Proc. Nail. Acad. Sci. USAVol. 82, pp. 7565-7569, November 1985Biochemistry

Inhibition of the Mg(II)ATP-dependent phosphoprotein phosphataseby the regulatory subunit of cAMP-dependent protein kinase*

(enzyme regulation/phosphorylation-dephosphorylation/glycogen cascade system)

STEWART R. JURGENSENt, P. BOON CHOCKt, SUSAN TAYLORt, JACKIE R. VANDENHEEDE§,AND WILFRIED MERLEVEDE§tSection on Metabolic Regulation, Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20205;tDepartment of Chemistry D-006, University of California at San Diego, La Jolla, CA 92093; and §Afdeling Biochemie, Faculteit Geneeskunde, KatholiekeUniversiteit Leuven, B-3000 Leuven, Belgium

Communicated by E. R. Stadtman, July 23, 1985

ABSTRACT We report potent inhibition of theMg(II)-ATP-dependent protein phosphatase, Fc-M, by theregulatory subunit dimer of type II cAMP-dependent proteinkinase, RI'. The protein kinase catalytic subunit has no effecton phosphatase activity and is unable to substitute for kinase FAin the kinase FA- and Mg(II)ATP-mediated phosphatase acti-vation reaction. Phosphatase inhibition was investigated as afunction of RII concentration. The results suggest that RII bothinhibits the active phosphatase and inhibits phosphatase acti-vation. The inhibition is shown to be noncompetitive withrespect to substrate (phosphorylase a). The potential physio-logical significance of this inhibition is discussed in terms ofphosphorylation/dephosphorylation cascade systems involvingthis kinase and phosphatase.

In recent years, increasing attention has been focused onuncovering the role of protein phosphatases in regulating thestate ofphosphorylation of various proteins and enzymes thatcontrol an array of cellular processes. It is known thatreversible, protein phosphorylation/dephosphorylation is animportant general mechanism of regulating diverse cellularprocesses in response to various physiological stimuli (1, 2).The identification of protein kinases acting in protein

phosphorylation cascades and the mechanisms by which theyare regulated via second messengers such as cAMP and Ca2'have preceded similar knowledge of the protein phosphatasesinvolved. Recently, Ingebritsen and Cohen (3, 4) have at-tempted to categorize a variety of protein phosphatase formsidentified in different systems as being one of four differentclasses of enzymes. In the nomenclature of Ingebritsen andCohen (3, 4), type 1 protein phosphatase has been shown tobe inhibited by two different heat-stable protein inhibitorstermed inhibitor 1 and inhibitor 2 (5-9).A Mg(I)*ATP-dependent protein phosphatase has been

isolated fr6m rabbit skeletal muscle and characterized (10,11). This enzyme is a major phosphorylase phosphatase inskeletal muscle and it also readily dephosphorylates the ,Bsubunit of phosphorylase kinase as well as glycogen synthase(10, 12), suggesting an important role in the glycogen cascadesystem. The enzyme, isolated in its inactive form, is activatedby kinase FA (also identified as glycogen synthase kinase 3)in the presence of Mg(II)-ATP (13, 14). Inhibitor 2, alsotermed modulator, M, has been identified as a modulator ofthis phosphatase (9). It is now considered to be a subunit ofthe enzyme that has been designated Fc-M. Recent work onthe mechanism of activation of this phosphatase by kinase FAhas implicated phosphorylation/dephosphorylation of mod-ulator in the interconversion of active and inactive phospha-tase (15). A similar enzymatic activity has been isolated from

rabbit skeletal muscle by a procedure involving an acetone-precipitation step (16, 17). This enzyme consisted of acomplex between a 38-kDa catalytic subunit, and the 31-kDamodulator, M. Activation of the inactive phosphatase couldbe accomplished either with trypsin treatment in the presenceof Mn(II) or by treatment with kinase FA and Mg(II)-ATP,resulting in phosphorylation of the modulator subunit. AMg(II)-ATP-dependent protein phosphatase has also beenreconstituted from type 1 protein phosphatase and inhibitor2 (18, 19). The inactive complex was activated by glycogensynthase kinase 3 and Mg(II)-ATP, which resulted in phos-phorylation of inhibitor 2 on threonine and caused inhibitor2 to dissociate from the active phosphatase.

