Apr 10, 2015 - a protein termed G-substrate (see preceding paper) as the phosphate acceptor. ... In the preceding paper (21), we described the purification.
THEJOURNALOF BIOLOGICAL CHEMISTRY Val. 256. No. 7, Issue of April 10, pp. 3494-3500.1981 Prrnted in U.S.A.
A Specific Substratefrom Rabbit Cerebellum for Guanosine 3’5’Monophosphate-dependent ProteinKinase 11. KINETIC STUDIES ON ITS PHOSPHORYLATION BY GUANOSINE 3’5’-MONOPHOSPHATE-DEPENDENT PROTEIN KINASES* AND ADENOSINE 3’:5’-MONOPHOSPHATE-DEPENDENT (Received for publication,July 18, 1980, and in revised form, October 8, 1980)
Dana W. Aswad$ and Paul Greengard From the Depurtment of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510
Kinetic studies on the activity of purified cGMP-de- evolved from a common ancestral gene (6). pendent protein kinase and catalytic subunit of CAMPDespite the similarities between these two enzymes, it dependent protein kinase havebeen carried out using seems unlikely that they serve to phosphorylate the same a protein termed G-substrate (see preceding paper)as substrates in vivo. A number of studies suggest that CAMP the phosphate acceptor. Each enzyme catalyzed the and cGMPmediate distinct physiological processes(see Refs. phosphorylation of 2.0-2.1 mol of 32P/mol of G-sub- 13 and 14 for reviews). Moreover, there are a number of in strate, with phosphorylation occurring primarily at vitro studies in which selective phosphorylation of a specific threonine residues. When phosphorylationwas carried protein or proteins by cGMP has been demonstrated (10, 15out in the simultaneouspresence of t h e two enzymes, 20). In thepreceding paper (21), we described the purification to a value of and characterization of one such protein, termed G-substrate, the stoichiometry increased only slightly, 2.4, suggesting that both enzymes phosphorylated the which has been shown to undergo selective endogenous phossame two sites. Initial rate studies on the phosphoryphorylation in rabbitcerebellar cytosol in the presence of low lation of G-substrate by cGMP-dependent protein kiconcentrations of cGMP (18). nase yieldeda K , of 0.21 p~ and a V,, of 2.2 pmol/min/ In the present paper, we have carried out detailed kinetic mg. Similar studies with t h e CAMP-dependent protein kinase yielded a K,,, of 5.8 PM and a V,, of 2.3 pmol/ studies on the phosphorylation of G-substrate by purified ex- cGMP-dependent and CAMP-dependent protein kinases with min/mg.cGMP-dependentproteinkinasethus hibited a high degree of specificity towards this sub- the following goalsin mind. Previous studies of this and other strate which was apparently based on selective sub- apparently specific substrates for cGMP-dependent protein strate binding rather than catalytic efficacy. The activ-kinase have been carried out inrelatively crude systems such ity of cGMP-dependent protein kinase towards G-sub- as cytosol (18),membranes (15,17,19, 20), ribosomes (lo), strate w a s maximal at pH 7.5-8.0 a n d a Mg2’ concen- and intact lymphocytes (16). It thus seemed important to tration of 1-3 mM. Activity declined sharply at high demonstrate that selective phosphorylation by cGMP-deionic strength (>20 mM KCl). pendent protein kinase could occur in a purified system in order to rule out that theapparent selectivity was an artifact of the in vitro system used or that factors other than the A number of recent papers have focused on the similarities protein kinase were involved in determining substrate speciand/or differences between the properties of CAMP-dependficity. Given the ability to demonstrate this selectivity in a ent and cGMP-dependent protein kinases (1-11). The two enzymes are similar in several respects such as hydrodynamic purified system, it seemed desirable to determine its kinetic shape, amino acid content, and the ability to undergo auto- basis, i.e. whether it was primarily a K,,, or a Vmaxeffect. phosphorylation (4, 6). A number of proteins, including his- Finally, until now, the best substrate for cGMP-dependent tones (1,9), cardiac troponin-I (7, a), phosphorylase b kinase, protein kinase, and the substrate most often used t o characglycogen synthetase, pyruvate kinase, fructose 1,6-diphospha- terize this enzyme, was histone H2B. Histones are highly basic tase ( 4 , and hormone-sensitive lipase (5) can be phosphory- and therefore somewhat atypical proteins. Moreover, they lated in vitro by either enzyme. (However, the phosphoryla- occur predominantly in the nucleus, whereas cGMP-dependtion of these proteins, measured in vitro, is generally more ent protein kinase is primarily a cytosolic enzyme. Therefore, rapid with the CAMP-dependent protein kinase.) Studies on it seemed important to study the characteristics of this enzyme the phosphorylation of synthetic peptides also indicate a using G-substrate,a protein which is more typical in its degree of parallelism in substrate specificity (4,9, 12). Based physical properties and which may have more physiological on these similarities, it has been suggested that cGMP-de- relevance for the action of cGMP. pendent and CAMP-dependent protein kinases may have
EXPERIMENTAL PROCEDURES
Chemicals
* This work was supported by United States Public Health Service Grants DA-01627, MH-17387, and NS-08440 and a grant from the McKnight Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Recipient of a Muscular Dystrophy Association Postdoctoral Fellowship and a National Institutes of Health Postdoctoral Fellowship, 3-F32-NS-05304. Presentaddress, Department of Psychobiology, University of California, Irvine, California 92717.
