Eur. J. Biochem. 90, 585-593 (1978)
Mechanism of Activation of Protein Kinase I from Rabbit Skeletal Muscle The Equilibrium Parameters of Ligand Interaction and Protein Dissociation Jurgen HOPPE, Riidiger LAWACZECK, Erwin RIEKE, and Karl G. WAGNER Gesellschaft fur Biotechnologische Forschung, Abteilung Molekularbiologie, Braunschweig-Stockheim (Received May 12, 1978)
Protein kinase I from rabbit skeletal muscle binds adenosine 3' :5 '-monophosphate (CAMP)and ATP with high affinity; cAMP promotes and ATP retards dissociation (activation) of the tetrameric enzyme. The interrelationship of ligand interaction with protein dissociation has been probed by quantitative ligand binding, using the filter assay technique, and by computer simulation of binding curves in terms of Scatchard plots. A comparison of the experimental and computed binding data strongly confirms the supposition that the dimeric regulatory subunit (R2) binds cAMP cooperatively. Mainly from ATP binding it is further concluded that the interaction of the two catalytic subunits with R2 is also strongly cooperative, whereas the binding of ATP to the holoenzyme is noncooperative. The underlying random model, possessing low-affinity and high-affinity sites not only for ATP but also for CAMP, allows the determination of a minimal set of equilibrium parameters required for description and also an estimation oftheir magnitude. It further provides a basis for an explanation of the different behaviour of protein kinases I and I1 reported in the literature.
Regulation of CAMP-dependent protein kinases obeys the general rule that protein molecules endowed with catalytic activity interact with inhibitory proteins which bind the effector (CAMP). In the more thoroughly studied cases the inhibitory subunits were revealed to be dimeric proteins with two binding sites; hence, the inhibited holoenzyme is a tetrameric protein formed in the following way : 2C (active) + Rz S R z C (inactive) ~ . This equilibrium is primarily governed by cAMP as the free dimeric regulatory subunit binds the second messenger with high affinity. However, activation by cAMP is modulated by ATP and it is the nature of this modulation which leads to two classes of CAMPdependent protein kinases. In type I the holoenzyme has two high-affinity sites for ATP, thus ATP shifts the equilibrium to the right and acts as an antagonist of CAMP. Modulation of type I1 enzymes occurs This is paper 4 of a series; paper 3 appeared earlier in this journal [ll]. Part of this work was presented at a congress of the Deutsche Gesellschuft fur Biologische Chemie, Miinchen 1976 [6] and at the 12th FEBS Meeting, Dresden 1978 [19]. Abbreviations. CAMP, adenosine 3 ' : 5'-monophosphate; R and C, regulatory and catalytic subunit of CAMP-dependent protein kinase; Mes, 2-(N-morpholino)ethanesulfonic acid. Enzyme. Protein kinase (EC 2.7.1.37).
through phosphorylation of the dimeric regulatory subunit. As phosphorylation enhances its affinity for CAMP, this modulation is synergistic to the action of cyclic AMP [l - 51. The aim of the present work was the analysis of the equilibria of ligand binding and protein dissociation of enzyme type I from rabbit skeletal muscle. This system is composed of the tetrameric protein and the two antagonistic ligands cAMP and ATP, each of which have two binding sites. The protein dissociationassociation equilibrium can also be considered as the interaction of two protein ligands (monomeric catalytic subunits) with the dimeric regulatory subunit. In order to elucidate this rather complicated interrelation of interaction, binding of the ligands was determined in a series of experiments performed mainly with one labelled ligand. The magnitude and cooperativity of the equilibrium constants were estimated by comparison with simple model binding curves that were simulated by computer calculations. There have been earlier attempts to devise models for CAMP-dependent protein kinases [7,8]; especially Ogez and Segel [7] recently simulated Hill plots and compared available cAMP binding data on the basis of several proposals. The results of the preceding papers of this series [9- 111, however, facilitated the
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choice of the present model and a series of CAMP and ATP binding data could be used for its evaluation.
EXPERIMENTAL PROCEDURE CAMP-dependent protein kinase type I from rabbit skeletal muscle was purified as described previously [12]. The binding of radioactive CAMP or ATP was determined by filter assays as described in the previous papers [9- 121. The binding data of the present work are mean values from duplicates or triplicates experiments. [8-3H]cAMP, [8-3H]ATP, [14C]ATP and [y32P]ATP were purchased from Amersham Buchler GmbH; CAMPand ATP were obtained from Boehringer, Mannheim. Preparation of R Subunit 2 mg pure protein kinase holoenzyme after chromatography on hydroxyapatite [12] were placed onto a column (1 x 6 cm) of histone-Sepharose 4B (prepared according to [13]) which had been equilibrated with 5 mM Mes, 15 mM 2-mercaptoethanol, p H 6.5. Elution was performed with a linear gradient (total volume of 200 ml) from zero to 0.7 M KSCN in the above buffer. Protein kinase type I was known to dissociate at high concentrations of KSCN (about 0.4 M [14]). It was found that the R subunit eluted at higher concentrations of KSCN (0.6 M) than the C subunit (0.3 M). Pure fractions of R subunit were collected and concentrated with hollow fibers SHF 36 from Serva Feinbiochemica. KSCN was removed by dialysis against 5 mM Mes, 100 mM NaCl, 15 mM 2-mercaptoethanol pH 6.5. The R subunit migrates as a single band in polyacrylamide gel electrophoresis containing dodecylsulphate corresponding to a molecular weight of 48000 [2,12]. Computer Simulation of the Binding Data The basis of our computer simulation of the CAMP and ATP binding to protein kinase I were the reaction schemes outlined in Fig.3 and 5, respectively (see below). The law of mass action for the various species and the conservation of mass were used to calculate the concentrations of all products involved. For the purpose of a rough estimation the equilibrium constants KA, KT,Kp together with the coupling (a, p) and cooperativity factors (a, b ) were varied while the initial enzyme and nucleotide concentrations were kept constant in the range of the experimental values. By comparison with the experimental data values for KA, KT, CI and fi could be reasonably estimated. The cooperativity factors a and b were chosen in such a manner that the final variation in K P describing the association-dissociation equilibrium of the holoen-
A Model for the Activation of Protein Kinase
zyme resulted in the same tendencies as observed in the equivalent experiments (cf. Fig.4 and 6). At this point it should be stressed that our intention was rather to show trends than to determine numerical values for the thermodynamic parameters. Without loss of generality these tendencies will help to understand the underlying mechanism and its regulation.
RESULTS AND DISCUSSION The system to be described consists of the protein kinase type I (from rabbit skeletal muscle) and its two ligands CAMP and ATP, while the substrate protein to be phosphorylated is not considered. The following entities exist as possible protein species of the kinase: the holoenzyme R ~ C Zthe , partially dissociated holoenzyme R2C, the dimeric regulatory subunit Rz, and the free catalytic subunit C. The ATP-Binding Sites Two types of binding sites for ATP have to be considered: the substrate binding site in the catalytic subunit and two binding sites of high affinity on the holoenzyme (RzCZ)[1,2]. For the high-affinity site a Kd in the range of 50 - 100 nM was determined [2,9] and phosphotransferase activity or ATPase activity could not be detected 1151. ATP stabilizes the formation of the holoenzyme, lowers the affinity of the enzyme towards CAMP [l, 161 and counteracts the dissociation of the holoenzyme by NaCl [17]. ATP does not bind to the free regulatory subunit [l,161, nor is ATP interaction with the holoenzyme competitive with CAMP [91. Mapping the two ATP sites (at R z C ~and C, respectively) with various analogues of ATP [9, IS] strongly suggested that this nucleotide does not interact with the CAMP site. All the evidence collected, including affinity labelling of the ATP site [19] (and unpublished results of J. Hoppe) are in favor of the supposition that the high-affinity site for ATP at the holoenzyme RzC2 is congruent with the ATP catalytic site at C. However, this congruency holds only for the adenine subsites, the ribose and triphosphate moieties obviously occupy subsites which either experience a conformational change when C associates with R2 or are directly affected by the R subunit [18]. If the latter is true, specific contributions of R are responsible for the significant increase in binding affinity and this change of the ATP site accounts for the inhibition of phosphotransferase activities [19]. Under conditions were ATP binding to the holoenzyme is determined (filter assay cf. Experimental Procedure), no measurable binding of ATP or incorporation of phosphate into the free C subunit was detected [20] (and J. Hoppe, unpublished results).