In this investigation, we report an inhibition of theMg(II)-ATP-dependent protein phosphatase by type IIcAMP-dependent protein kinase. cAMP-dependent proteinkinase, when substituted for kinase FA, failed to activate thephosphatase. However, its inclusion in the activation reac-tion with kinase FA resulted in inhibition of the phosphatase.The inhibition is characterized and is shown to be due todissociated regulatory subunit dimer RII, dissociated fromtype II cAMP-dependent protein kinases holoenzyme R2C2.

EXPERIMENTAL PROCEDURESMaterials. [y32P]ATP was purchased from New England

Nuclear. Phosphorylase b was isolated from rabbit skeletalmuscle and converted to 32P-labeled phosphorylase a asdescribed (20, 21). Phosphatase Fc-M and kinase FA wereisolated as described (10, 13). Two different preparations ofphosphatase Fc-M were tested for inhibition by RI' withsimilar findings. However, most experiments were per-formed by using a preparation with lower specific activity(=1,500 units/mg) compared to that described previously(10, 15). This preparation was devoid of other knownphosphorylase phosphatases, since the expression of phos-phatase activity required activation by kinase FA andMg(II).ATP. The phosphatase activity unit is defined as theamount of enzyme that releases 1 nmol of [32P]phosphate permin at 30°C from 32P-labeled phosphorylase a (2 mg/ml).Bovine heart type II cAMP-dependent protein kinase, puri-fied as described (22), was kindly donated by Emily Shacter(National Heart, Lung, and Blood Institute). RI' and thecatalytic subunit C were isolated from porcine heart andpurified to near homogeneity as described (23).Enzyme Assays. Assays of phosphorylase phosphatase

activity were performed by using an organic extractionprocedure as described (15) or with a modified extractionprocedure (24). Dephosphorylation of phosphorylase a in theassay was usually 10% or less.

*Presented in part at the 75th Annual Meeting of the AmericanSociety of Biological Chemists, St. Louis, MO, June 3-7, 1984 (seeref. 34).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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The concentration of phosphorylase was determined byusing the absorbance index of AIro = 13.1 (25). The concen-tration of other proteins was determined by the method ofBradford (26).

RESULTSIdentification of RII as a Phosphatase Inhibitor. The

Mg(II).ATP-dependent phosphatase Fc-M is activated bykinase FA in the presence of Mg(II).ATP (13, 14). We foundthat bovine heart (type II) cAMP-dependent protein kinase,when substituted for kinase FA, and in the presence ofcAMPand Mg(II).ATP, was unable to activate the phosphatase.However, including cAMP-dependent protein kinase in theactivation-preincubation with kinase FA resulted in a CAMP-dependent inhibition of phosphatase activity. The isolatedsubunits of the type II cAMP-dependent protein kinase fromporcine heart were used to test whether phosphatase inhibi-tion derived from C or RI.' C did not inhibit phosphataseactivity when it was included with kinase FA and Mg(II).ATPduring Fc-M activation (Fig. 1). However, RYI did inhibit thephosphatase. Inhibition by RI' was slightly enhanced by theaddition of cAMP, which by itself had no effect on phospha-tase activity. The RI' preparation contained some boundnucleotide derived from the affinity purification step in whichcGMP was used to elute the protein. The addition of cAMP(4 /iM in assay) should assure near saturation of the cAMPbinding sites of RI' and also cause exchange of any boundcGMP for cAMP. Control experiments showed that cGMPalone at 4 ,uM had no effect on phosphatase activity. Theinhibition of phosphatase by added RI was reversed by theaddition of an excess of C, which caused the reformation ofthe protein kinase holoenzyme RI'C2. Dissociation of thereformed holoenzyme by addition of cAMP restored theFcM inhibitory activity. The inhibitory activity of RYI washeat labile and was destroyed by heating at 90'C for 5 min.

