cyclic-amp-dependent protein kinase 11

Exogenous

substrate

stimulates

autodephosphorylation

of

cyclic-AMP-dependent protein

kinase 11

Bj0rn T.

GJERTSEN, Beathe FAUSKE

and

Stein

0.

D0SKELAND*

Cell Biology Research Group, Department of AnatomyandCell Biology, UniversityofBergen,Arstadveien 19, N-5009Bergen, Norway

The autophosphorylated regulatory subunit (32P-RII) of

cyclic-AMP-dependent protein kinase II was efficiently

de-phosphorylatedby its C subunit in the absence of added ADP, provided that Mg/ATP and a standard protein kinase peptide

substrate were present. This raises the possibility that

auto-dephosphorylationcould be significant in the intact cell. Only the

cyclic-AMP-complexed free form of 32P-RII was efficiently

dephosphorylated, indicating that the autodephosphorylation

was intermolecular. Autodephosphorylation of 32P-RII in the

INTRODUCTION

Enzymescaninprinciple catalyseareaction inboth theforward

andthereversedirection. This is also thecaseforthecyclic-AMP

(cAMP)-dependentprotein kinase (cA-PK), which catalyses the reversible reaction:

MgATP+protein=P-protein+MgADP (1)

Thisenzymeexists in twomain isoenzyme forms, cA-PKI and cA-PKII, and hasa pivotalrole in mediating the effects of the secondmessengercAMP byphosphorylationofkey cellproteins.

The type II isoenzyme undergoes an autophosphorylation

re-action,whereaspecific serine residue in the'hinge'region of the

regulatory moiety (RII) is phosphorylated by the catalytic (C) moiety. For general reviews see Beebe and Corbin (1986),

Edelmanetal.(1987), Tayloretal.(1990), McKnight(1991)and D0skelandetal. (1993).

The protein-kinase-catalysed reaction (eqn. 1) is considered virtually irreversible within the cell, because the forward reaction is faster thanthereverse reaction (Shizuta et al., 1975; Rosen and Erlichmann, 1975; D0skeland et al., 1984), and because of the low proportion of intracellular MgADP relative to

MgATP.

The present study reports the novel finding that, for

auto-phosphorylated RII, reversal of the kinase reaction did not require added ADP,provided that the Csubunitwas supplied

with standard peptide substrate and MgATP. This raises the

possibility that thereactioncanbe ofsignificance in the intact cell,i.e.evenwhenMgATPpredominatesrelativetoMgADP. A mechanism is proposed for the coupling between

auto-dephosphorylation of P-RII and phosphorylation of peptide substrate, involving a transient C-MgADP complex formed during C-catalysed phosphorylation of standard peptide substrates.

presence ofMgATP and kemptideoccurred withformation of

[y-32P]ATP, suggesting transfer of 32p of phospho-RII to a

transient C*(MgADP) complex formed during the forward kinase reaction with peptide as substrate.

Autodephosphoryl-ation promoted by phosphorylation of exogenous substrates couldoperatealsofor other kinasesconformingtoamechanism

whereMgADP remains boundto the active site after the other product(phosphorylatedsubstrate) has left thecatalytic complex.

EXPERIMENTAL

Materials

[y-32P]ATP and [5',8-3H]cAMP were from The Radiochemical

Centre, Amersham, Bucks., U.K. ATP-regenerating system

(phosphocreatine and creatine kinase) was from Boehringer,

Mannheim, Germany. Microcystin-LR, phosphate-acceptor heptapeptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly, 'kemptide') and all unlabelled nucleotides were from Sigma, St. Louis, MO,

U.S.A. Reagents for the Bradford (1976) protein assay and a

mixed globulin protein standard wereobtained from Bio-Rad,

Richmond, CA, U.S.A. A synthetic peptide corresponding to residues 5-24 of the heat-stable cA-PK inhibitor protein (Scott et al., 1986) was generously given by Dr. J.Scott and Dr.E.G.Krebs, University of Washington, Seattle,WA, U.S.A. C subunit and cA-PKIIwerepreparedfrom bovinemyocardium,

essentiallyas describedbySugdenetal.(1976)and Ogreidand D0skeland (1980), respectively.

