Proc. Nat. Acad. Sci. USAVol. 71, No. 10, pp. 4037-4041, October 1974

Guanosine 3 ':5'-Monophosphate-Dependcnt Protein Kinases in Mammalian Tissues(cyclic AMP/protein kinase modulator)

J. F. KUO

Department of Pharmacology, Emory University, Atlanta, Georgia 30322

Communicated by I. C. Gunsalus, August 1, 1974

ABSTRACT The mammalian protein kinase activitystimulated preferentially by low concentrations of guano-sine 3':5'-monophosphate (cyclic GMP), but not by adeno-sine 3' :5'-monophosphate (cyclic AMP), was readilyassayed in a modified incubation system that contained aneutral phosphate buffer, protein kinase modulator, andarginine-rich histone. Cyclic GMP-dependent proteinkinase activity assayed under these conditions was abouttwo to three orders of magnitude higher than that pre-viously detected. The enzyme activity occurred in allguinea pig and rat tissues (lung, heart, aorta, brain,liver, ileum, adipose, and pancreatic islets) examined. Theactivity can be separated from the cyclic AMP-dependentprotein kinase activity, also present in the same tissues,by means of either Sephadex G-200 gel filtration or am-monium sulfate fractionation. The cyclic GMP-dependentenzyme preparations had Ka values for cyclic GMP rangingfrom 0.03 to 0.12 pAM, compared to the Ka values for cyclicAMP ranging from 0.6 to 3.8 puM. The presence of phos-phate and protein kinase modulator was essential formaximal cyclic GMP-dependent enzyme activity.The occurrence of high levels of cyclic GMP-dependent

protein kinase activity in mammalian tissues clearlysuggests that it may serve as a "target" enzyme for cyclicGMP, mediating many biological effects of this cyclicnucleotide.

After the discovery of cyclic AMP-dependent protein kinasesin mammalian muscle (1), liver (2), as well as in many othermammalian tissues (3, 4), this class of protein kinase wassubsequently found in all tissues from a wide variety of speciesand phyla throughout the animal kingdom (5, 6). Anotherclass of protein kinases specifically activated by cyclic GMPwas later found to be present in every one of many arthropodtissues examined (5, 6). Only trace amounts of cyclic GMP-dependent protein kinase activity, however, were detectedin mammalian tissues (5, 7). It has been proposed (4-6),as an unifying hypothesis, that many, if not all, of the actionsof cyclic AMP and cyclic GMP may be mediated throughthe regulation of cyclic AMP-dependent and cyclic GMP-dependent protein kinases, respectively.In most mammalian tissues and cells, the content of cyclic

GMP is about 3-10% of that of cyclic AMP (8-12). In lungand cerebellum, however, the cyclic GMP content is as highas 30-70% of that of cyclic AMP (8, 11, 12). Moreover, forexample, the cyclic GMP content in lung slices stimulated bycholinergic agonists could reach levels that were comparableto or even higher than those of cyclic AMP (13). Because ofthe existence of cyclic GMP, and particularly its increase inspecific mammalian tissues in response to stimulation by

cholinergic agonists (10-14), as well as by insulin (15) and byestrogens (16), it would seem that a "target" enzyme forcyclic GMP is likely to be present in mammalian tissues atlevels that could be considered functionally significant. Inthis report I present experimental evidence that a cyclic GMP-dependent protein kinase activity does indeed occur at highlevels in all guinea pig and rat tissues examined. Previousfailures in detecting the mammalian cyclic GMP-dependentprotein kinases in significant quantities were largely due tothe fact that the assay conditions used earlier were not suit-able for the mammalian enzymes, even though they wereshown to be optimal for arthropod cyclic GMP-dependentenzymes.

EXPERIMENTAL PROCEDURE

Materials. ['y-32P]ATP was purchased from New EnglandNuclear Corp. Cyclic GMP, 8-bromo cyclic GMP, and cyclicAMP were obtained from Boehringer Mannheim; mixedhistone (Type II) and arginine-rich histone (Type IV) werefrom Sigma.