Inhibition of Active Phosphatase and of Phosphatase Acti-vation. Inhibition of phosphatase Fc-M by RI', added eitherbefore initiating activation of phosphatase or after phospha-tase activation, was compared (Fig. 2). In assays 1-3 in Fig.2A, RII and cAMP were added immediately before initiatingthe activation of Fc-M. In assays 4-6, addition of RI' wasmade 4.5 min after initiating activation. There was signifi-cantly less phosphatase inhibition when RI' was added afteractivating the phosphatase (assay 6) compared to when it wasadded before initiating phosphatase activation (assay 3). Thissuggests that RI' inhibits both the activation of Fc-M and thecatalytic activity of the active phosphatase.

Previous results have shown that different steady statelevels of phosphatase activity occur when different concen-trations of kinase FA are used during Fc-M activation (10).The inhibitory effect of RI'2 was investigated with activation

% activity0 20 40 60 80 100

A p

C

D

FG

Control+ C+ RI2'+ R' + cAMP+ RI' + C+ R' + C + cAMP+ R' (heated) + cAMP

FIG. 1. Inhibition of phosphatase FC'M by RI'. Fc-M was acti-vated with kinase FA and Mg(II)-ATP for 5 min in the presence or

absence of RI', C, and cAMP as indicated and then was assayed foractivity in a subsequent 5-min assay. The final concentrations ofassay components were: Fc-M, 0.3 Ag/ml; FA, 0.4 ,g/ml; RI', 31 nM(3.3 ,ug/ml); C, 94 nM (3.9 gg/ml); cAMP, 4 AM; and phosphorylasea, 1 mg/ml (10.5 /iM).

% inhibition

ControlControl+ RI'

ControlControl+ R2'

+ cAMP+ cAMP+ cAMP+ cAMP+ cAMP+ cAMP

FIG. 2. (A) Comparison of phosphatase Fc*M inhibition by RI'added either before or after activation of phosphatase. In assays 1-3,phosphatase Fc-M was activated for 5 min with kinase FA andMg(II)-ATP. R2' and cAMP were added immediately before initiatingthe activation of Fc-M. At 5 min, 32P-labeled phosphorylase a wasadded, and phosphatase activity was assayed. In assays 4-6, Fc-Mwas also activated for 5 min; however, the addition of RI' (or buffercontrol) was made 4.5 min after initiating phosphatase activation.The average of control assays (with or without cAMP) was used asthe baseline activity. The values shown for assays in the presence ofR2I are the average of duplicate assays. Typically, duplicates vary by2% or less from their average. The final concentrations of RI' andcAMP were 50 nM (5.3 )ug/ml), and 4 ,uM, respectively. Theconcentrations of Fc-M, FA, and phosphorylase a in the assay were0.3 Ag/ml, 0.4 /Lg/ml, and 1 mg/mil (=10 1uM), respectively. (B)Comparison of phosphatase inhibition by R2I at two different levelsof activation. Phosphatase Fc-M was activated for 5 min with kinaseFA and Mg(II)-ATP in the presence or absence of cAMP and RYI.32P-labeled phosphorylase a was then added, and phosphataseactivity was assayed. In assays 1-3, 400 ng/ml of kinase FA (finalassay concentration) was used to activate Fc-M completely. Inassays 4-6, the kinase FA was reduced to 80 ng/ml, which results in72% of the maximal activation of the phosphatase. The average ofduplicate control assays in the presence of cAMP was used as thebaseline activity. The values shown for assays in the presence of R'are the average of duplicates. The final assay concentrations ofcomponents other than FA were the same as in A except for RII,which was at 100 nM (10.6 Ag/ml).