Determination of

[H]cAMP

binding

to R; assayfor

kemptide

Determination of[3H]cAMPbindingtoRIorRIIwasbasedon

measurement of their binding capacity for [3H]cAMP and

separation by using specific antibodies (Ekangeret al., 1989). The C-subunitconcentration was estimated by mixing various

concentrations withpurified RIof knownconcentration (in the absenceofcAMP)andtitrating theresultingfree kinaseactivity by assaying its phosphotransferase activity at 30°C in 50mMpotassiumphosphate/15mMHepes/1OmMmagnesium

acetate/70,M phospho-acceptor peptide (kemptide)/0. mM

[y-32P]ATP (2mCi/mmol)/ 0.5 mM EGTA /1 mM dithio-erythritol.Thisestimatewas15%lower than the estimate based on protein content and Mr=40000, suggesting either that the

Abbreviations used:cAMP,cyclic AMP; cA-PKI, cA-PKII,cAMP-dependent proteinkinasetype andtype11respectively;RI, RII,theregulatorysubunits ofcA-PKI and cA-PKII respectively; C,thecatalytic subunit ofcA-PKIor cA-PKII; kde, rateconstant forpseudo-first-order dephosphorylation; Kd,

apparent equilibrium dissociation constant; C(MgADP), C subunit with MgADP bound to its active site as a result of direct binding of MgADP; C*(MgADP), C subunit with bound MgADPformed insitufrom bound MgATP upon peptide phosphorylation; kemptide, thephosphate-acceptor

heptapeptide Leu-Arg-Arg-Ala-Ser-Leu-Gly.

Table 1 Test of the

ability

of selected substances tomodulatethe

C-subunit-catalysed

dephosphorylation

of32P-RII

Pre-autophosphorylated32P-cA-PKIIwassubjected todephosphorylationin thepresence of the Csubunit of cA-PK and theagentsindicated inthe first column of the Table.Other conditionswere

asdescribed in theExperimental section. The initial rate ofdephosphorylationwasdetermined and isexpressedrelative to the rate observed in thepresence ofMgATPandkemptide.The apparent Km value for MgADP andthe K1 value ofMgATP (with30,uM or100,uM ADP)arealso shown.

Substance tested Dephosphorylation rate

[Concentration(s)] (relativetothat withMgATP/kemptide) Range(n)

5 mM Mg+1 uM-*3 mMADP 5 mM MgADP+1FM -4mMATP 5 mM Mg+ 1 mM GDP 5 mM Mg+3 mMATP+ 0.3 mM kemptide 1.5FM H-8 +MgATP/kemptide 100 FM H-8 + MgATP/kemptide 10FM KT-5720 + MgATP/kemptide 1 mg/mlPolylysine +MgATP/kemptide 1FM cA-PK inhibitor peptide + MgATP/kemptide 10FM Microcystin-LR +MgATP/kemptide

protein standard was not accurate for the C subunit when the Bradford (1976)assay was used,or thatsomeofthe C-subunit

protein wascatalytically inactive.

Theamount ofphospho-acceptor peptide (kemptide)present

in a sample was estimated byallowing the phosphorylation to

proceed to apparent completion. The assay was at 30°C in 50 mM potassium phosphate / 15 mM Hepes / 10 mM

mag-nesium acetate/ 0.3 mM

[y-32P]ATP

(1 mCi/mmol) /0.5 mM

EGTA

/0.1

mM

EDTA/l

mM

dithioerythritol/100

nM C sub-unit.Sampleswereremoved after20,30and40minofincubation. Completeness of substrate phosphorylation wasindicated

by

a

plateau ofincorporation asa function of incubation time.

Assay for

autodephosphorylatlon

of cA-PKII

ThecA-PKIIholoenzymewasroutinely autophosphorylated by preincubation for 15min at 0°C in 15 mM Hepes/NaOH,

pH7.2,

containing

20mMKCI, 1 mMEDTA,0.3 mMEGTA,

0.3mg/ml BSA and 0.15mg/ml of soybean trypsin inhibitor, with 2.5 mM magnesium acetate and 2

,uM [y-32P]ATP

(60Ci/mmol) ina volume of0.25ml. Excess radioisotope was

removed by passing the preincubation mixture through a

Sephadex

G-25 column (1.2ml bed

volume)

equilibrated with

50 mM potassium phosphate (pH 7.2)/l mM sodium

pyro-phosphate/20mMNaF/5mMEDTA/5mMEGTA. Fractions (60

Fl)

werecollected, andthe three firstflow-through fractions

were pooled. The amount of32P-RII recovered was the same

whether or not

2,uM

microcystin was included during

pre-incubation andinthecolumn-equilibration buffer.