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J. Hoppe, R. Lawaczeck, E. Rieke, and K. G. Wagner
This could be explained by the small ATPdse activity found with the C subunit [15]. Since direct determination of a binding constant is not possible we chose the Km value (10 pM) as a measure of the affinity of ATP towards the free catalytic subunit. This assumption seems to be reasonable as calculations based on initial velocity rates [21,22] showed that the Kmvalue reflects a true binding constant. The CAMP-Binding Site
Cyclic AMP binds to the protein kinase with high affinity and the site has been explored very thoroughly (for review cf. [23]). All the features of this binding, so far reported, and the assay conditions strongly suggest that the sites involved belong to the free regulatory subunit. It is an interesting question, for the model to be elaborated, whether the undissociated holoenzyme also has binding sites for CAMP. If cAMP promotes dissociation of the protein kinase (cf. the discussion below), then these possible binding sites have to be of lower affinity than those at the free regulatory subunit. Evidence that cAMP can bind to sites on R species which still carry the catalytic subunit is seen from studies with agarose-immobilized cAMP reported in the preceding paper or this series [11]. Direct evidence for two different sites for cAMP is demonstrated in the second paper of this series [lo]; spin-labelled CAMP is strongly immobilized by the protein kinase under conditions where binding promotes the dissociation of the enzyme. However, in the presence of ATP, which promotes the association of the enzyme, part of the spin-labelled cAMP exists in a less immobilized state, which is clearly distinguished from free unbound cAMP and highly immobilized CAMP. A further approach was performed with the sirnultaneous binding of [14C]ATPand [3H]cAMP and conditions similar to those described in the legend of Fig. 4 (data not shown). ATP was present at a constant concentration (54 pM or 5 pM), while that of cAMP was varied, Both series of binding data revealed that at low cAMP concentration the total amount of nucleotide bound (ATP plus CAMP) exceeds the expected value (two nucleotides per kinase molecule) by about 10%. As detection of ATP binding to the free catalytic subunit can be excluded, this finding is indicative of cAMP binding by R sites which are not free from the catalytic subunit and ATP, i.e. complexes such as RzCZ(ATP)z(cAMP)should be formed.
Fig. 1. The interrelation of c A M P binding and enzyme dissociation. In the dimeric model (RC) the regulatory subunit is indicated as a shadowed square and the catalytic subunit as a (large) circle. The small circle is an occupied CAMP site of high affinity, the triangle is one of low affinity. At the edges of the square the equilibrium constants (throughout dissociation constants) are indicated for the respective interactions. KP is the protein dissociation constant of the non-liganded protein (RC); K A is the dissociation constant of an R . cAMP complex; a KP is the protein dissociation constant of an RC complexed with CAMP,a being larger than 1 indicates that CAMP binding promotes protein dissociation. As the different dissociation-association steps occur in a closed system, the equilibrium constants are interdependent (this can be easily shown by writing down the mass equations and forming the respective quotients). As a consequence the dissociation of cAMP from RC . cAMP has the equilibrium constant tl K A ,which indicates (a > 1) that binding of cAMP to RC is weaker than to free R. Hence, tl is termed a coupling constant according to the terminology of Segel[7], coupling protein dissociation with cAMP binding
described in the following sections was performed on the general assumption that all interaction events occur randomly. It was further assumed that there is no direct interaction between ATP and cAMP binding; hence only the interaction (coupling according to the terminology of Segel [7]) of ATP or cAMP binding and binding of the catalytic subunit (protein association or dissociation) have to be considered. The coupling of cAMP binding and protein dissociation for a dimeric enzyme is illustrated in Fig. 1. Throughout the equilibrium constants are defined as dissociation constants, the index A refers to CAMP, P to the catalytic subunit and T to ATP. In the closed equilibrium system the coupling factor a describes both the difference in the binding affinity of cAMP towards R and RC and the difference in the protein dissociation RC and RC(cAMP). In other words c1 describes the coupling of cAMP binding with protein dissociation (cf. [71).
The Noncooperative Model
cAMP Binding towards the Isolated Free Regulatory Subunit
The present enzyme-ligand system can be described with a dimeric protein (Rz) possessing two sites for each of the three ligands: catalytic subunit (C), cAMP and ATP (mediated by C). Construction of the models
Binding of cAMP by protein kinase I showed Hill coefficients greater than one [2] : the simulation of these binding curves by computer calculation [7] demonstrated that noncooperative models are not
588
A Model for the Activation of Protein Kinase
sufficient to describe this situation. Cooperativity could be the basis of either cAMP binding or dissociation of the two catalytic subunits from Rz or of both events. An experimental approach to study the co0.10
I
I
operativity of cAMP binding alone can be performed with the isolated dimeric regulatory subunit. Hence, as described in Experimental Procedure, CAMP-free regulatory subunit was isolated by the elution of the holoenzyme from a histone-Sepharose column with a KSCN gradient. Fig.2 indicates the binding of tritiated cAMP by the regulatory subunit in the form of a Scatchard plot. It is obvious that cAMP binding by Rz is cooperative, i.e. the occupation of one site by cAMP provides the second site with higher affinity (cf. also conclusions). The Cooperative Model
0
1 2 r Fig.2. Binding of cAMP to the isolated regulatory subunit. r is defined as the concentration of bound cAMP molecules divided by the total R concentration, C A is the free cAMP concentration (Scatchard plot). Binding was determined by the filter assay in 2 mM potassium phosphate of pH 6.7 and 1.5 mM 2-mercaptoethanol at 23 "C; the concentration of the regulatory subunit was 6.4 nM based on cAMP binding
The introduction of additional factors for cooperative interaction enhances the complexity of the models significantly. Extention of the model outlined in Fig. 1 and using the same terminology leads to the concept in Fig. 3. This model considers cooperative properties of both cAMP binding and C dissociation. Cooperativity is expressed by the interaction factors a and b (the terminology of [7]) and values smaller than 1 result in positive cooperativity. Anticipating the results described below, the simulation of CAMP-bindingdata by computer calculations
Fig. 3. Cooperative tetrumeric model for c A M P binding and kinase activation. This is an extension of the dimeric model described in Fig. 1 and the same terminology is used. The extension to a tetrameric and cooperative model is performed with several simplifications, which are described in the text. The tetrameric model includes two identical sites both for cAMP and for C at the dimeric regulatory subunit Rz. If these sites were independent (noncooperative), a factor of 4 would be sufficient to describe the higher probability of occupation for a ligand, when both sites at R2 are free, relative to the situation when one site is already occupied (cf. [27]). As dissociation constants are used this factor of 4 describes the reduced probability of the second step. According to the terminology of Segel [7], cooperativity is expressed by interaction factors: a for the binding of cAMP molecules, b for the interaction of catalytic subunits C with Rt. For both processes positive cooperativity was assumed, i.e. the affinity of the second step (e.g. C to RzC or cAMP to Rz . CAMP) is higher than for the first step. In our scheme with dissociation constants, this fact is expressed by adding interaction factors (a or b) smaller than 1 to the equilibrium constant of the second step. Application of the equilibrium constants ( K pfor protein dissociation, K A for cAMP binding) and the factor t(, coupling of cAMP binding with protein dissociation has been explained in the legend of Fig. 1
589
J. Hoppe, R. Lawaczeck, E. Rieke, and K. G. Wagner