of Fc-M at two different concentrations of kinase FA. Inassay§ 1-3 in Fig. 2B, 400 ng of kinase FA per ml was used;this concentration completely activated the phosphatase. Inassays 4-6, kinase FA was reduced to 80 ng/ml, whichresulted in 72% of the maximal activation ofthe phosphatase.At the lower level of FcM activation, there was a greaterdegree of inhibition by RI'. In light of the decreased phos-phatase inhibition by RI' at the higher level of kinase FA, weconsidered the possibility that kinase FA was converting RYIto a less (or non) inhibitory form via a phosphorylationreaction. This idea was tested in the following experiment.RI' was preincubated with kinase FA and Mg(II)-ATP for 10min at 30'C and then FC-M was added, thereby initiatingphosphatase activation. This pretreatment of RI' did notcause any decrease of its phosphatase inhibitory activity.Similar preincubation of RI' with Mg(II)-ATP alone also hadno effect on phosphatase inhibition. These results indicatethat under the conditions used to activate Fc-M, kinase FA isnot converting RI' to a less inhibitory form.

It should be pointed out that glycogen synthase kinase 3(kinase FA) and glycogen synthase kinase 5 have been shownto phosphorylate RI' in vitro (27). However, in those exper-iments, in which the concentration of RI' was about 1.5mg/ml, phosphorylation by glycogen synthase kinase 3

B =

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occurred slowly with an incorporation of about 0.3 mol of Piper mol of subunit after 2 hr of incubation. The rate ofphosphorylation was even further reduced to <0.1 mol of Piper mol of subunit in 2 hr, if RI' was dephosphorylated firstat all sites containing endogenous covalently bound phos-phate. In the present investigation, the RI' concentrationsused to inhibit phosphatase Fc-M were considerably lower.In the experiment presented in Fig. 1, for example, theconcentration of RI' was 3.3 Ag/ml. The concentration ofkinase FA used to activate phosphatase Fc-M was alsosomewhat less than the concentration used in the RI' phos-phorylation experiments mentioned above. This makes itunlikely that significant phosphorylation of RI' can occurduring the activation-preincubation of Fc-M.

Effect of RI' on Kinetics of Phosphatase Activation. The timecourse of the activation of phosphatase by kinase FA andMg(II)-ATP was determined in the presence and absence ofRI' and cAMP (Fig. 3A). The degree ofphosphatase inhibitionby RI' changed during the time course of activation. Thepercent inhibition decreased as a function of activation time,asymptotically approaching a final level of inhibition (Fig.3B). This observation may be explained by RI' inhibiting theactivation of the phosphatase as well as causing partialinhibition of the activated enzyme.

RI' Concentration-Dependence of Phosphatase Inhibition.Phosphatase inhibition curves determined under severalconditions are plotted as a function of RI' concentration inFig. 4A. At a given concentration of RI', the inhibition curveshifts towards greater inhibition, when a lower concentrationof kinase FA was used (curve with open circles) compared towhen a higher concentration was used (curve with opensquares). In these assays, RII was added immediately beforeinitiating the activation of Fc-M. The degree of inhibition byRI' was significantly reduced when Fc-M was activated firstfor 10 min and then RI' was added (curve with open triangles).The phosphatase inhibition data describing each of these

A

CM 600EU)

C400-

200

0 5 10 15 20 25B

60

0

40

--- 20

0 5 10 15 20 25

Time, min

FIG. 3. (A) Time course ofphosphatase activation in the presenceand absence of RI'. Fc-M was activated with kinase FA andMg(II)-ATP in the presence of cAMP and in the presence (i) orabsence (o) of RI'. Aliquots of the activation mixture taken at timeintervals during the activation were assayed in a 1-min assay.Concentrations of components in the assay were: Fc-M, 0.25 Ag/ml;RI', 100 nM (10.6 Ag/ml); cAMP, 4 AM; and phosphorylase a, 2mg/ml(21 AM). The assay concentration of kinase FA was 80 ng/ml,which results in about 70% of the maximal activation of phosphataseFcLM. (B) The percentage of phosphatase Fc-M inhibition by RII (inA) during the time course of activation. The percentage of Fc-Minhibition is plotted versus the time of activation.