Methods. Crude protein kinase modulator from guineapig lung and other tissues was prepared through the steps ofboiling of the extracts and precipitation of the factor from5% trichloroacetic acid (17-19).

Cyclic GMP-dependent protein kinase activity was pre-pared from guinea pig lung and was separated from the cyclicAMP-dependent enzyme activity also present in the sametissue by two different methods as follows.Method I: Fresh lungs (15 g) collected from two male guinea

pigs (each weighing about 560 g) were homogenized in 6 ml of50 mM potassium phosphate buffer (pH 7.0) at 40 for 10 secin an Omni-Mixer (Sorvall) at maximum speed. The homog-enate was centrifuged for 15 min at 30,000 X g. The super-natant fluid (crude extract) was filtered through two layers ofglass wool to remove fat and was then loaded onto a SephadexG-200 column (3.2 X 50.0 cm); the gel had been previouslyequilibrated with 50 mM phosphate buffer (pH 7.0). Theprotein kinase activity was eluted from the column with thesame buffer; the flow rate was 0.5 ml/min, and the fractionsize was 4 ml.Each fraction was assayed for enzyme activity in the

absence and presence of 0.3 uM concentration of either cyclicGMP or cyclic AMP. Three peaks of protein kinase activitywere obtained (Fig. 1). The material in peaks 1 and 3 wasused as the sources of cyclic AMP-dependent kinase activity,while that of peak 2 was used as the source of cyclic GMP-dependent enzyme activity. The enzyme fraction from othertissues of guinea pigs and rats were similarly prepared.

4037

Abbreviations: Cyclic GMP, guanosine 3': 5'-monophosphate;cyclic AMP, adenosine 3': 5'-monophosphate.

Proc. Nat. Acad. Sci. USA 71 (1974)

30 ~=A Basal1 0.3jjM cAMP2.5p- 03jjM cGMP -25 >

0/

Peak 2 2OW

1.5- 1 /1W~~~~0 1~~~~~~~~~1

Z 1.0 Peak3 10

0.5-

0 0.

0 15 20 25 30 35 40 45 50 55

FRACTION NUMBER

FIG. 1. Chromatography of crude extracts of guinea pig lungon Sephadex G-200 column. The enzymatic activity in 0.02-mlaliquots was assayed under the standard incubation conditions as

described in the text. When present, the concentration of cyclicnucleotides was 0.3 MM. Peak 1 consisted of fractions 20-27; peak2, fractions 28-34; and peak 3, fractions 44-50.

Method II: Two fresh guinea pig lungs were homogenizedin 20 ml of 50 mM potassium phosphate buffer (pH 7.0). Tothe crude extract was added solid ammonium sulfate to a

concentration corresponding to 20 g/100 ml of the enzyme

solution. The precipitate thus formed was recovered bycentrifugation and dissolved in 4 ml of the extraction buffer.The resultant solution was dialyzed overnight against thesame buffer. This preparation, designated fraction 1, was usedas the source of cyclic AMP-dependent enzyme activity.Next, additional ammonium sulfate was added to the super-

natant to a concentration corresponding to 25 g/100 ml of theoriginal crude extract. The precipitate formed was similarlyrecovered, dissolved, and dialyzed. This preparation was

designated fraction 2, and was used as the source of cyclicGMP-dependent enzyme activity.The standard assay system for protein kinase activity,

designed especially for detecting cyclic GMP-dependentactivity, contained, in a final volume of 0.02 ml, potassiumphosphate buffer (pH 7.0), 10 Mmoles; theophylline, 0.5Mmole; arginine-rich histone, 40 Mg; magnesium chloride, 2Mmoles; [y-32P]ATP, 1 nmole, containing about 1.2 X 106cpm; crude protein kinase modulator (guinea pig lung), 60,Mg; and appropriate amounts of protein kinase fractions andcyclic nucleotides as indicated. The reaction was carried outfor 10 min at 300. The [32P]histone formed was recoveredaccording to the procedure reported elsewhere (5). One unitof enzyme activity is defined as that amount of enzyme thattransferred 1 pmole of 82p from ['y-32P]ATP to recoveredhistone in 10 min at 300 under the assay conditions.