*
Histone H2B was purchased from Worthingt,on Biochemicals and used without further treatment. It had a purity of 50-70% as judged by SDSI-gel electrophoresisin 15%polyacrylamide. All other chemicals were obtained from the sources indicatedin the preceding paper (21). I The abbreviations used are: SDS, sodium dodecyl sulfate; EGTA, ethylene glycol bis(Saminoethy1 ether)-N,N,N’,N’.-tetraacetic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid.
3494
Studies Kinetic Purification
of
on cGMP-dependent Protein Kinase
G-substrate
G-substrate was purified through stage6 from2,000 rabbit cerebella as described in the preceding paper (21).Approximately7 mgof protein was obtained with a purity of 62% as measured by densitometric scanning of a 12% polyacrylamide SDS-gel. All of the experiments reported here were carried out with a single preparation of G substrate (Fig. I), although similar results were obtained in early experiments with different preparations of G-substrate and protein kinases.
3495
3.0 ml of scintillation fluid. Data were corrected for blanks in which histone was deleted from the reaction mixture. Blanks varied from 25 pmol/paper.
Miscellaneous Methods [y-:12P]ATPwas synthesized according to Glynn and Chappel (23). Protein was determined by the method of Lowry el al. (24). SDS-gel electrophoresis,autoradiography,and identification of phosphorylated amino acid residues were described in the preceding paper (21). RESULTS
Purification
cGMP-dependent Protein Kinase and CAMPdependent Protein Kinase cGMP-dependent protein kinase was purified to homogeneity from bovine lung according to Walter et al. (11) and stored a t -15°C in 5 mM KPO, (pH 7.0), 0.5 mM EDTA, 30 mM P-mercaptoethanol, and 45% glycerol. The enzyme migrated as a single band of 81,000 daltons when subjected to SDS-gel electrophoresis in 10% polyacrylamide. The specific activity varied from 2.5-4.0 pmol/min/mg when assayed against histone H2B (0.4 mg/ml) at 2.5 mM Mg'+ as described below. Catalytic subunit of CAMP-dependent protein kinase was purified from bovine heart according to Beavoet al. (22) and stored at 4°C in 300 mM KPO, (pH 7.0). The enzyme migrated as a single band at 39,000 daltons when subjectedtoSDS-gelelectrophoresisin 10% polyacrylamide. The specific activity was 10.2 pmol/min/mg when assayed against histone H2B (0.4 mg/ml) a t 2.5 mM Mg" as described below. of
Comparison of Phosphorylation of G-substrate by cGMPdependent a n d cAMP-dependent ProteinKinases-Samples of G-substrate, having a purity of 62%,were phosphorylated by either the cGMP-dependent or the CAMP-dependent protein kinase, and then subjected to SDS-polyacrylamide gel electrophoresis, followed by autoradiography (Fig. 1). Even though several minor protein bands were present in the preparation of G-substrate used, essentially all of the :mPmigrated withthe 23,000-dalton-protein stainingband regardless of whichkinase was used. Fig. 2 showsthatboth enzymes catalyzed a maximal incorporation of 2.0-2.1 mol of '"P/mol of G-substrate. When phosphorylationwas carried out for 30 min in the simultaneous presence of both enzymes, the stoichiometry rose slightly to 2.4. This modest increaseindicated that both enzymes phosphorylated primarily the same two Enzyme Assays sites. The extra 0.3 mol of "'P incorporated may have been Initial Rates Using G-substrate as thePhosphateAcceptorAssays were carried out in 1.5-ml polyethylene microfuge tubes (Sar- due to slow phosphorylation of some additional site or sites staedt 690) in a reaction volume of 100 pI containing the following which became evidentonly at thehigher total kinase concencomponents: 10 mM Hepes (pH 7.5). 0.1 mM EGTA, 2.5 mM MgClr, tration used in the additivity experiment.