20
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1
2
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0
1
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Fig.4. A comparison of calculated and experimental Scatchard curves f o r the binding of c A M P to protein kinase I . The calculation (A) is based on the model of Fig. 3 with the following fixed parameters: K A0.1 ~ M , 1000, M a 0.01 and b 0.0025; K p was varied: ( I ) 5 nM, (2) 10 nM, (3) 20 nM, (4) 50 nM, (5) 0.1 pM, (6) 0.3 pM, (7) 0.6 pM, (8) 1 pM, (9) 5 pM. The experimental data (B) were obtained by the filter assay in 25 mM potassium phosphate of pH 6.7, 2 mM magnesium acetate and 1.5 mM 2-mercaptoethanol at 23 “ C .The protein kinase concentration was 0.15 pM based on cAMP binding (the same concentration was also included into the computations). Binding of tritiated cAMP was determined in the presence of (A) 53.6 pM ATP, (A) 4.9 pM ATP, (0) 1 M NaC1, (0) 1 mg/ml total histones from calf thymus or (0) no additions. For definition of r and CA see the legend of Fig.2
did not succeed for a noncooperative variant of the model shown in Fig.3 (a and b equal 1). We also got negative results considering positive cooperativity for protein dissociation alone (b < 1). When positive cooperativity for cAMP binding alone was considered (a < 1) the simulated curves showed a slight resemblance to the experimental curves. However, a rather good resemblance was obtained with positive cooperativity in both processes. Cooperativity is justified for cAMP binding from the results described above with the Rz subunit and for the protein dissociation step from results obtained by dissociation of the enzyme on Sepharose-immobilized CAMP derivatives (cf. the preceding paper [11I). To make the model simple, a was set constant irrespective of whether one deals with the free regulatory subunit (R2) or with the holoenzyme (RzC2). Also the interaction factor b for the two sites of C on Rz was taken as constant irrespective of whether cAMP has bound to the respective R subunit or not. As a further simplification, one species of the central complex of
Fig. 3 was not considered. R2C can bind one molecule of CAMP in two different ways, either to the R subunit which carries a C subunit or to the R subunit without a C. The latter state binds cAMP with an affinity higher than the former; the ratio is defined as a which is in our model 1000. Thus neglection of the weaker binding species seems to be justified. On the basis of this model Scatchard plots were simulated by computer. In Fig.4 a set of simulated binding curves are presented ; the protein concentration was kept constant and the equilibrium constants for cAMP binding (KA,a, a) and the protein cooperative factor (b) were fixed as indicated in the legend of Fig.4. The protein dissociation constant (KP)was varied from 5 nM to 5 pM. The shape of the curves changes from high positive cooperativity of cAMP binding at high Kp values to noncooperative behavior and to negative cooperativity at low KP values. A check of this model with appropriate binding experiments is also indicated in Fig.4. The binding of tritiated cAMP was determined in the presence of
590
A Model for the Activation of Protein Kinase
Fig. 5. Model for ATP binding and kinase activation. The filled circles represent occupied high-affinity sites, triangels low-affinity binding sites for ATP. For the application and explanation of the equilibrium parameters and the factor 4 see the legends of Fig. 1 and 3. KT is the dissociation constant for ATP with the free catalytic subunit. b represents the factor which couples ATP binding and protein dissociation. The higher affinity of ATP towards the holoenzyme is reflected in the fact that this step occurs with b KT and is smaller than 1. ATP binding (to the holoenzyme) is assumed to be noncooperative
different agents which are known to affect the dissociation equilibrium of the protein kinase. Histone and sodium chloride are reported to promote kinase dissociation [17,24], whereas the effect of ATP, as discussed above, will reduce protein dissociation. The qualitative correspondence of the experimental and simulated curves is rather good. The cooperative feature of the curves increases when the protein dissociation is favored, but almost vanishes at a high ATP concentration which promotes protein association. Comparing the simulated curves with the experimental ones allows an estimation of the protein dissociation constant, provided the other parameters are reasonable. From the experiment of Fig.4 (in the absence of other agents) a value of about 0.1 pM can be extracted for Kp which results in 1 nM for the association of the second C subunit (4 b K p ) . The present model is also able to describe the increase in the binding affinity with decreasing enzyme concentration (data not shown). At the same time binding cooperativity increases. The Interaction with A T P
In order to describe the interaction of ATP with the protein kinase, a model with similar features to that of cAMP interaction was designed. However, ATP binding was taken to be noncooperative (Fig. 5). This is obvious for ATP binding to the monomeric catalytic subunit ; the experiments and calculations,
described below, justify noncooperativity also for the two ATP sites of the holoenzyme. For the binding of ATP to the free catalytic subunit an equilibrium constant of 10 pM (related to K , [18]) was assumed. The coupling factor p was assumed to be 0.01. As in the model of Fig. 3 the species RzC C(ATP) is neglected relative to the species RzC(ATP) C (central species) which is justified as binding of ATP to a C subunit associated with R is assumed to occur with 100-fold higher probability relative to binding to a free C subunit (p value). The cooperativity factor (b)for protein dissociation is the same as that in the cAMP binding model. For computation of the binding curves the protein dissociation constant (KP)was varied between 10nM and 10 pM, and the simulated curves are shown in Fig.6. Unlike the binding curves for CAMP, ATP binding occurs with high affinity and non-cooperatively when the protein dissociation constant is low: increasing this constant leads to a reduction in the affinity and to an increase in the overall cooperativity. Exactly these properties are observed in the experimental binding curves also shown in Fig. 6 . Protein dissociation was again promoted by addition of sodium chloride. The qualitative correspondence of the experimental and the simulated curves is rather good.
+ +
The Simultaneous Binding of C A MP and A T P Fig. 7 shows experimental data on ATP binding in the presence of various constant concentrations of
591
J. Hoppe, R. Lawaczeck, E. Rieke, and K. G. Wagner
A \
x
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30
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1C
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0
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Fig. 6. A comparison of calculated and experimental Scaichard curves for the binding of ATP to protein kinase I . The calculation (A) is based on the model of Fig.5 with the following fixed parameters: KT 2.5 pM; 0.01; b 0.0025; Kp was varied: (1) 10 pM, (2) 1 pM, (3) 10 nM. The experimental data (B) were obtained by the filter assay in 25 mM potassium phosphate pH 6.7, 2 mM magnesium acetate and 1.5 mM 2-mercaptoethanol at 23 "C.The binding of tritiated ATP was determined in the presence of (0)1.0 or (A) 0.5 M NaCl or (0)no additions. cT is the concentration of free ATP; for explanation of r see legend of Fig.2. The kinase concentration was 0.15 pM (also included into the calculations) I
I
0
1 r
2
Fig. 7. Binding of ATP in the presence of CAMP.The protein kinase concentration and the experimental conditions are the same as indicated in the legend of Fig. 6, which also contains the explanation of CT (for r see legend of Fig.2). The cAMP concentration was (0)zero, (0)0.3 pM, (A) 0.5 FM or (0)1.0 pM
CAMP. It was mentioned above that, under the conditions of the filter assay applied, only the highaffinity ATP binding by the holoenzyme can be detected. The simulation of these curves would necessitate calculations on the basis of an equilibrium scheme which includes both models, that for ATP and for cAMP binding. This would be a rather formidable task; hence we tried a qualitative interpretation of the binding features from the properties of the two models outlined above (Fig. 3 and 5).