RESULTS

Three distinct peaks of protein kinase activity were obtainedwhen the crude extracts of guinea pig lung were chroma-tographed on a Sephadex G-200 column (Fig. 1). Peaks 1 and3 contained a protein kinase activity that was stimulated bycyclic AMP, whereas cyclic GMP at the same concentration(0.3 MM) had little effect. The peak 2 enzyme, on the otherhand, was stimulated to a much greater extent by 0.3 ,uMcyclic GMP than by the same concentration of cyclic AMP,indicating that a cyclic GMP-dependent protein kinaseactivity originally present in the tissue extracts was enrichedin this peak. The cyclic GMP-stimulated protein kinaseactivity, although lower than that stimulated by cyclic AMP,was readily detectable in the crude extracts (Table 1). Separa-tion of cyclic GMP-dependent enzyme activity from the

TABLE 1. Preparation of cyclic GMP-dependent and cyclicAMP-dependent protein kinase activity from guinea pig lungs

Protein kinaseactivity Ka values

Total (units X 10-3) (AM)pro- Cy- Cy- Cy- Cy-tein cylic clic clic clic

Fraction (mg) None GMP AMP GMP AMP

Method ICrude extracts 295 67 121 150Sephadex G-200Peak 1 90 16 18 40 2.1 0.06Peak 2 36 5 23 10 0.03 0.6Peak 3 25 2 3 8 1.8 0.08

Method IICrude extracts 320 62 122 158(NH4)2S04 stepFraction 1 110 21 30 45 3.6 0.09Fraction 2 45 13 32 20 0.05 0.9

Two fresh guinea pig lungs (about 15 g) were used as thestarting material for enzyme preparation by either Method I orMethod II. When present, the concentration of the cyclicnucleotides was 0.3 ,uM. Activity was assayed under the standardconditions, as described in the text and the legend of Fig. 1.

cyclic AMP-dependent enzyme by means of Sephadex G-200chromatography (Method I), as shown in Fig. 1, is sum-marized in Table 1. The cyclic GMP-dependent enzymeactivity could also be largely separated from the cyclic AMP-dependent enzyme by a simple ammonium sulfate fractiona-tion step (Method II). Thus, fraction 1 consisted of an activitypreferentially stimulated by cyclic AMP, whereas fraction2 consisted of an activity preferentially stimulated by cyclicGMP (Table 1).The histone phosphorylation catalyzed by peak 2 enzyme

proceeded linearly, as a function of incubation time, for atleast 10 min (Fig. 2). The enzyme activity was stimulated, in

20

a 0 ControlX A Cyclic AMP /0a 0 Cyclic GMPo A 8-Br Cyclic GMPS Aa-

0~~~~~~

E 10 0

Aa) 0~~~~~

20~~~~~~a_ ~~~00 .

0 5 10Incubation time (min)

FIG. 2. Cyclic GMP-dependent protein kinase activity as afunction of incubation time. An aliquot (0.02 ml, containing 20/g of protein) of peak 2 enzyme (Fig. 1) was incubated in theabsence and presence of 0.3 M&M concentration of cyclic GMP,8-bromo cyclic GMP, or cyclic AMP. Assay conditions were asdescribed in the text.

4038 Biochemistry: Kuo

Mammalian Cyclic GMP-Dependent Protein Rinases 4039

(Cyclic nucleotide) x 1 6 M

FIG. 3. Double-reciprocal plots of cyclic GMP-dependentprotein kinase activity as a function of cyclic nucleotide concen-

trations. Aliquots (0.02 ml, containing 20kAg of protein) of peak 2enzyme were used as the source of cyclic GMP-dependent pro-

tein kinase activity. The concentrations of the cyclic nucleotidesused ranged from 0.033 to 2 MM.