G-substrate (0.1-20 p ~ as, indicated, aftercorrection for purity), cGM1'-dependent protein kinase (0.02-0.05 pg/ml) or CAMP-dependent protein kinase catalytic subunit (0.03-0.30 pg/ml), and 100 p~ [y-'"I']ATP(200-600 cpm/pmol).Forassays of cGMP-dependent protein kinase, 1p~ cGMP wasincluded. Prior toassay, both enzymes were diluted from their storage solutions into10 mM Hepes (pH 7.5), 0.1 mM EGTA, 60 mM P-mercaptoethanol (for the cGMP-dependent protein kinase only), and 1 mg/ml of bovine serum albumin. Dilutedenzyme was added, in avolume of 10 pl, to the assay mixture a t a stage where allcomponents, except the ATP, were present. After adding the enzyme, the tubes were preincubated a t 30°C for 2 min. The reaction was then initiated by adding the ATP in a volume of 25 pl and incubation was carried out for 1-3 min a t 3OOC. The reaction was stopped by adding 0.8 ml of7% ice-cold trichloroacetic acid, followed by 20 pl of a 5 mg/ml solution of bovine serum albumin to provide carrier. All subsequent operations were carried out at 4°C. After5 min, the tubes werecentrifuged in a Beckman microfuge B for 1 minand the supernatantwas removed by aspiration. The pelletwasdissolved in 100 pl of 0.1 N NaOH and immediately reprecipitatedwith 0.8 ml of 7 8 trichloroacetic acid. The above procedures, from precipitation through redissolving in NaOH, were carried out a total of three times. After dissolving the final pellet in 0.1 N NaOH, 1.0 ml of Aquasol (New England Nuclear) was added andradioactivitywasdetermined by liquid scintillationcounting. Data were corrected for a blank, -1 pmol, in which substrate was omitted from the reaction mixture. Incubation times andenzyme concentrations were varied such that the incorporation of ."P never exceeded 0.16 mol of :"P/mol of G substrate. Under theseconditions, it was found that linear rateswere obtained over the entire range of substrate concentrations studied. Experimental points are, in most cases, an averageof duplicate assays. This trichloroacetic acid method of processing assays was used because it provided a 10-fold greater sensitivity than thephosphocellulose paper method described in the preceding paper (21). Initial Rates Using Histone H2B as the Phosphate AcceptorAssays were carried out in a reaction volume of 100 p1 containing the following components: 10 mM Hepes (pH 7.5), 0.1 mM EGTA, MgCI? (as indicated), 0.4 mg/ml of histone H2B, 0.1 pg/ml of cGMP-dependent protein kinase plus 1 p~ cGMP or0.1 pg/ml of CAMP-dependent protein kinase catalyticsubunit,and 100 p~ [y-'"PIATP (200-400 cpm/pmol). Reactions were carried outfor 5 min a t 30°C as described above. T o terminate the reaction, 10 pl of 1 N HrSO, were added and the tubeswere placed on ice. Samples of 25 pl were then spotted onto strips of phosphocellulose paper (1.2 X 2.5 cm). The papers were washed with 10 liters of 1% acetic acid, and individually counted in
FIG. 1. SDS-polyacrylamide gel electrophoresis of G-substrate after phosphorylation by cCMP-dependent and CAMPdependent protein kinases. Phosphorylation was carried out under standard conditions (see "Experimental Procedures") with 0.84 mg/ ml of G-substrate (628 pure), and0.005 mg/ml of cGMP-dependent protein kinase plus 1 p~ cGMP, or 0.05 mg/ml of CAMP-dependent protein kinase catalyticsubunit, in the presence of 100 p~ [ y '"PIATP. The reaction was carried out for 10 min a t 30°C and stopped by adding 50 pI of 3 X concentrated SDS-sample buffer containing 10% P-mercaptoethanol, followed by heating in boiling water for 2 min. Samples of 60 pl were subjected to SDS-gel electrophoresis in 12% polyacrylamide. The gel was stained with Coomassie blue, dried, and subjected to autoradiography. Protein-staining pattern of unphosphorylated G-substrate (Lane A ) and autoradiograms of G-substrate phosphorylated with cGMP-dependent protein kinase (Lane B ) and CAMP-dependent protein kinase (Lane C ) are shown.