The

dephosphorylation

reactionwasstartedbyadding20

Fl

of the

pooled 32P-labelled

cA-PKII fractions to 1.3 ml of

buffer

(15

mM Hepes/NaOH, pH 7.2, 20 mM KCI, 1 mM EDTA, 0.3 mM EGTA, 0.3mg/ml of BSA, 0.15mg/ml of soybean trypsin

inhibitor)

containing routinely6 mMmagnesiumacetate, 30

FM

cAMP and 50-100 nMof thecatalytic subunit ofcA-PK. Variousconcentrations ofADP, ATP, ATP-regeneratingsystem

(phosphocreatine and creatine kinase) and other agents to be tested (Table 1) were present in some incubations. The

de-phosphorylation reactionwasarrested by spotting150

Ful

samples

on to

filter-paper

discs

(25

mm

diameter),

which were

immedi-ately

transferredto 10% (w/v)

trichlor-oacetic

acidwith 5 mM

pyrophosphate. After washing

(10

ml/filter) six times in 5%

trichloroacetic acid, the filters were treated sequentially with

0.07 -0.79(Km=11 FM) 0.78 -0.04(K=14FM) 0.02 1.0 0.42 0.07 0.19 0.04 0.14 1.0 Km8-14(n= 3)

Ki

11-19(n=3) 0.00-0.04 (n =2) 0.38-0.45 (n=2) 0.05-0.08 (n=2) 0.17-0.21 (n =2) 0.03-0.05 (n=2) 0.11-0.17 (n=2) 0.95-1.06 (n=2)

ethanol and acetone, dried, and counted for radioactivity in a

liquid-scintillation counter.

Determination of

pseudo-first-order rate

constants

for

dephosphorylafton of 32P-RII:

estimat!On

of the fractional

occupancy

of the

binding

site

governing

the cAMP effect on

autodephosphorylation

When the initial rate ofdephosphorylation of 32P-RII obeyed

apparentfirst-orderkinetics, the operationalrate constant

(kde.)

for the dephosphorylation process was calculated

simply

from the relation:

kde=ln2/tl

where

tL

is the time (in seconds) required for 50%

dephosphorylation of the phosphoprotein

initially

present.

Assuming that cAMP accelerated the dephosphorylation of

P-RII by binding to a site, and acted proportionally with the fractional occupancyof thissite, it follows that:

Fractionaloccupancy=

[kde(x)

-kde(0)/[kde(max.

cA)

-kde(0)]

where kde(O) is the rate constant for dephosphorylation in the absence of added cAMP, kde(x) that in the presence of

con-centration x of cAMP, and kde(max.cA) that at maximally effective cAMP concentration.Asimilarapproach has been used

to estimate the occupancy of one binding site governing the

behaviourof anotherbinding site (D0skelandetal., 1987).

Determination of

[nP]ATP

produced

during

the

autodephosphorylatlon

of

32P-RII

Pre-autophosphorylation

was asdescribed

above,

exceptthat the

concentration of cA-PKII wasincreased 5-fold and thespecific radioactivityof[y-32P]ATPwasincreased2-fold.[y-32P]ATPwas removed from the preincubation by passing it twice through SephadexG-25 columns, each twice the routine size. Since the desalted preincubation was diluted only 3-fold in the

de-phosphorylation reaction, the second columnwas equilibrated

withonly5mMNaF,5mMpyrophosphateand 2 mM each of EGTAandEDTA, toavoid excessive chelation of

Mg2+ during

the

ensuing autodephosphorylation

reaction. Less than 2% of the

32p

inthe sample subjected to

autodephosphorylation

was

solublein0.6-M HCl04.

C subunit, 300,M kemptide and 0.5 mM ATP. The ATP-regenerating system and other ingredients were as described above. The reaction was also performed in the absence of kemptide, ATP or ATP-regenerating system, both with and without 3 mM ADP. In one series of experiments the

auto-dephosphorylation wascompared between the incubations con-taining MgADP + complete ATP-regenerating system and those

containing MgADP with phosphocreatine and boiled creatine

kinase. The reactions were arrested by addition ofHCl04 and

separated by h.p.l.c. essentiallyasdescribed by D0skeland et al. (1992), by using a LiChrosorb RP-18 UltroPac column (4 mm x 250 mm) with 2

%

(v/v) methanol, 110mMpotassium

phosphate(pH 6.9)and0.3 mMtetrabutylammoniumhydrogen

sulphate as the mobile phase. Fractions of volume 0.3 ml were

collected and assayed for radioactivity.

To test whether the labelled substance co-chromatographing

withauthentic ATPwas[y-32P]ATP,the peak h.p.l.c.fractionsof presumed [32P]ATP were tested for ability to transfer 32P to

peptide substrate under standard protein kinaseassay conditions

with 0.1 mM ATP, 150 nM C subunit and 300

,#M

kemptide.