a decreasing order of potency, by 0.3 ,uM concentration of thefollowing cyclic nucleotides; 8-bromo cyclic GMP > cyclicGMP > cyclic AMP. It has been shown earlier (20) thatthe 8-bromo derivative of cyclic GMP is more effectivethan its parent compounds in stimulating an, arthropod(lobster) cyclic GMP-dependent protein kinase. The reactionalso proceeded linearly either in the absence or presence ofadded cyclic nucleotides when increasing amounts of thepeak 2 enzyme were added (data not shown). The linearity ofthe reaction with respect to incubation time and amount ofenzyme, under the experimental conditions, was also seen

with all other enzyme fractions mentioned in Table 1.In order to further ascertain the cyclic nucleotide specificity

of the peak 2 enzyme, a kinetic analysis of stimulation bycyclic GMP and cyclic AMP was performed (Fig. 3). The Kafor cyclic GMP was found to be 0.03 uM, whereas that forcyclic AMP was found to be 0.6 MM, 20-fold higher than theformer. These results clearly indicate that the peak 2 enzymespecifically required cyclic GMP for its activation. The Ka

values for cyclic GMP and cyclic AMP of the peak 2 enzyme

are quite comparable to the corresponding values reportedearlier for the cyclic GMP-dependent class of protein kinasespurified from various arthropod tissues (5, 6). The enzyme

activity associated with fraction 2, prepared according toMethod II, was also shown to have a Ka for cyclic GMP(0.05 MM) that was much lower than the Ka for cyclic AMP(0.9 MM). The Ka values of cyclic AMP-depenident enzymefractions from the same tissue, together with the Ka valuesof cyclic GMP-dependent enzyme fractions, are summarizedin Table 1.The protein kinase assay system used earlier contained

acetate buffer and mixed histone (4-6). When the fraction 2enzyme (prepared according to Method II and enriched withcyclic GMP-dependent protein kinase activity, Table 1) was

assayed under these conditions, very little stimulation bycyclic GMP was detected (Table 2). Cyclic GMP also hadlittle effect when arginine-rich histone was substituted formixed histone. In incubations containing phosphate buffer,cyclic GMP stimulated the enzyme activity when mixed

TABLE 2. Effects of buffers and substrate proteins onthe protein kinase activity stimulated

by cyclic GMP or cyclic AMP

Protein kinase activity (units)

Cyclic CyclicBuffer and substrate protein None GMP AMP

Acetate bufferMixed histone 16.3 23.0 26.1Arginirue-rich histone 16.1 25.7 22.9

Phosphate bufferMixed histone 18.8 32.3 28.2Arginine-rich histone 16.8 40.1 28.1

The ammonium sulfate fraction 2 (Table 1), containing 62,ugof protein, was incubated in either 50 mM potassium phosphatebuffer (pH 7.0) or 50 mM sodium acetate buffer (pH 5.9). Theamount of either histone used was 40 Mg, and the amount ofprotein kinase modulator included in the incubation was 60 .g.When present, the concentration of either cyclic nucleotide was0.3 MM. Assay conditions, except for those mentioned above,were the same as described in the text.

histone was used as substrate, and there was an even greaterstimulation when arginine-rich histone was substituted (Table2).

It has been shown earlier that protein kinase modulatordepresses mammalian and arthropod cyclic AMP-dependentprotein kinases stimulated by cyclic AMP when assayed withacetate buffer and arginine-rich histone (19). In contrast, themodulator augments arthropod cyclic GMP-dependent pro-tein kinase stimulated by cyclic GMP under the same condi-tions (19). Similar effects of the modulator were obtained inthe present studies with guinea pig enzyme preparationsassayed in phosphate, instead of acetate, buffer. Thus, theprotein kinase modulator depressed cyclic AMP-dependentprotein kinase activity associated with peaks 1 and 3 (Fig. 1)in the absence and presence of either cyclic AMP or cyclicGMP (Fig. 4). The modulator augmented the cyclic GMP-dependent protein kinase activity associated with peak 2 (Fig.1) in the presence of cyclic GMP, but, conversely, depressedthe activity in the presence of cyclic AMP, alone or in theabsence of both cyclic nucleotides.The importance of phosphate for cyclic GMP-dependent