a l
on cGMP-dependent Protein Kinase
Studies Kinetic
3496
with G-substrate as thephosphate acceptor) decreased significantly in a concentration-dependent manner. This phenomenon has been noted previously (25) with histone asthe phosphate acceptor and is presumed to be due to self-aggregation of the purified catalytic subunit. Control experiments with the cGMP-dependent protein kinase indicated that the specific activity towards G-substrate was independent of enzyme concentration up to 0.1 pg/ml, the highest concentration tested. Therefore, it would seem most appropriate to compare a V,,, of 2.2 pmol/min/mg in experiments 1 and 2 with the value of 2.3 pmol/min/mg for experiment 3 alone. The average value for the K , obtained with the cGMPdependent protein kinase, 0.21 p ~ is, 27-fold lower than the , with the CAMP-dependent average value, 5.8 p ~ obtained g o protein kinase. Although there was some variation in the K , 5 IO 15 20 25 30 values, there seems no doubt that the more rapid phosphorylation of G-substrate by cGMP-dependent than by CAMPMinutes FIG. 2. Time course and stoichiometry of phosphorylation dependent protein kinase, seen in Fig. 3, was due primarily to of G-substrate. Phosphorylation was carried out under standard differences in the K,,, values of the two enzymes. conditions with 1.2 mg/ml of G-substrate (62% pure), 200 pM [ y Further Characterization of cGMP-dependent Protein Ki"'PIATP, and either0.017 mg/ml of cGMP-dependent protein kinase nase Activity Using G-substratetheasPhosphate Acceptorplus 1 ~ L cGMP M ).-".( or 0.04 mg/ml of CAMP-dependentprotein kinase (A-A). Phosphorylation was carried out for the indicated Detailed characterization of purified cGMP-dependent protimes and the reaction was stopped by addition of 7% trichloroacetic tein kinase has previously been carried out with either hisacid. 32P incorporation into the trichloroacetic acid precipitate was tones (9,26-28) or synthetic peptides (9) as phosphate accepmeasured as described under "Experimental Procedures." tors. Histones are highly basic, somewhat atypical proteins and it is not known if they serve in vivo as substrates for In thepreceding paper, we showed that phosphorylation of cGMP-dependent protein kinase. Moreover, synthetic pepG-substrate by cGMP-dependent protein kinase occurs exclu- tides, by virtue of their small size, lack the secondary and sively onthreonine residues. To determine which amino acids tertiary structure of an intact protein which may have signifwere phosphorylated by the CAMP-dependent protein kinase, icant effects on enzymatic properties. Therefore, it seemed G-substrate (fully phosphorylated by the CAMP-dependent important todetermine optimal conditions for cGMP-dependprotein kinase) was hydrolyzed for 4 h in 6 N HC1 and then ent protein kinase activity using G-substrate as thephosphate subjected to electrophoresis at pH 1.9. Of the 32Precovered acceptor. Fig. 6 shows the effect of pH on the activity of cGMPfrom the electrophoresis sheet, 26% stayed at the origin (unhydrolyzed material), 8% ran as phosphoserine, 41% ran as dependent protein kinase. The enzyme activity exhibited a phosphothreonine, and 25% ran as P,. These results are com- fairly broad pH optimum with maximal activity occurring patible with the interpretation that CAMP-dependent protein between pH 7.5 and 8.0. Of interest in this experiment was the kinase phosphorylates primarily the same two sites on the G- finding that the enzyme activity measured with Tris buffer substrate asdoes the cGMP-dependent protein kinase. It may was somewhat lower than with Hepes buffer (see also Table be significant, however, that no phosphoserine was detected 11). To determine the effect of ionic strength, enzyme activity when the cGMP-dependent protein kinase was used(21).This suggests that, while the site specificity of the two enzymes does overlap to a large degree, there may be some additional r I 1 I I phosphorylation at sites which differ for the two enzymes. The relative rates of phosphorylation of G-substrate by cGMP-dependent and CAMP-dependent protein kinases were studied by initial rate kinetics. Fig. 3 shows one such experiI50 