Comparing the antagonistic effect of the two ligands, CAMP and ATP, on the protein dissociation equilibrium it is obvious that cAMP is more effective with the parameters of the present concept. The efficiency of cAMP (a= 1000) exceeds that of ATP (lip = 100) by a factor of 10. As cAMP binds cooperatively and the protein dissociation is also cooperative, one can assume that in the presence of cAMP the reaction mixture predominantly consists of the species R ~ C and Z Rz(cAMP)z. Increasing the cAMP concentration reduces the RZCZconcentration and hence the available binding sites for ATP. As ATP binding is only measured with the RC subunits and this interaction is noncooperative, the shape of the curves shown in Fig. 7 reveal exactly the behaviour described. The affinity constant (slope) of ATP binding is hardly affected, which would have been expected by simply reducing the number of binding sites. It may be possible that at higher ATP concentrations than used in the present experiments the curvature will become biphasic, because high ATP concentration will counteract cAMP and drive the protein equilibrium towards the holoenzyme.
CONCLUSIONS The efforts of the present work to approximate ligand-enzyme binding data of a dissociating tetrameric protein by computed binding curves lead to a rather good correspondence both on the qualitative and the quantitative level and help in the understanding of the underlying molecular interactions. Together
592
with previous experimental evidence, it is possible to outline the protein kinase I/ligand system using a minimal number of interaction events and their coordinate equilibrium parameters. The random model proposed implies that cAMP binding occurs at sites of different affinities. The preceding papers offer evidence for weak cAMP binding towards the holoenzyme [lo, 111 and also the simultaneous binding of labelled ATP and cAMP described. Evidence for the cooperativity of cAMP binding is directly deduced from the results with the isolated dimeric subunit (cf. Fig. 2 and [16]) and is also in accord with model computations ;approximation of binding curves did not succeed with noncooperative binding either in the present work or in that of Ogez and Segel [7]. As Fig. 4 demonstrates, positive cooperativity can be suppressed or even shifted to negative cooperativity when the protein dissociation constant decreases, whereas an increase in Kp has the opposite effect. The former can be verified by addition of ATP and the latter by addition of NaC1. The decrease of the Hill coefficient reported by Hofmann et al. [2] due to the presence of ATP is explained by the properties of the present model. In other words the effect of ATP on the cAMP binding behaviour can be simply described by a decrease in the spontaneous protein dissociation. The dependency of the features of the Scatchard plots for cAMP binding of Fig.4 upon the protein dissociation constant also explains some pecularities of protein kinase I1 from bovine heart. The protein dissociation constant is obviously lower than that of protein kinase I [17]. In this case our model predicts a reduced affinity for cAMP and reduction in cooperativity. The results of Hofmann et al. [2] apparently fit well to this assumption. It was further reported that phosphorylation of the regulatory subunit of enzyme type I1 results in an increase in the affinity for cAMP and in the cooperativity. These consequences would be expected from the curves of Fig.4 provided that phosphorylation of the regulatory subunit increases protein dissociation. The findings of Rosen et al. [3] that phosphorylation decreases the rate of association of the protein subunit are consistent with an increased protein dissociation constant. The preceding studies on affinity chromatography [ l l ] revealed that binding of only one cAMP ligand (immobilized on Sepharose) by the tetrameric protein kinase led to the dissociation of both catalytic subunits, which indicates that the protein dissociation steps are cooperative. The results of the present work support this, as a good correspondence with the experimental data could only be obtained with positive cooperativity in this step. Apparently positive COoperativity is governed or mediated by the dimeric regulatory subunit both with respect to cAMP binding and association of the two catalytic subunits. Obvi-
A Model for the Activation of Protein Kinase
ously the latter subunits do not mediate cooperativity. These ideas are also in accord with the assumption, that there is no direct coupling of ATP and cAMP binding, which was not included into the present model and for which no evidence was indicated. The influence of ATP interaction upon that of cAMP and vice versa is mediated by the protein dissociation. Recent findings of Schwechheimer and Hofmann [16] on the property of isolated CAMP-free regulatory subunit are rather difficult to reconcile with the present model. They reported dissociation constants for cAMP which were dependent upon the protein concentration in a similar way to that found for the holoenzyme [25]. The regulatory subunit was isolated by urea dissociation and is obviously monomeric; we also isolated this subunit by dissociation with KSCN (cf. Experimental Procedure) and also observed that cAMP binding was dependent upon protein concentration (J. Hoppe, unpublished results) ; however Ki values for R2C2 were higher than those found for the regulatory subunit (at the same R concentration). An explanation for the behaviour of the CAMP-free monomeric subunit may arise from their observation [16] that dimeric subunits are partially formed through the addition of CAMP. Recently Peters et al. [26] reported a considerable increase in phosphotransferase activity upon dilution of the isolated C subunit. They attributed this phenomenon to the formation of aggregates. A similar mechanism may also hold for the isolated R subunit. This may be the reason for the concentration dependence of affinity and cooperativity of binding of CAMP. However, with the holoenzyme only the formation of the dimeric regulatory subunit was reported upon interaction with CAMP. Thus, the monomeric CAMP-free R subunit, obtained by treatment with urea or KSCN, is obviously not a physiological entity. This work has been supported by the Deutsche Forschungsgemeinschaft (Wa 91) and the Funds der Chemischen Industrie. We are grateful to Dr V. Wray for linguistic advice, to Mrs R. Jahne for typing the manuscript and to Mr E. Kiihne for preparing the graphs.
REFERENCES 1. Beavo, J. A., Bechtel, P. J. & Krebs, E. G. (1975) Adv. Cyclic Nucleotide Res. 5,241 -251. 2. Hofmann, F., Beavo, J. A., Bechtel, P. J. & Krebs, E. G. (1975) J. Biul. Chem. 250, 7795 - 7801. 3. Rosen, 0. M. & Erlichmann, 3 . (1975) J . Biuf. Chem. 250, 7788 - 7794. 4. Rangel-Aldao, R. & Rosen, 0. M. (1976) J . Biol. Chem. 251, 3375 - 3380. 5. Haddox, M. K., Newton, N. E., Hartle, D. K. & Goldberg, N. D. (1972) Biuchem. Biuphys. Res. Cummun. 47,653-666. 6. Hoppe, J., Rieke, E. & Wagner, K. G. (1976) Hoppe-Seyler's 2. Physiol. Chem. 357, 264. 7. Ogez, J. R. &Segel, I. R. (1976)J. Biol. Chem. 251,4551 -4556.
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16. 17. 18.
Biophys. Acta 364, 250-259. Hoppe, J., Marutzky, R., Freist, W. & Wagner, K. G. (1977) Eur. J . Biochem. 80, 369 - 373. Hoppe, J., Rieke, E. & Wagner, K. G. (1978) Eur. J. Biochem. 83,411-417. Rieke, E., Hoppe, J. & Wagner, K. G. (1978) Eur. J . Biochem. 83,419-426. Hoppe, J. & Wagner, K. G. (1977) FEBS Lett. 74, 95-98. Corbin, J. D., Brostrom, C. O., King, C. A. & Krebs. E. G. (1972) J . Biol. Chem. 247, 7790-7798. Huang, L. C. & Huang, C. (1975) Biochemistry, 14, 18-24. Shizuta, Y., Bechtel, P. J., Beavo, J. A. & Krebs, E. G. (1978) Adv. Cyclic Nucleotide Res. 9, 756. Schwechheimer, K. & Hofmann, F. (1977) J . Bid. Chem. 252, 7690 - 7696. Corbin, J. D., Keely, S . L. & Park, C. R. (1975) J . Biol. Chem. 250,218-225. Hoppe, J., Freist, W., Marutzky, R. & Shaltiel, S. (1978) Eur. J . Biochem. 90,427 - 432.