protein kinase activity is demonstrated in Fig. 5. Phosphatestimulated the activity of the freshly prepared Sephadex G-200 peak 2 enzyme (Fig. 1) in a concentration-related manner.A stimulatory effect of phosphate on a cyclic GMP-dependentenzyme activity prepared from rat cerebellum has been re-ported (21). Most of the cyclic GMP-dependent activity dis-appeared when the same enzyme preparation was stored for1 week at -20°. The activity, however, was nearly fully re-stored upon addition of phosphate (Fig. 5). The optimal ac-tivation concentration of phosphate for both fresh and storedenzyme preparations was found to be around 40-50 mM;higher concentrations resulted in a lower enzyme activity.The basal enzyme activity, on the other hand, was progres-sively depressed by increasing concentrations of phosphate.It should be mentioned that storage of the same enzymepreparation for more than 2 weeks resulted in a completeloss of the cyclic GMP-dependent activity, which could nolonger be restored by added phosphate.

Proc. Nat. A cad. Sci. USA 71 (1974)

Proc. Nat. Acad. Sci. USA 71 (1974)

Peak 1

t 60cII

e 50-

W 401

30

2 204 I0'

z

-10' 5! ,ae5*

0 30 60 90. 0 30 60 90 0 30 60 90

PROTEIN KINASE MOCULATOR pg)

PIG. 4. Stimulatory anid inhibitory effects of protein kinase

modulator on pr6tein kinase preparations obtained from guineapig lung. Aliquots (0.02 ml) of envymes of peahli.1, 2, and 3 (Fig.1), containing 56, 30, And 31 Pg of protein, respectively, were

incubated in the presence of various concentrations of proteinkinase modulator, as indicated. When present, the concentrationof the cyclic nucleotides was 0.3 uM. The standard assay condi-tions were as described in the text.

Several tissues from guinea pig and rat were also examinedfor the occurrence of cyclic GMP-dependent protein kinaspactivity. The tissues were homogenized, and the extracts 'werechromatographed on Sephadex G-200 columns according tothe procedure (Method I) described for guinea pig lung.Three distinct protein kinase peaks, qualitatively similar tothose seen with guinea pig lung preparation (Fig. 1), were in-Variably obtained for all tissue samples studied; the materialin peaks 1 and 3 was exclusively cyclic AMP-dependent,whereas that in peak 2 was largely cyclic GMP-dependent.The data pertaining to cyclic GMP specificity of the Peak 2enzyme activity are summarized in Table 3. The data obtained

20

±elWl fresh Enzyme<a10n --Stored Enzyme

1 , I I ,

20 80 100 120POTASSIUM PHOSPHATE (rnM)

FIG. 5. Stimulation by potassium phosphate-f cyclic GMP-dependent protein0inase activity. Peak 2 enzyme (Fig. 1),containing 30 /Ag of protein, was incubated in the absence andpresence of 0.3 IAM cyclic GMP and various concentrations ofpotassium phosphate buyer (pH 7.0), as indicated. The freshenzyme refers to a-preparation whose activity was assayed within2 days after the enzyme was prepared from the fresh tissue. Thestored enzyme refers to the same preparation that was stored for7 days at -20° before the enzyme activity was assayed.

TABLE 3. Cyclic GMP-dependent protein kinase activityisolated from variotis guinea pig and rat tissues

Specific proteinkinase activity

(units/mg of protein)

0.3 O.:t. K. values

cyclic cyclic Cyclic CyclicSpecies and tissue None GMP AMP GMP AMP

Guinea pigLung 140 638 280 0.03* 0.6*Heart (whole) 180 350 210 0.05 1.2Aorta (thoracic) 80 285 110 0.06 0.8Brain (whole) 125 280 140 0.12 3.8Liver 60 i05 80 0.09 1.0Ileum 40 85 50 0.02 0.8

RatEpididymal fat pad 30 88 45 0.06 1.0Pancreatic islet 145 658 216 0.05 1.2

Tissues collected from two to six animals were quickly homoge-nized, and the crude extracts were chromatographed on SephadexG.200 coluinns according to-the procedure described in the text forguinea pig lung. Enzyme fractions preferentially stimulated bycyclic GMP under the standard assay conditions were pooled(corresponding to peak 2, Fig. 1) and were used as the source ofenzyine.- Assay conditions were as described in text. The concen-tration of cyclic nueleotides used was 0.2 MM, instead of 0.3 AM.