ment in whichphosphorylation was studied at a concentration of 0.5 p~ G-substrate. Under these conditions, G-substrate was phosphorylated approximately 9 times faster by the IO0 cGMP-dependent protein kinase than by the CAMP-dependent protein kinase. To determine the kinetic basis of this specificity, initial rates were studied over a wide range of G50 substrate concentration. Figs. 4 and 5 show typical Lineweaver-Burk plots for the cGMP-dependent and CAMP-dependent protein kinases, respectively. A summary of the ki0 netic constants obtained in a total of four separate experi0.25 0.50 0.75 1.0 1.25 ments is presented in Table I. Minutes The V,,, values obtained with both enzymes werestrikingly similar. The average value of 2.2 pmol/min/mg obtained with FIG. 3. Initial rate kinetics for phosphorylation of G-subthe cGMP-dependent protein kinase is close to the average strate. Phosphorylation assays were carried out under standard and either value of 1.9 pmol/min/mg obtained with the CAMP-depend- conditions with 0.5 PM G-substrate, 100 PM [Y-~~PIATP, p g / d of cGMP-dependent protein kinase plus 1 PM cGMP ent protein kinase. It should be noted, however, that theVmaX 0.025 (M or )0.025 p g / d of CAMP-dependent protein kinase values obtained in experiments 3 and 4 do not agree as closely (A-A), for the times indicated. The rates have been normalized to as those obtained in experiments 1 and 2. We subsequently equal concentrations of catalytic subunits using molecular weights of found that, at concentrations above 0.03 pg/ml, the specific 81,OOO and 39,000 for the cGMP-dependent and CAMP-dependent activity of the CAMP-dependent protein kinase (measured protein kinases, respectively. c
I
I
I
I
I
0
- 4
.
cGMP-dependent Kinetic Studies on Protein Kinase
3497
FIG. 4. Lineweaver-Burk plot for phosphorylationof G-substrate by cGMP-dependent protein kinase. Initial rate assays were carried out under standard conditions. The datashown here iare from experiment 2 in Table I.
I
I
I
1
I
I
was measured a t varying concentrations of KC1 added to the standard assay buffer (Fig. 7). Optimal activity was found a t 10 mM KCl, but this representedonly a 12%increase over the basal level. Further addition of KC1 caused a sharp decline in enzyme activity to 50% of maximum a t 80-100 mM KCl. A similar degree of inhibition was seen with other salts (see Table 11). The effect of Mg2+ on the activity of cGMP-dependent protein kinase was of particular interest. It had previously beenshown that optimal activity of this enzymetowards histone H2B was achieved at 50-75 mM Mg"+ (9, 28-30), an unphysiological concentration. As shown in Fig. 8, optimal activity towards G-substrate was achieved at 1-3 mM MgZ+. As the Mg2+ concentration was increased over the range of 10-100 mM, enzyme activity exhibited a steady decline (data not shown). No evidence for a second optimum a t high Mg2+ was observed. In the complete absence of Mg", the enzyme exhibited about 50% of optimal activity regardless of whether cGMP was presentor not. In the absence of cGMP,the addition of small amounts of Mg2+suppressed this basal activity, resulting in a stimulation ratio(+cGMP/-cGMP) of greater than 20-fold a t 1 mMMg". For comparison, the effect ofMg'+ onenzymeactivity towards histone H2Bis shown in Fig. 9. The results aresimilar to those reported by other laboratories (9, 28-30), i.e. a local optimum of activity occurred a t 1-10 mM Mg2+and a second broader optimumoccurred a t 50-75 mM Mg'. Stimulation by cGMP was low a t 1-10 mM Mg"+, due to a high basal activity I
I
6
7
I
1
8
9
2.0
I / Veloclty
0.8
0 0.1
0.2
0.3
0.4
PH
0.5
FIG. 6. pH dependence of cGMP-dependent protein kinase activity. Initial rates were measured with 1.0 PM G-substrate as the
I/ [G-Substrate] (pM")
FIG. 5. Lineweaver-Burk plot for phosphorylation of G-substrate by CAMP-dependent protein kinase. Initial rate assays were carried out under standard conditions. The datashown here are from experiment 3 in Table I.
phosphate acceptor under standardconditions, except that thebuffer was altered to consist of: A-A, 20 mM potassium 1,4 piperazinediethanesulfonic acid; w, 20 mM K-Hepes; H, 20 mM Tris-
c1.