19. Wagner, K. G., Rieke, E., Lawaczeck, R. & Hoppe, J. (1978) Proc. 12th FEBS Meet. 4, in the press. 20. Bechtel, P. J., Beavo, J. A. & Krebs, E. G. (1977) J. Biol. Chem. 252, 2691 -2697. 21. Moll, G . W. & Kaiser, E. T. (1977) J . Biol. Chem. 252, 30073011. 22. Matsou, M., Chang, L., Huang, C. & Villar-Palasi, C. (1978) FEBS Lett. 87, 77-79. 23. Jastorff, B. (1976) in Eucaryotic Cell Function and Growth (Dumont, J. E., Brown, B. L. & Marshall, N. L., eds) Plenum Press, New York and London. 24. Diskeland, S. O., Ueland, P. M. &Hag& H. J. (1977) Biochem. J . 161,653-665. 25. Beavo, J. A., Bechtel, P. J. & Krebs, E. G . (1974) Proc. Natl Acad. Sci. U.S.A. 71, 3580-3583. 26. Peters, K. A,, Demailles, J. A. & Fischer, E. H. (1977) Biochemistry, 16, 5691 - 5697. 27. Tanford, G. (1961) Physicai Chemistry of Macromolecules, John Wiley, New York.
J. Hoppe and K. G. Wagner, Abteilung Molekularbiologie, Gesellschaft fur Biotechnologische Forschung mbH, Mascheroder Weg 1, D-3300 Braunschweig-Stockheim, Federal Republic of Germany
R. Lawaczeck, Institut fur Physikalische Chemie der Julius-Maximihdns-Universitat Wiirzburg, Markusstrasse 9- 11, D-8700 Wurzburg, Federal Republic of Germany E. Rieke, E. Merck AG, Postfach 4119, D-6100 Darmstadt, Federal Republic of Germany
Mechanism of Activation of Protein Kinase I from Rabbit Skeletal Muscle The Equilibrium Parameters of Ligand Interaction and Protein Dissociation Jurgen HOPPE, Riidiger LAWACZECK, Erwin RIEKE, and Karl G. WAGNER Gesellschaft fur Biotechnologische Forschung, Abteilung Molekularbiologie, Braunschweig-Stockheim (Received May 12, 1978)
Protein kinase I from rabbit skeletal muscle binds adenosine 3' :5 '-monophosphate (CAMP)and ATP with high affinity; cAMP promotes and ATP retards dissociation (activation) of the tetrameric enzyme. The interrelationship of ligand interaction with protein dissociation has been probed by quantitative ligand binding, using the filter assay technique, and by computer simulation of binding curves in terms of Scatchard plots. A comparison of the experimental and computed binding data strongly confirms the supposition that the dimeric regulatory subunit (R2) binds cAMP cooperatively. Mainly from ATP binding it is further concluded that the interaction of the two catalytic subunits with R2 is also strongly cooperative, whereas the binding of ATP to the holoenzyme is noncooperative. The underlying random model, possessing low-affinity and high-affinity sites not only for ATP but also for CAMP, allows the determination of a minimal set of equilibrium parameters required for description and also an estimation oftheir magnitude. It further provides a basis for an explanation of the different behaviour of protein kinases I and I1 reported in the literature.
Regulation of CAMP-dependent protein kinases obeys the general rule that protein molecules endowed with catalytic activity interact with inhibitory proteins which bind the effector (CAMP). In the more thoroughly studied cases the inhibitory subunits were revealed to be dimeric proteins with two binding sites; hence, the inhibited holoenzyme is a tetrameric protein formed in the following way : 2C (active) + Rz S R z C (inactive) ~ . This equilibrium is primarily governed by cAMP as the free dimeric regulatory subunit binds the second messenger with high affinity. However, activation by cAMP is modulated by ATP and it is the nature of this modulation which leads to two classes of CAMPdependent protein kinases. In type I the holoenzyme has two high-affinity sites for ATP, thus ATP shifts the equilibrium to the right and acts as an antagonist of CAMP. Modulation of type I1 enzymes occurs This is paper 4 of a series; paper 3 appeared earlier in this journal [ll]. Part of this work was presented at a congress of the Deutsche Gesellschuft fur Biologische Chemie, Miinchen 1976 [6] and at the 12th FEBS Meeting, Dresden 1978 [19]. Abbreviations. CAMP, adenosine 3 ' : 5'-monophosphate; R and C, regulatory and catalytic subunit of CAMP-dependent protein kinase; Mes, 2-(N-morpholino)ethanesulfonic acid. Enzyme. Protein kinase (EC 2.7.1.37).
through phosphorylation of the dimeric regulatory subunit. As phosphorylation enhances its affinity for CAMP, this modulation is synergistic to the action of cyclic AMP [l - 51. The aim of the present work was the analysis of the equilibria of ligand binding and protein dissociation of enzyme type I from rabbit skeletal muscle. This system is composed of the tetrameric protein and the two antagonistic ligands cAMP and ATP, each of which have two binding sites. The protein dissociationassociation equilibrium can also be considered as the interaction of two protein ligands (monomeric catalytic subunits) with the dimeric regulatory subunit. In order to elucidate this rather complicated interrelation of interaction, binding of the ligands was determined in a series of experiments performed mainly with one labelled ligand. The magnitude and cooperativity of the equilibrium constants were estimated by comparison with simple model binding curves that were simulated by computer calculations. There have been earlier attempts to devise models for CAMP-dependent protein kinases [7,8]; especially Ogez and Segel [7] recently simulated Hill plots and compared available cAMP binding data on the basis of several proposals. The results of the preceding papers of this series [9- 111, however, facilitated the
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choice of the present model and a series of CAMP and ATP binding data could be used for its evaluation.
EXPERIMENTAL PROCEDURE CAMP-dependent protein kinase type I from rabbit skeletal muscle was purified as described previously [12]. The binding of radioactive CAMP or ATP was determined by filter assays as described in the previous papers [9- 121. The binding data of the present work are mean values from duplicates or triplicates experiments. [8-3H]cAMP, [8-3H]ATP, [14C]ATP and [y32P]ATP were purchased from Amersham Buchler GmbH; CAMPand ATP were obtained from Boehringer, Mannheim. Preparation of R Subunit 2 mg pure protein kinase holoenzyme after chromatography on hydroxyapatite [12] were placed onto a column (1 x 6 cm) of histone-Sepharose 4B (prepared according to [13]) which had been equilibrated with 5 mM Mes, 15 mM 2-mercaptoethanol, p H 6.5. Elution was performed with a linear gradient (total volume of 200 ml) from zero to 0.7 M KSCN in the above buffer. Protein kinase type I was known to dissociate at high concentrations of KSCN (about 0.4 M [14]). It was found that the R subunit eluted at higher concentrations of KSCN (0.6 M) than the C subunit (0.3 M). Pure fractions of R subunit were collected and concentrated with hollow fibers SHF 36 from Serva Feinbiochemica. KSCN was removed by dialysis against 5 mM Mes, 100 mM NaCl, 15 mM 2-mercaptoethanol pH 6.5. The R subunit migrates as a single band in polyacrylamide gel electrophoresis containing dodecylsulphate corresponding to a molecular weight of 48000 [2,12]. Computer Simulation of the Binding Data The basis of our computer simulation of the CAMP and ATP binding to protein kinase I were the reaction schemes outlined in Fig.3 and 5, respectively (see below). The law of mass action for the various species and the conservation of mass were used to calculate the concentrations of all products involved. For the purpose of a rough estimation the equilibrium constants KA, KT,Kp together with the coupling (a, p) and cooperativity factors (a, b ) were varied while the initial enzyme and nucleotide concentrations were kept constant in the range of the experimental values. By comparison with the experimental data values for KA, KT, CI and fi could be reasonably estimated. The cooperativity factors a and b were chosen in such a manner that the final variation in K P describing the association-dissociation equilibrium of the holoen-
A Model for the Activation of Protein Kinase
zyme resulted in the same tendencies as observed in the equivalent experiments (cf. Fig.4 and 6). At this point it should be stressed that our intention was rather to show trends than to determine numerical values for the thermodynamic parameters. Without loss of generality these tendencies will help to understand the underlying mechanism and its regulation.