* Taken from Table 1 and Fig. 3.

from various mammalian preparations in the present studiesare comparable to those observed with arthropod cyclic GMP-dependent enzymes reported elsewhere (5, 6). The tissue levelsof mammalian cyclic GMP-dependent enzyme activity ob-served under the modified assay procedure (Table 3) wereestimated to be 2-3 orders of magnitude higher than thoseobtained using the previous assay procedure (4-6). Resultssimilar to those in Table 3 were also obtained when proteinkinase modulator prepared from any other guinea pig andrat tissues was substituted for that prepared from guinea piglung (data not shown). These observations suggest that a rigidtissue- or species--specificity does not exist for the interactionof cyclic GMP-dependent protein kinase with protein kinasemodulator.

DISCUSSIONOne apparent difference between the mammalian and arthro-pod cyclic GMP-dependent protein kinase is the relative in-stability of the mammalian enzyme. This could account forthe previous Idifficulties in demonstrating cyclic GMP-dependent enzyme activity in mammalian tissues. The cyclicGMP-dependetit activity disappeared, for example, after theenzyme preparations (crude extracts, Sephadex peak 2, andammonium sulfate fraction 2) from guinea pig lung werestored for more than two weeks at -20°. Moreover, little orno cyclic GMP-dependent activity was found either in guineapig lung (and other tissues) stored for 10 days at -20°, orin frozen tissues obtained commercially (Pel-Freez).

It had been shown earlier that cyclic GMP-dependent pro-tein kinases occur at very high levels in arthropod tissues, andthat their activity can be readily assayed under the incubationconditions shown to be optimal for both arthropod and

4040 Biochemistry: KU'o''

Mammalian Cyclic GMP-Dependent Protein Kinases 4041

mammalian cyclic AMP-dependent enzymes (5, 6). It is clearfrom the present studies that mammalian cyclic GMP-de-pendent enzyme activity can be easily detected only if phos-phate and protein kinase modulator are both present in theincubation system, and a specific protein, such as arginine-richhistone, is used as substrate. Under these conditions, the cyclicAMP-dependent activity (representing a major portion ofthe total cyclic nucleotide-stimulated activity) is depressedwhereas the cyclic GMP-dependent activity (apparently theminor class of protein kinase) is greatly enhanced. The proteinkinase modulator may function as a physiological regulator ofcyclic nucleotide-dependent protein kinases, thus balancingthe effects of the two cyclic nucleotides. It has been shownthat protein kinase modulator occurs in every mammaliantissue examined (19). Its unique ability to regulate proteinkinase activity is an intrinsic characteristic of the native modu-lator molecule, and is not an artifact of the harsh procedure(i.e., boiling followed by precipitation with trichloroaceticacid) used for its preparation (J. F. Kuo,' T. P. Lee, and P.Greengard, to be published). The substantially high levels ofcyclic GMP-dependent protein kinase activity assayed in thepresence of protein kinase modulator may therefore reflectthe native physiological conditions present in the tissues.The demonstration of high levels of this enzyme activity

in every one of the mammalian tissues examined further sup-ports the contention that cyclic GMP-dependent proteinkinase functions as a "target" enzyme for mediating variousbiological effects of cyclic GMP.

Note Added in Proof. Procedures for preparation of cyclicGMP-dependent protein kinase from rat pancreatic islets bySephadex G-200 chromatography (22) and some propertiesof the enzyme from various mammalian tissues (23) will ap-pear elsewhere. Casnellie and Greengard (24) recently showeda cyclic GMP-dependent phosphorylation of endogenous sub-strate proteins in membranes of mammalian smooth muscle.

I thank Janice Gay Patrick for her expert technical asistance.This work was supported by a USPHS Grant HL-15696 and by agrant from Georgia Heart Association. I am a recipient of Re-search Career Development Award GM-50165 from USPHS.This is publication number 1212 from Division of Basic HealthSciences, Emory University.

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Proc. Nat. Acad. Sci. USA 71 (1974)