three curves can be accounted for by a single bindinginteraction between RI' and phosphatase Fc-M. Fig. 4Bpresents a computer fit of the data based on such anassumption. The computer-generated binding isotherm is inreasonable agreement with the data. The amplitude of theinhibition was reduced when RI' was added after activationof the phosphatase because the activated phosphatase wasonly partially inhibited by RI' binding. Addition of RI' beforeinitiating activation of the phosphatase (curve with opensquares) resulted in an increased amplitude of inhibitionbecause activation of the phosphatase was inhibited as wellas the activated enzyme activity. The apparent Kds for thebinding interaction calculated from these two curves (curveswith open triangles and squares) are in reasonable agreement,89 and 112 nM, respectively. When the kinase FA concen-tration was reduced 80%, there was a shift in the inhibitioncurve (curve with open squares to curve with open circles)and a decrease in the apparent Kd to 42 nM. This suggests anantagonism between the binding of RI' and of kinase FA to thephosphatase. These data are insufficient to distinguish be-tween mutual exclusion and partial antagonism. Neverthe-less, by assuming a mutual exclusion mechanism, one canestimate the value of Kd for the R1I'Fc-M complex to be -24nM. This value is in reasonable agreement with the estimatedK, (15 nM) for RI' inhibition of the phosphatase catalyticsubunit isolated by the ethanol treatment procedure (seebelow). This mechanism also gives an estimate of the Kd (':3nM) for kinase FA binding to phosphatase Fc-M.The spontaneously active type 1 phosphatase was partially

purified following a procedure using ethanol treatment (28),except that the final gel filtration step was omitted. This type1 phosphatase could not be further activated by kinase FA andMg(II)-ATP. As shown in Fig. 4A, the addition of RI' alsoresulted in inhibition of this phosphatase (curve with solidcircles). Inhibition occurred at a low concentration of addedRI', but the maximum degree of inhibition achieved was onlyabout 30-35%. The inhibitory activity of RI' was shown againto be heat labile; it was destroyed by heating for 5 min at 90TC.The inhibition of type 1 phosphatase caused by 50 nM RI' wasnot affected by the addition of a molar excess of kinase FA(data not shown). This indicates that even at high concen-trations of FA, there is no direct interaction between kinaseFA and RI' that has an effect on the ability of RI' to inhibit type1 phosphatase. This is of interest in light of the observations(i) of decreased inhibition of FC-M when higher concentra-tions of kinase FA were used and (ii) of decreased inhibitionof Fc-M when RI' was added after, as compared to before,Fc-M activation. In another experiment (not shown) RI' (50nM) was preincubated for different times from 0-10 min at30°C with the spontaneously active type 1 phosphatase beforeadding substrate to start the assay. This preincubation did notaffect the ability of RI' to inhibit the phosphatase. Similarpreincubation of the phosphatase alone did not alter itsactivity. This result indicates that there is not a slow timedependence for the formation of an inhibited phosphataseafter addition of RI'.

It is known that RI' may contain covalently bound phos-phate at several different sites (27, 29). Thus, it might bepossible for RI' to inhibit phosphatase by binding at thephosphatase substrate binding site, in which case the inhibi-tion by RI' would be expected to be competitive with respectto phosphorylase a. However, the inhibition of phosphataseby RI' was observed to be noncompetitive. The data from aninitial rate kinetic experiment performed at different concen-trations of RI' is shown in Fig. 5. In the presence of RI' adecrease in the Vm,_ was observed with no effect on the Kmfor phosphorylase a. In addition, inhibition ofphosphatase byRI' occurred with the concentration of phosphorylase a inlarge excess over the concentration of RI'. Together, thesedata indicate that the inhibition is neither due to RI' binding