RESULTS AND DISCUSSION The system to be described consists of the protein kinase type I (from rabbit skeletal muscle) and its two ligands CAMP and ATP, while the substrate protein to be phosphorylated is not considered. The following entities exist as possible protein species of the kinase: the holoenzyme R ~ C Zthe , partially dissociated holoenzyme R2C, the dimeric regulatory subunit Rz, and the free catalytic subunit C. The ATP-Binding Sites Two types of binding sites for ATP have to be considered: the substrate binding site in the catalytic subunit and two binding sites of high affinity on the holoenzyme (RzCZ)[1,2]. For the high-affinity site a Kd in the range of 50 - 100 nM was determined [2,9] and phosphotransferase activity or ATPase activity could not be detected 1151. ATP stabilizes the formation of the holoenzyme, lowers the affinity of the enzyme towards CAMP [l, 161 and counteracts the dissociation of the holoenzyme by NaCl [17]. ATP does not bind to the free regulatory subunit [l,161, nor is ATP interaction with the holoenzyme competitive with CAMP [91. Mapping the two ATP sites (at R z C ~and C, respectively) with various analogues of ATP [9, IS] strongly suggested that this nucleotide does not interact with the CAMP site. All the evidence collected, including affinity labelling of the ATP site [19] (and unpublished results of J. Hoppe) are in favor of the supposition that the high-affinity site for ATP at the holoenzyme RzC2 is congruent with the ATP catalytic site at C. However, this congruency holds only for the adenine subsites, the ribose and triphosphate moieties obviously occupy subsites which either experience a conformational change when C associates with R2 or are directly affected by the R subunit [18]. If the latter is true, specific contributions of R are responsible for the significant increase in binding affinity and this change of the ATP site accounts for the inhibition of phosphotransferase activities [19]. Under conditions were ATP binding to the holoenzyme is determined (filter assay cf. Experimental Procedure), no measurable binding of ATP or incorporation of phosphate into the free C subunit was detected [20] (and J. Hoppe, unpublished results).
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J. Hoppe, R. Lawaczeck, E. Rieke, and K. G. Wagner
This could be explained by the small ATPdse activity found with the C subunit [15]. Since direct determination of a binding constant is not possible we chose the Km value (10 pM) as a measure of the affinity of ATP towards the free catalytic subunit. This assumption seems to be reasonable as calculations based on initial velocity rates [21,22] showed that the Kmvalue reflects a true binding constant. The CAMP-Binding Site
Cyclic AMP binds to the protein kinase with high affinity and the site has been explored very thoroughly (for review cf. [23]). All the features of this binding, so far reported, and the assay conditions strongly suggest that the sites involved belong to the free regulatory subunit. It is an interesting question, for the model to be elaborated, whether the undissociated holoenzyme also has binding sites for CAMP. If cAMP promotes dissociation of the protein kinase (cf. the discussion below), then these possible binding sites have to be of lower affinity than those at the free regulatory subunit. Evidence that cAMP can bind to sites on R species which still carry the catalytic subunit is seen from studies with agarose-immobilized cAMP reported in the preceding paper or this series [11]. Direct evidence for two different sites for cAMP is demonstrated in the second paper of this series [lo]; spin-labelled CAMP is strongly immobilized by the protein kinase under conditions where binding promotes the dissociation of the enzyme. However, in the presence of ATP, which promotes the association of the enzyme, part of the spin-labelled cAMP exists in a less immobilized state, which is clearly distinguished from free unbound cAMP and highly immobilized CAMP. A further approach was performed with the sirnultaneous binding of [14C]ATPand [3H]cAMP and conditions similar to those described in the legend of Fig. 4 (data not shown). ATP was present at a constant concentration (54 pM or 5 pM), while that of cAMP was varied, Both series of binding data revealed that at low cAMP concentration the total amount of nucleotide bound (ATP plus CAMP) exceeds the expected value (two nucleotides per kinase molecule) by about 10%. As detection of ATP binding to the free catalytic subunit can be excluded, this finding is indicative of cAMP binding by R sites which are not free from the catalytic subunit and ATP, i.e. complexes such as RzCZ(ATP)z(cAMP)should be formed.
Fig. 1. The interrelation of c A M P binding and enzyme dissociation. In the dimeric model (RC) the regulatory subunit is indicated as a shadowed square and the catalytic subunit as a (large) circle. The small circle is an occupied CAMP site of high affinity, the triangle is one of low affinity. At the edges of the square the equilibrium constants (throughout dissociation constants) are indicated for the respective interactions. KP is the protein dissociation constant of the non-liganded protein (RC); K A is the dissociation constant of an R . cAMP complex; a KP is the protein dissociation constant of an RC complexed with CAMP,a being larger than 1 indicates that CAMP binding promotes protein dissociation. As the different dissociation-association steps occur in a closed system, the equilibrium constants are interdependent (this can be easily shown by writing down the mass equations and forming the respective quotients). As a consequence the dissociation of cAMP from RC . cAMP has the equilibrium constant tl K A ,which indicates (a > 1) that binding of cAMP to RC is weaker than to free R. Hence, tl is termed a coupling constant according to the terminology of Segel[7], coupling protein dissociation with cAMP binding
described in the following sections was performed on the general assumption that all interaction events occur randomly. It was further assumed that there is no direct interaction between ATP and cAMP binding; hence only the interaction (coupling according to the terminology of Segel [7]) of ATP or cAMP binding and binding of the catalytic subunit (protein association or dissociation) have to be considered. The coupling of cAMP binding and protein dissociation for a dimeric enzyme is illustrated in Fig. 1. Throughout the equilibrium constants are defined as dissociation constants, the index A refers to CAMP, P to the catalytic subunit and T to ATP. In the closed equilibrium system the coupling factor a describes both the difference in the binding affinity of cAMP towards R and RC and the difference in the protein dissociation RC and RC(cAMP). In other words c1 describes the coupling of cAMP binding with protein dissociation (cf. [71).