TABLEI Summary of kinetic constants for phosphorylation of G-substrate by cGMP-dependent andCAMP-dependent protein kinases Values of K,,, and VmaXwere calculated by a computer program 7). In calculating the molar catalytic constant for cGMP-dependent which performed a least squares weighting on a linear transformation protein kinase, the average VmaX from experiments 1 and 2 was used. of the original data. The standard error within a given experiment is For CAMP-dependent protein kinase, only the Vmaxfrom experiment indicated. AU experiments were carried out under standard conditions 3 was used for reasons explained in the text. Subunit molecular using 2.5 mM MgC12 and 100 PM ATP. Molar catalytic constants weights of 81,000and 39,000 were used for cGMP-dependent protein (turnover numbers) were calculated for saturating concentrations of kinase and CAMP-dependent protein kinase, respectively. both G-substrate and ATP, and a KC1 concentration of 10 m~ (Fig. Kinase
Experiment
Kinase concentration
cGMP-dependent
I
CAMP-dependent
2 3 4
0.02 0.04 0.03 0.30
Range of G-substrate concentrations CLM
0.10-1.0
0.33-2.0 2.1-18.2 0.26-23.4
Molar catalytic constant
K", PM
pmol/min/mg
0.15 & 0.01 0.26 f 0.01 4.9 rt 0.2 6.6 & 0.4
2.38 k 0.01 2.03 -+ 0.04 2.30 -C 0.05 1.45 IC_ 0.04
min"
247 124
cGMP-dependent on Studies Kinetic Protein Kinase
3498
of the enzyme, but stimulation increased markedly in a progressive manner at thehigher Mg2+concentrations. With 1 p~ G-substrate as the phosphate acceptor and a Mg2+concentration of 2.5 mM, the K , of cGMP-dependent . protein kinase for ATP was found to be 22.0 & 1.1p ~ Glass and Krebs (9) reported the Kn2 for ATP to be 20-26 p M regardless of whether histone H2B or synthetic peptideswere used as substrate. Thus, the K , of cGMP-dependent protein kinase for ATP appears to be relatively independent of the phosphate acceptor used in the assay. Table I1 shows the effect of several compounds often encountered in the purification or assay of enzymes related to cyclic nucleotide metabolism. Inhibition of cGMP-dependent protein kinase activity was seen with 0.1 M Na-acetate and 0.1 M NaC1, and was similar to thatseen in Fig. 7 with 0.1 M KC1, indicating that the inhibition observed with all of these salts
2.5 2 .o
2.5
fl
0
5
0
15
IO
20
CMgC121 (mM) FIG. 8. Effect of MgC12 on cGMP-dependent protein kinase activity using 1.0 p~ G-substrate as the phosphate acceptor. Initial rates were measured under standard conditions, except that the MgC1, concentration was varied as indicated. Assays were carried and ) absence (M of) I PM cGMP. out in the presence (M
3
14
0
I
1
0.1
0.2
0.3
[KC11
(M)
I
0.4
0.5 I
0
20
1
1
I
40
60
80
1
100
FIG. 7. Effect of KC1 oncGMP-dependent protein kinase activity. Initial rates were measured with 1.0 p~ G-substrate as the phosphate acceptor under standard conditions, except that KC1 was added to the assay at theindicated concentration.
FIG. 9. Effect of MgCI2 on cGMP-dependent protein kinase activity using 25 PM histone H2B as the phosphate acceptor.