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100

.

cc$Cu

R"', nM

C0

:._nc

R"', nM

FIG. 4. (A) Phosphatase inhibition as a function of RI' concentration. FcM was activated for 5 min in the presence of RYI, cAMP, kinaseFA (final concentration, 80 ng/ml), and Mg(II)-ATP, and activity was determined in a subsequent 5-min assay (o). The inhibition curve obtainedwhen Fc-M was activated as described above, except that the concentration of kinase FA was increased 5-fold to 400 ng/ml, is shown (o). Therewas no inhibition observed when the R2' was first heated for 5 min at 90'C (a). The inhibition curve when Fc-M was activated with kinase FA(final concentration, 400 ng/ml) and Mg(II).ATP for 10 min prior to addition of RI' which was followed by further incubation for 5 min and thenassayed is indicated by -A-. The assay concentrations of other components were: Fc*M, 0.25 ,g/ml; cAMP, 4 ,uM; and phosphorylase a, 10,uM. The inhibition curve for ethanol-treated type 1 phosphatase activity is shown (o). The inhibitory activity of RI' was destroyed by heatingfor 5 min at 90°C (see "X"). The cAMP and phosphorylase a concentrations in these assays were the same as above. (B) Computer fit ofphosphatase inhibition by R2I based on the assumption of a single binding site. Symbols show data from A. Curves were generated by a computerfit based on a single-binding-site assumption. The apparent Kd for the binding interaction and the amplitude of inhibition were variables in thedata fitting.

at the substrate binding site nor due to RI' forming a complexwith the substrate and thus reducing its effective concentra-tion.

DISCUSSION

In this investigation, we show that the regulatory subunit oftype II cAMP-dependent protein kinase, RI', is a potentinhibitor of the phosphatase Fc-M. This inhibition is distinctfrom the inhibition of this phosphatase by the heat-stableprotein inhibitors, inhibitor 1 and inhibitor 2. The inhibitoryactivity of RI' is both heat labile and requires the dissociatedregulatory subunit. Inhibition by RII is reversed by reformingthe protein kinase holoenzyme RIIC2.

Gergely and Bot (30) have also reported inhibition ofphosphorylase phosphatase, isolated by using ethanol treat-ment as in ref. 28, by the regulatory subunit of cAMP-

0

1 /phosphorylase a, uM-'

FIG. 5. Double reciprocal plot of Fc-M inhibition by RII. Fc-Mwas activated for 5 min in the presence of different concentrations ofRI' and then assayed with different concentrations of substrate(phosphorylase a). Curves: o, double reciprocal plot determined inthe absence of RI'; o, in the presence of 50 nM RI'; A, in the presenceof 100 nM RI'; and *, in the presence of 400 nM RI'. Theconcentration of Fc-M was 0.25 ,ug/ml and of kinase FA was 0.4

Ag/ml. The Km for phosphorylase a calculated from the interceptwith the x axis is 5.5 ,M (subunit concentration).

dependent protein kinase isolated from rabbit skeletal mus-cle. In that report, however, higher concentrations of regu-latory subunit were required for inhibition of phosphataseactivity. These workers suggested that phosphatase inhibi-tion by the protein kinase regulatory subunit resulted from itsinteraction with substrate, phosphorylase a. However, in thepresent investigation, inhibition occurred with phos-phorylase a concentrations in large excess over the concen-tration of RI'. In addition, the kinetic experiment presentedin Fig. 5 demonstrates that inhibition by RI' follows a simplenoncompetitive inhibition pattern. If the inhibition werederived from an interaction between RI' and phosphorylasea, one would not obtain the observed linear plot (Fig. 5) whenRI' was present in the reaction mixture. These results indicatethat the phosphatase inhibition by RII is not a substrate-directed effect.The results indicate that RI' inhibits both the active