The Noncooperative Model
cAMP Binding towards the Isolated Free Regulatory Subunit
The present enzyme-ligand system can be described with a dimeric protein (Rz) possessing two sites for each of the three ligands: catalytic subunit (C), cAMP and ATP (mediated by C). Construction of the models
Binding of cAMP by protein kinase I showed Hill coefficients greater than one [2] : the simulation of these binding curves by computer calculation [7] demonstrated that noncooperative models are not
588
A Model for the Activation of Protein Kinase
sufficient to describe this situation. Cooperativity could be the basis of either cAMP binding or dissociation of the two catalytic subunits from Rz or of both events. An experimental approach to study the co0.10
I
I
operativity of cAMP binding alone can be performed with the isolated dimeric regulatory subunit. Hence, as described in Experimental Procedure, CAMP-free regulatory subunit was isolated by the elution of the holoenzyme from a histone-Sepharose column with a KSCN gradient. Fig.2 indicates the binding of tritiated cAMP by the regulatory subunit in the form of a Scatchard plot. It is obvious that cAMP binding by Rz is cooperative, i.e. the occupation of one site by cAMP provides the second site with higher affinity (cf. also conclusions). The Cooperative Model
0
1 2 r Fig.2. Binding of cAMP to the isolated regulatory subunit. r is defined as the concentration of bound cAMP molecules divided by the total R concentration, C A is the free cAMP concentration (Scatchard plot). Binding was determined by the filter assay in 2 mM potassium phosphate of pH 6.7 and 1.5 mM 2-mercaptoethanol at 23 "C; the concentration of the regulatory subunit was 6.4 nM based on cAMP binding
The introduction of additional factors for cooperative interaction enhances the complexity of the models significantly. Extention of the model outlined in Fig. 1 and using the same terminology leads to the concept in Fig. 3. This model considers cooperative properties of both cAMP binding and C dissociation. Cooperativity is expressed by the interaction factors a and b (the terminology of [7]) and values smaller than 1 result in positive cooperativity. Anticipating the results described below, the simulation of CAMP-bindingdata by computer calculations
Fig. 3. Cooperative tetrumeric model for c A M P binding and kinase activation. This is an extension of the dimeric model described in Fig. 1 and the same terminology is used. The extension to a tetrameric and cooperative model is performed with several simplifications, which are described in the text. The tetrameric model includes two identical sites both for cAMP and for C at the dimeric regulatory subunit Rz. If these sites were independent (noncooperative), a factor of 4 would be sufficient to describe the higher probability of occupation for a ligand, when both sites at R2 are free, relative to the situation when one site is already occupied (cf. [27]). As dissociation constants are used this factor of 4 describes the reduced probability of the second step. According to the terminology of Segel [7], cooperativity is expressed by interaction factors: a for the binding of cAMP molecules, b for the interaction of catalytic subunits C with Rt. For both processes positive cooperativity was assumed, i.e. the affinity of the second step (e.g. C to RzC or cAMP to Rz . CAMP) is higher than for the first step. In our scheme with dissociation constants, this fact is expressed by adding interaction factors (a or b) smaller than 1 to the equilibrium constant of the second step. Application of the equilibrium constants ( K pfor protein dissociation, K A for cAMP binding) and the factor t(, coupling of cAMP binding with protein dissociation has been explained in the legend of Fig. 1
589
J. Hoppe, R. Lawaczeck, E. Rieke, and K. G. Wagner
20
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Fig.4. A comparison of calculated and experimental Scatchard curves f o r the binding of c A M P to protein kinase I . The calculation (A) is based on the model of Fig. 3 with the following fixed parameters: K A0.1 ~ M , 1000, M a 0.01 and b 0.0025; K p was varied: ( I ) 5 nM, (2) 10 nM, (3) 20 nM, (4) 50 nM, (5) 0.1 pM, (6) 0.3 pM, (7) 0.6 pM, (8) 1 pM, (9) 5 pM. The experimental data (B) were obtained by the filter assay in 25 mM potassium phosphate of pH 6.7, 2 mM magnesium acetate and 1.5 mM 2-mercaptoethanol at 23 “ C .The protein kinase concentration was 0.15 pM based on cAMP binding (the same concentration was also included into the computations). Binding of tritiated cAMP was determined in the presence of (A) 53.6 pM ATP, (A) 4.9 pM ATP, (0) 1 M NaC1, (0) 1 mg/ml total histones from calf thymus or (0) no additions. For definition of r and CA see the legend of Fig.2
did not succeed for a noncooperative variant of the model shown in Fig.3 (a and b equal 1). We also got negative results considering positive cooperativity for protein dissociation alone (b < 1). When positive cooperativity for cAMP binding alone was considered (a < 1) the simulated curves showed a slight resemblance to the experimental curves. However, a rather good resemblance was obtained with positive cooperativity in both processes. Cooperativity is justified for cAMP binding from the results described above with the Rz subunit and for the protein dissociation step from results obtained by dissociation of the enzyme on Sepharose-immobilized CAMP derivatives (cf. the preceding paper [11I). To make the model simple, a was set constant irrespective of whether one deals with the free regulatory subunit (R2) or with the holoenzyme (RzC2). Also the interaction factor b for the two sites of C on Rz was taken as constant irrespective of whether cAMP has bound to the respective R subunit or not. As a further simplification, one species of the central complex of
Fig. 3 was not considered. R2C can bind one molecule of CAMP in two different ways, either to the R subunit which carries a C subunit or to the R subunit without a C. The latter state binds cAMP with an affinity higher than the former; the ratio is defined as a which is in our model 1000. Thus neglection of the weaker binding species seems to be justified. On the basis of this model Scatchard plots were simulated by computer. In Fig.4 a set of simulated binding curves are presented ; the protein concentration was kept constant and the equilibrium constants for cAMP binding (KA,a, a) and the protein cooperative factor (b) were fixed as indicated in the legend of Fig.4. The protein dissociation constant (KP)was varied from 5 nM to 5 pM. The shape of the curves changes from high positive cooperativity of cAMP binding at high Kp values to noncooperative behavior and to negative cooperativity at low KP values. A check of this model with appropriate binding experiments is also indicated in Fig.4. The binding of tritiated cAMP was determined in the presence of
590
A Model for the Activation of Protein Kinase
Fig. 5. Model for ATP binding and kinase activation. The filled circles represent occupied high-affinity sites, triangels low-affinity binding sites for ATP. For the application and explanation of the equilibrium parameters and the factor 4 see the legends of Fig. 1 and 3. KT is the dissociation constant for ATP with the free catalytic subunit. b represents the factor which couples ATP binding and protein dissociation. The higher affinity of ATP towards the holoenzyme is reflected in the fact that this step occurs with b KT and is smaller than 1. ATP binding (to the holoenzyme) is assumed to be noncooperative
different agents which are known to affect the dissociation equilibrium of the protein kinase. Histone and sodium chloride are reported to promote kinase dissociation [17,24], whereas the effect of ATP, as discussed above, will reduce protein dissociation. The qualitative correspondence of the experimental and simulated curves is rather good. The cooperative feature of the curves increases when the protein dissociation is favored, but almost vanishes at a high ATP concentration which promotes protein association. Comparing the simulated curves with the experimental ones allows an estimation of the protein dissociation constant, provided the other parameters are reasonable. From the experiment of Fig.4 (in the absence of other agents) a value of about 0.1 pM can be extracted for Kp which results in 1 nM for the association of the second C subunit (4 b K p ) . The present model is also able to describe the increase in the binding affinity with decreasing enzyme concentration (data not shown). At the same time binding cooperativity increases. The Interaction with A T P
In order to describe the interaction of ATP with the protein kinase, a model with similar features to that of cAMP interaction was designed. However, ATP binding was taken to be noncooperative (Fig. 5). This is obvious for ATP binding to the monomeric catalytic subunit ; the experiments and calculations,
described below, justify noncooperativity also for the two ATP sites of the holoenzyme. For the binding of ATP to the free catalytic subunit an equilibrium constant of 10 pM (related to K , [18]) was assumed. The coupling factor p was assumed to be 0.01. As in the model of Fig. 3 the species RzC C(ATP) is neglected relative to the species RzC(ATP) C (central species) which is justified as binding of ATP to a C subunit associated with R is assumed to occur with 100-fold higher probability relative to binding to a free C subunit (p value). The cooperativity factor (b)for protein dissociation is the same as that in the cAMP binding model. For computation of the binding curves the protein dissociation constant (KP)was varied between 10nM and 10 pM, and the simulated curves are shown in Fig.6. Unlike the binding curves for CAMP, ATP binding occurs with high affinity and non-cooperatively when the protein dissociation constant is low: increasing this constant leads to a reduction in the affinity and to an increase in the overall cooperativity. Exactly these properties are observed in the experimental binding curves also shown in Fig. 6 . Protein dissociation was again promoted by addition of sodium chloride. The qualitative correspondence of the experimental and the simulated curves is rather good.