TABLEI1
Initial rates were measured under standard conditions, except that the MgC12concentration was varied as indicated. Assays were carried and absence (M of) 1 p~ cGMP. out in the presence (W)
Effect of some selected compounds on the activity of cGMPdependent protein kinase Enzyme assays were carried out under initial rate conditions, as described under “ExperimentalProcedures,” using 0.02pg/ml enzyme plus 1 p~ cGMP, 1.0 PM G-substrate, and 2.5 mM MgCL. Data are averages of duplicate assays. Control samples (no test compound added) had a specific activity of 2.06 pmol/min/mg. Care was taken to assure that the pH of the assay mixture was maintained at 7.5 when test compounds were added. IBMX, 3-isobutyl-1-methylxanthine; PMSF, phenylmethylsulfonyl fluoride. Compound tested
1. Sodium azide 2. Na-fluoride 3. Na-pyrophosphate 4. IBMX 5. PMSF 6. Sucrose 7. NaCl
8. Na-acetate 9. K-phosphate 10. Tris-chloride 11. Ammonium chloride
cGMP-dependent Concentration protein kinase activity ”%. of control 3 mM 104
10 mM
2 mM 1 mM 25 pM 0.02 M 0.10 M 0.02 M 0.10 M 0.02 M 0.10 M 0.01 M 0.02 M 0.02 M
106 69 89 99 102 96 105 50 96 55 89 83 91
hlqCI,] (mM)
was an ionic strength effect. At concentrations of 20 mM or less, several compounds caused significant inhibition. Na-pyrophosphate, a potent phosphatase inhibitor (31), produced 31%inhibition at a concentration of 2 m ~The . natureof this effect was not investigated further, but is probably due to chelation of Mg2+ which was present at 2.5 mM inthese experiments. Tris-C1 (pH 7.5), at 20 mM, produced a 17% inhibition, confirming the effect noted in Fig. 6. DISCUSSION
The results presented here show that G-substrate can be phosphorylated by either cGMP-dependentor CAMP-dependent protein kinase, but that a substantially greater degree of specificity is associated with phosphorylation by the cGMPdependent protein kinase. The results indicate that a high degree of substrate specificity is inherent in the mechanism by which the cGMP-dependent protein kinase molecule interacts with the G-substrate, and thus help to explain, in principle, how cGMP and CAMPcan carry out separatephysiological functions, even within the same cell. This conclusion is supported by the recent work of Glass and Krebs (9) who used synthetic peptides, modeled after the phosphorylation sites on histone H2B, to demonstrate that the two kinases have different specificity determinants which are dictated, at least
cGMP-dependent Studies Kinetic on Protein Kinase
3499
in part, by the primary sequence surrounding the phosphor- histoneH2B as thesubstrate. We have observedalarge variation in both specific activity and cGMP dependence of ylated residues. of commercial The substratepreference exhibited towards G-substrateby enzyme activity using different lots and sources the cGMP-dependent protein kinase relative to the CAMP- histone H2B as substrate. This variation is probably due to dependent protein kinasewas apparently due to selective varying degrees of contamination of H2B by other histone fractions. Walton and Gill (32) recently demonstrated that substrate binding rather than to a difference incatalytic efficacy. This wasreflectedin the finding that the K, of several fractions of histone and poly-L-arginine caused a rise in basalenzyme activity (-cGMP) andalso adecrease in total ) cGMP-dependent protein kinase for G-substrate (0.21p ~was lack of stimulation by cGMP 27-fold lower than the K, of the CAMP-dependent protein enzyme activity (+cGMP). Since (32,33) orincomplete , the catalytic constant was roughly the has also been associated with proteolysis kinase (5.8 p ~ )while same for the two enzymes. A similar,though less pronounced, removal of bound cyclic nucleotide (27, 28), these findings difference in substrate binding has been reported using histone emphasize the importance of using a well characterized subH2B as the phosphate acceptor (9). In that study, theK, for strate when evaluating the activityof cGMP-dependent prohistone H2B with cGMP-dependent protein kinase (1.5 pM) tein kinase. Tris-C1, a buffer commonly used in enzyme assays, exhibited was found to be 3.5 times lower than the K,,, with CAMP, the molar catalytic a weak but reproducible inhibition of cGMP-dependent prodependent protein kinase (5.3 p ~ )while tein kinase activity at 0.02 M . This inhibition may have been constants were virtually the same with thetwo enzymes. since NH&l also produced mild The only other protein for which kinetic constants with due to the amino cation, both enzymes have been determined under comparable con- inhibition, while other monovalent cations, such as Na' and A ditions is cardiac troponin-I (7). The K,,, values of the two K', producednoinhibition at the same concentration. enzymes