phosphatase and the activation of Fc0M by kinase FA andMg(II)'ATP. This conclusion is supported by the fact that (i)a higher degree of inhibition was observed when RI' wasadded before initiating Fc-M activation compared to thatwhen RI' was added after Fc-M has been activated (Figs. 2Aand 4A); (ii) when RI' was added prior to Fc M activation, thepercent inhibition decreases with time until reaching a finallevel (Fig. 3B); and (iii) preincubation of RI' with kinase FAand Mg(II)-ATP causes no decrease in the RI' inhibitorycapacity. Binding of RI' to both active and inactive forms ofphosphatase Fc-M could affect both the activity of theactivated phosphatase and the activation of the phosphataseby kinase FA and Mg(II)-ATP.The direct inhibition of phosphatase Fc0M by the regula-

tory subunit, RI', of type II cAMP-dependent protein kinasemay be an important regulatory feature of phosphorylation/dephosphorylation cascade systems involving this kinase andphosphatase. Significant phosphatase inhibition is observedwith concentrations of isolated RI' on the order of 100 nM.Cellular concentrations ofcAMP-dependent protein kinase ina variety of tissues have been estimated to range from 0.2 to0.7 AM (20, 31). However, compartmentalization within thecell may raise local concentrations of protein kinase to evenhigher levels. Recent reports have indicated that compart-mentalization of cAMP and cAMP-dependent protein kinase

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4 cAMPR-2C 2C + R" (cAMP)4

1 1ATP ADP I

IlI

I-P

IIP i H0

Kinase FAFcM; FcMa //

FIG. 6. Scheme for the synchronous regulation of cAMP-depen-dent protein kinase and the Mg(II).ATP-dependent phosphatase.FcMj and FcMa, inactive and active forms of Fc-M; I and I-P,dephosphorylated and phosphorylated forms of the interconvertiblesubstrate I; RP'C2, cAMP-dependent protein kinase holoenzyme; C,protein kinase catalytic subunit; R2'(cAMP)4, protein kinase regula-tory subunit dimer with bound cAMP.

may be involved in segregating responses to different stimuli,which are mediated through cAMP-dependent protein kinase(32). We are currently investigating the possible inhibition ofthe Mg(II)-ATP-dependent phosphatase by the regulatorysubunit of other types of cAMP-dependent protein kinase.The role envisaged for the coordinate regulation of type II

cAMP-dependent protein kinase and this phosphatase issummarized in Fig. 6. This represents the situation for amonocyclic cascade utilizing these enzymes as converterenzymes that respectively phosphorylate and dephosphoryl-ate the interconvertible substrate I. Increased cAMP con-centration dissociates the protein kinase into its regulatoryRYI and catalytic C subunits. C catalyzes phosphorylation ofI, and simultaneously RI' inhibits the activated phosphataseand the activation of the phosphatase. This inhibition con-tributes to the increased fractional phosphorylation of I. Sucha synchronous regulatory mechanism provides both signalenhancement and an enhanced sensitivity to the effector,cAMP. That is, a lower concentration of cAMP will berequired to achieve an intermediate level of fractional phos-phorylation, and changes in fractional phosphorylation willbe more sensitive to changes in cAMP.

This effect may be multiplied further due to the fact thatphosphatase Fc-M has a broad substrate specificity anddephosphorylates many of the phosphorylation sites of thephosphoproteins involved in the glycogen cascade system.Furthermore, the coincident activation of protein kinaseactivity and inhibition of protein phosphatase activity re-duces the ATP consumption of this system, thus making itmore energetically efficient (33).

We thank Earl R. Stadtman for his advice and encouragementduring this work and Emily Shacter for her interest and helpfulsuggestions. J.R.V. and W.M. are grateful for support from the"Fonds voor Geneeskundig Wetenschappelijk Onderzoek" and"Onderzoeksfonds Katholieke Universiteit Leuven." J.R.V. is a

Senior Research Associate of the "National Fonds voor Weten-schappelijk Onderzoek."

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