (PS)-cAMP[S] is no longer able to activate the phosphotransferase reaction. Furthermore, other factors which alter the properties of the cGMP-binding sites may influence the stimulatory effect of (PS)-cAMP[S] on the catalytic activity of cGMP kinase. One known factor is the autophosphorylation of cGMP kinase. This modification abolishes positive cooperative binding of cGMP [5] and lowers the concentration of cAMP required to activate the phosphotransferase activity of the enzyme [6, 71. In contrast to the native enzyme, (PS)-cAMP[S] as well as

88

0 100 500 1000

B

0.005 -

0 400 500 1000 I . . I . I

I I . . # J 500 2000

(PSI-CAMPIS] (VMI

Fig. 5. Dixon plots of the kinetics of the inhibitory eflect of ( P S ) - cAMP(SJ. The inhibition of CAMP-stimulated (A) or cGMP- stimulated (B) peptide phosphorylation was determined as described in Methods. The competition with ATP (C) was determined in the presence of 10 pM cGMP

cGMP and cAMP stimulated the phosphorylation of the peptide substrate by an autophosphorylated enzyme (Fig. 2). A double-reciprocal replot yielded KA values of 0.5 yM, 25 pM and 300 pM for cGMP, cAMP and (PS)-cAMP[S]. Maximal velocity was identical whether cGMP or cAMP was present and amounted to 8.3 pmol phosphate transferred min- ’ (mg enzyme)- ’. (PS)-cAMP[S] stimulated the enzyme only to an activity of 2.1 ymol min-’ mg-’. This low velocity rate is probably not only due to an inefficient or incomplete activation of the catalytic center of the enzyme but also due to the inhibitory effect of (PS)-cAMP[S] on the ATP-binding site.

DISCUSSION

Previously it has been suggested that the (PS) stereomer of cAMP[S] interacts slightly differently with type I and type I1 CAMP-dependent protein kinase [12]. (PS)-cAMP[S] has been classified as an agonist of cAMP when the type I holoenzyme was used and as a partial agonist in the case of the type I1 enzyme. The (PI?)-stereomer of cAMP[S] was identified as an apparently pure antagonist with both types of enzyme. In this study (PR)-cAMP[S] acted always as a pure antagonist of cGMP or cAMP irrespective of the different functions of cGMP-dependent protein kinase studied. In contrast, (PS)- cAMP[S] showed characteristics of a partial agonist which stimulated cGMP-dependent protein kinase activity if the

enzyme was used. Under regular assay conditions, i.e. in the presence of ATP and a peptide substrate, only the antagonistic properties of (PS)-cAMP[S] could be demonstrated. These results clearly show that the interaction of (PS)-cAMP[S] with cGMP-dependent protein kinase is not identical to its interaction with CAMP-dependent protein kinase. Its effects on cGMP kinase resemble to a certain extent those reported for the type I1 enzyme of CAMP kinase which is activated only to 85% by (PS)-cAMP[S] [12]. The distinct interaction with cGMP and cAMP kinases is probably based on a different activation mechanism of both enzymes and not on differences in the structure of the binding sites, which show sequence homology of about 35% between both enzymes [13]. The activation of cAMP kinase is caused by dissociation of the holoenzyme into the regulatory and the free catalytic subunits, whereas cGMP kinase is activated without dissociation of the holoenzyme. The different efficiency of (PS)-cAMP[S] to activate cGMP kinase clearly demonstrate that the interaction between the regulatory, cGMP-binding domain and the cata- lytic center is affected (a) by the occupation of the cGMP- binding sites, (b) by the occupation of the two substrate sites by Mg.ATP and the protein substrate and (c) by the phosphorylation state of the enzyme itself. These consider- ations provide further evidence that the phosphorylation status of the enzyme might be important for the activation of cGMP-dependent protein kinase.

The work was supported by a grant from Deutsche Forschungsge- meinschaft and Fonds der chemischen Industrie.

REFERENCES 1. Lincoln, T. M. & Corbin, J. D. (1983) Adv. Cyclic Nucleotide Res.

2. Mackenzie, C. N. 111 (1982) J. Biol. Chem. 257, 5589-5593. 3. Hofmann, F., Gensheimer, H. P. & Goebel, C. (1983) FEBS Lett.

4. Corbin, J. D. & Dmkeland, S. 0. (1983) J . Biol. Chem. 258,

5. Hofmann, F., Gensheimer, H. P. & Goebel, C. (1985) Eur. J .

6. Foster, J. L., Guttmann, J. & Rosen, 0. M. (1981) J . Biol. Chem.

7. Hofmann, F. & Flockerzi, V. (1983) Eur. J . Biochem. 130, 599-

8. Hofmann, F. & Gensheimer, H. P. (1983) FEBS Lett. 151, 71 -

9. Eckstein, F., Simonson, L. P. & Bar, H. P. (1974) Biochemistry

10. De Wit, R. J. W., Hoppe, J., Stec, W. J., Baraniak, J. & Jastorff, B. (1982) Eur. J . Biochem. 122, 95-99.

11. O’Brian, C. A,, Roczniak, S. O., Bramson, H. N., Baraniak, J., Stec, W. J. &Kaiser, E. T. (1982) Biochemistry 21,4371 -4376.

12. Van Haastert, P. J. M., Van Driel, R., Jastorff, B., Baraniak, J., Stec, W. J. & De Wit, R. J. W. (1984) J. Biol. Chem. 259,

13. Takio, K., Wach, R. D., Smith, S. B., Krebs, E. G., Walsh, K.

14. Glass, D. B., Masarachia, R. A,, Feramisco, J. R. & Kemp, B.

15. Johnson, R. A. & Walseth, T. F. (1979) Adv. Cyclic Nucleotide

16. Baraniak, J., Kinase, R. W., Lesiak, K. & Stec, W. J. (1979) J .

17. Van Haastert, P. J. M. & Kien, E. (1983) J. Bid. Chem. 258,

18. Walton, G. M. & Gill, G. N. (1980) J. Biol. Chem. 255, 1603-

15, 139-182.

164, 350-354.

11 391 - 11 397.

Biochem. 147, 361 -365.

256, 5029 - 5036.

603.

75.

13, 3806-3810.

10020 - 10024.

A. & Titani, K. (1984) J . Bid. Chem..23, 4207-4218.

E. (1978) Anal. Biochem. 87, 566-5575,

Res. 10, 135-167.

Chem. Soc. Chem. Commun. 940 - 942.

9636 - 9642.

autophosphorylation reaction or an autophosphorylated 1609.