for troponin-I were quite similar; 16.0 pM for the common feature of the overlapping substrate specificity of the cGMP-dependent protein kinase and 20.8 p~ for the CAMP- CAMP-dependent and cGMP-dependent proteinkinase is the basic residues onthe NH2dependent protein kinase. The preferential phosphorylation requirement for oneormore of troponin-I by CAMP-dependent protein kinase observed by terminal side of the phosphorylatable residue (9, 12; see also effect may thusbe due toweak these authors was due toa 12-fold greater V,, (&fold greater the following paper). The Tris molar catalytic constant) with the CAMP-dependent protein competitive inhibition at the lysine/arginine side chain reckinase thanwith the cGMP-dependent proteinkinase. It thus ognition site(s) on theenzyme. Although the function of G-substrate is not yet known, appears that substrate discriminationby these two enzymes several linesof evidence now suggest that it servesin viuo as may involve different mechanisms with different substrates. a substrate for cGMP-dependent protein kinase. First, endogIn their comparison of CAMP-dependent and cGMP-dependent protein kinases, Lincoln and Corbin (6) listed the enous phosphorylation of G-substrate in cerebellar cytosol is relative activity ratios of these two enzymes towards several selectively stimulated by low concentrations of cGMP (18). proteins generally believed to be in vivo substrates for the Second, G-substrate exhibitshighly selective binding in vitro CAMP-dependent protein kinase. These preference ratios, de- to the cGMP-dependent protein kinase (this study). Third, fined as molar specific activity of CAMP-dependent protein the rate of G-substrate phosphorylation measured in vitro with purified cGMP-dependent proteinkinase is rapidenough kinase/molar specific activity of cGMP-dependentprotein kinase, measured a t 0.1 p~ substrate, fell within the range of to be of physiological relevance (this study). Fourth, phos15-26 for four of the five proteins listed. (The one exception, phorylation of G-substrate is maximal at physiological levels troponin,had a preferenceratio of 2.) Using the kinetic of M$+ (this study). Fifth, G-substrate and cGMP-dependent protein kinase appear to be enriched in the same cell type, constants determined for G-substrate, we have calculated a cells of the cerebellum (34).Experiments reciprocal preference ratio (cGMP-dependent protein kinase/ namely the Purkinje with cerebellartissue slices are currentlyin progress to deterCAMP-dependent protein kinase) of38. Thus, the cGMPmine if the phosphorylation of G-substrate is regulated in dependentprotein kinase apparently exhibits at least the same level of substrate discrimination for its substrates as the intact cells in a cGMP-dependent manner. CAMP-dependent protein kinase does for its substrates. The high degree of substrate specificity exhibited bycGMPThe Mg'+ dependence of cGMP-dependent protein kinase dependent proteinkinase towards G-substrate suggested that activity differed markedly when using G-substrate and histone this substrate could be helpful in further elucidating those H2B as phosphate acceptors. We found, in confirmation of determinants of substrate specificity which distinguish the previous reports (28-30), that with histone H2B, cGMP-de- cGMP-dependent protein kinase from the CAMP-dependent pendent protein kinase activity was maximal at 60 mM, but protein kinase. In pursuit of this information, the amino acid that a lower optimum also occurred at 1-10 mM Mg2'. The sequences surrounding the two phosphorylation sites on GhighMg2+ requirement has been attributed to the special substrate have been determinedandarepresented in the properties of histones as substrates, rather than to an inherent following paper (35). requirement of the enzyme (9, 28). Our experiments with GAcknowledgments-We would like to thank Frank Wilson for substrate support this view, as only a single optimum at 1-3 mM Mg2' was observed. The Mg" dependence with G-sub- preparation of the CAMP-dependent protein kinase catalytic subunit and Drs. Doris J. Schlichter and Angus C. Nairn for helpful discusstrate was essentially identical to that previously observed sions and for aid in preparation of the cGMP-dependent protein a synthetic peptide cor- kinase used in this study. with Arg-Lys-Arg-Ser-Arg-Lys-Glu, responding to the cGMP-dependent protein kinase specific REFERENCES site on histone H2B (9). The inhibition of cGMP-dependent protein kinase activity towards G-substrateobserved a t high 1. Hashimoto, E., Takeda, M., Nishizuka, Y., Hamana, K., and Iwai, K. (1976) J.Biol. Chen. 251,6287-6293 Mgz+ concentrations was due, a t least in part, to an ionic 2. 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