+ +
The Simultaneous Binding of C A MP and A T P Fig. 7 shows experimental data on ATP binding in the presence of various constant concentrations of
591
J. Hoppe, R. Lawaczeck, E. Rieke, and K. G. Wagner
A \
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Fig. 6. A comparison of calculated and experimental Scaichard curves for the binding of ATP to protein kinase I . The calculation (A) is based on the model of Fig.5 with the following fixed parameters: KT 2.5 pM; 0.01; b 0.0025; Kp was varied: (1) 10 pM, (2) 1 pM, (3) 10 nM. The experimental data (B) were obtained by the filter assay in 25 mM potassium phosphate pH 6.7, 2 mM magnesium acetate and 1.5 mM 2-mercaptoethanol at 23 "C.The binding of tritiated ATP was determined in the presence of (0)1.0 or (A) 0.5 M NaCl or (0)no additions. cT is the concentration of free ATP; for explanation of r see legend of Fig.2. The kinase concentration was 0.15 pM (also included into the calculations) I
I
0
1 r
2
Fig. 7. Binding of ATP in the presence of CAMP.The protein kinase concentration and the experimental conditions are the same as indicated in the legend of Fig. 6, which also contains the explanation of CT (for r see legend of Fig.2). The cAMP concentration was (0)zero, (0)0.3 pM, (A) 0.5 FM or (0)1.0 pM
CAMP. It was mentioned above that, under the conditions of the filter assay applied, only the highaffinity ATP binding by the holoenzyme can be detected. The simulation of these curves would necessitate calculations on the basis of an equilibrium scheme which includes both models, that for ATP and for cAMP binding. This would be a rather formidable task; hence we tried a qualitative interpretation of the binding features from the properties of the two models outlined above (Fig. 3 and 5).
Comparing the antagonistic effect of the two ligands, CAMP and ATP, on the protein dissociation equilibrium it is obvious that cAMP is more effective with the parameters of the present concept. The efficiency of cAMP (a= 1000) exceeds that of ATP (lip = 100) by a factor of 10. As cAMP binds cooperatively and the protein dissociation is also cooperative, one can assume that in the presence of cAMP the reaction mixture predominantly consists of the species R ~ C and Z Rz(cAMP)z. Increasing the cAMP concentration reduces the RZCZconcentration and hence the available binding sites for ATP. As ATP binding is only measured with the RC subunits and this interaction is noncooperative, the shape of the curves shown in Fig. 7 reveal exactly the behaviour described. The affinity constant (slope) of ATP binding is hardly affected, which would have been expected by simply reducing the number of binding sites. It may be possible that at higher ATP concentrations than used in the present experiments the curvature will become biphasic, because high ATP concentration will counteract cAMP and drive the protein equilibrium towards the holoenzyme.
CONCLUSIONS The efforts of the present work to approximate ligand-enzyme binding data of a dissociating tetrameric protein by computed binding curves lead to a rather good correspondence both on the qualitative and the quantitative level and help in the understanding of the underlying molecular interactions. Together
592
with previous experimental evidence, it is possible to outline the protein kinase I/ligand system using a minimal number of interaction events and their coordinate equilibrium parameters. The random model proposed implies that cAMP binding occurs at sites of different affinities. The preceding papers offer evidence for weak cAMP binding towards the holoenzyme [lo, 111 and also the simultaneous binding of labelled ATP and cAMP described. Evidence for the cooperativity of cAMP binding is directly deduced from the results with the isolated dimeric subunit (cf. Fig. 2 and [16]) and is also in accord with model computations ;approximation of binding curves did not succeed with noncooperative binding either in the present work or in that of Ogez and Segel [7]. As Fig. 4 demonstrates, positive cooperativity can be suppressed or even shifted to negative cooperativity when the protein dissociation constant decreases, whereas an increase in Kp has the opposite effect. The former can be verified by addition of ATP and the latter by addition of NaC1. The decrease of the Hill coefficient reported by Hofmann et al. [2] due to the presence of ATP is explained by the properties of the present model. In other words the effect of ATP on the cAMP binding behaviour can be simply described by a decrease in the spontaneous protein dissociation. The dependency of the features of the Scatchard plots for cAMP binding of Fig.4 upon the protein dissociation constant also explains some pecularities of protein kinase I1 from bovine heart. The protein dissociation constant is obviously lower than that of protein kinase I [17]. In this case our model predicts a reduced affinity for cAMP and reduction in cooperativity. The results of Hofmann et al. [2] apparently fit well to this assumption. It was further reported that phosphorylation of the regulatory subunit of enzyme type I1 results in an increase in the affinity for cAMP and in the cooperativity. These consequences would be expected from the curves of Fig.4 provided that phosphorylation of the regulatory subunit increases protein dissociation. The findings of Rosen et al. [3] that phosphorylation decreases the rate of association of the protein subunit are consistent with an increased protein dissociation constant. The preceding studies on affinity chromatography [ l l ] revealed that binding of only one cAMP ligand (immobilized on Sepharose) by the tetrameric protein kinase led to the dissociation of both catalytic subunits, which indicates that the protein dissociation steps are cooperative. The results of the present work support this, as a good correspondence with the experimental data could only be obtained with positive cooperativity in this step. Apparently positive COoperativity is governed or mediated by the dimeric regulatory subunit both with respect to cAMP binding and association of the two catalytic subunits. Obvi-
A Model for the Activation of Protein Kinase
ously the latter subunits do not mediate cooperativity. These ideas are also in accord with the assumption, that there is no direct coupling of ATP and cAMP binding, which was not included into the present model and for which no evidence was indicated. The influence of ATP interaction upon that of cAMP and vice versa is mediated by the protein dissociation. Recent findings of Schwechheimer and Hofmann [16] on the property of isolated CAMP-free regulatory subunit are rather difficult to reconcile with the present model. They reported dissociation constants for cAMP which were dependent upon the protein concentration in a similar way to that found for the holoenzyme [25]. The regulatory subunit was isolated by urea dissociation and is obviously monomeric; we also isolated this subunit by dissociation with KSCN (cf. Experimental Procedure) and also observed that cAMP binding was dependent upon protein concentration (J. Hoppe, unpublished results) ; however Ki values for R2C2 were higher than those found for the regulatory subunit (at the same R concentration). An explanation for the behaviour of the CAMP-free monomeric subunit may arise from their observation [16] that dimeric subunits are partially formed through the addition of CAMP. Recently Peters et al. [26] reported a considerable increase in phosphotransferase activity upon dilution of the isolated C subunit. They attributed this phenomenon to the formation of aggregates. A similar mechanism may also hold for the isolated R subunit. This may be the reason for the concentration dependence of affinity and cooperativity of binding of CAMP. However, with the holoenzyme only the formation of the dimeric regulatory subunit was reported upon interaction with CAMP. Thus, the monomeric CAMP-free R subunit, obtained by treatment with urea or KSCN, is obviously not a physiological entity. This work has been supported by the Deutsche Forschungsgemeinschaft (Wa 91) and the Funds der Chemischen Industrie. We are grateful to Dr V. Wray for linguistic advice, to Mrs R. Jahne for typing the manuscript and to Mr E. Kiihne for preparing the graphs.
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J. Hoppe and K. G. Wagner, Abteilung Molekularbiologie, Gesellschaft fur Biotechnologische Forschung mbH, Mascheroder Weg 1, D-3300 Braunschweig-Stockheim, Federal Republic of Germany
R. Lawaczeck, Institut fur Physikalische Chemie der Julius-Maximihdns-Universitat Wiirzburg, Markusstrasse 9- 11, D-8700 Wurzburg, Federal Republic of Germany E. Rieke, E. Merck AG, Postfach 4119, D-6100 Darmstadt, Federal Republic of Germany