The reaction was allowed to run to apparent completion, as

ascertained bya plateau ofincorporationof32P into substrate

(achieved after less than 20 min of incubation). The degree of incorporation of 32P from [32P]ATP produced in the

auto-dephosphorylation reaction was compared with the

incorpor-ation using as phosphate donor [y-32P]ATP freshly obtained from the producer, and dilutedinh.p.l.c. mobile phase.

Autoradlography

of cA-PKII autophosphorylated in the absence

and

presence

of

kemptide and cAMP

cA-PKII holoenzyme (0.1 ,uM with respect to subunit

con-centration) was incubated at 0°C in 15mM Hepes/NaOH,

pH 7.2, 20 mM KCl, 1 mMEDTA, 0.3 mM EGTA, 0.3 mg/ml

BSA,0.15mg/ml soybean trypsin inhibitor,2.5 mMmagnesium acetateand4,uM[y-32P]ATP (60 Ci/mmol).Insomeincubations

cAMP(30 ,uM) orsubstrate peptide (300

4uM)

waspresent. The incubation volume was 100

,ll.

Samples of volume 20 ,ul were

removed, mixed first with 0.5 ml of

100%

trichloroacetic acid,

and then with5

/ug

each ofBSA, ovalbuminandsoybean trypsin inhibitor. The precipitate was collected by centrifugation, extracted with diethyl ether, and dissolved in 40,ul of10 mM Tris/phosphate, pH 8, with 9 M urea, 2 mM dithioerythritol and 1mMethylamine. This sample was diluted 1:1 insample buffer and subjected to SDS/PAGE (11

0%

total acrylamide

concen-tration) as described by Laemmli (1970). The washed and

subsequently dried gel was exposed to radiographic film

(/3-MAXfromAmersham).

RESULTS

Introductory experiments

The RIIsubunit of cA-PKII showedananomalous decrease in

its apparent cAMP affinity as a function of incubation time in tissue extracts supplemented with MgATP. It was considered

possible that this was due to loss of phosphate from the

autophosphorylation site of RII. However, neither inhibitors directed against phosphatases 1 and 2A (microcystin) or the

Ca2+/calmodulin-dependent phosphatase 2B (EGTA, W-7 and

trifluoperazine)norNaFpreventedthe decrease ofcAMPbinding

to RII [the nomenclature and inhibitor-sensitivity of protein phosphatases has been reviewed by Cohen

(1989)

and Bollen and Stalmans

(1992)].

In a further attempt to inhibit

phosphatases, the cA-PK substrate kemptide (500

1M)

was added to achieve anincreasing concentration ofthe presumed

phosphatase inhibitor phosphokemptide. Surprisingly, this further decreased the cAMP binding to RII. Decreased apparent cAMP affinity was also noted when kemptide was presented to purified cA-PKII incubated with MgATP. These findings led us to suspect that kemptide and MgATP enhanced the dephosphorylation of P-RII independently of phosphatases. This possibility was studied by using isolated cA-PKII.

The

C

subunit

of cA-PK catalyses dephosphorylation

of P-RII

In

the

presence

of saturating MgATP

and substrate

peptide

Autophosphorylated 32P-RII(cAMP)2 was indeed rapidly

dephosphorylated in the presence of kemptide, MgATP and

the C subunitof cA-PK (Figure 1). The initial reaction obeyed

apparentfirst-order kinetics (Figurela), and thefirst-orderrate constant for dephosphorylation was similar for all

32P-RII-(cAMP)2 concentrations (1-50 nM) tested (resultsnotshown),as

expectedfor a reaction in which the substrate concentration is far

belowits Km value. Thereactionwasnotsupportedby phospho-kemptide (Figure la), and requiredMg2+, andwasdependenton the C-subunit concentration (Figure lb). The effect of the C

, 0.25 ._-._ E Ca 0.1 cL o 1.0 c 0 0 LL. 0 1 2

Time ofincubation (min)

Figure 1 Dephosphorylation of32P-RII is catalysed by the C subunit of cA-PK in thepresenceofMgATPand kemptide

Panel(a) shows thedephosphorylationof32P-RII byC subunit(60 nM) in the presenceof kemptide (0.3 mM), with (U) or without (0) an ATP-regenerating system (4 mM phosphocreatine+10 units/ml creatinekinase;Cr-P/CPK). With 50FM kemptide(A) the

dephosphorylation rapidly abated. Phosphokemptide (O>) failed to support rapid dephosphorylation. Panel (b)shows the Mg2+requirement and the C-subunit concentration-dependence of thereaction.The cA-PKIIholoenzymewaspre-autophosphorylatedasdescribed

in the Experimental section, and 20,1 (containing 2.5pmol of 32P-RII) was exposed to

dephosphorylation by mixing with 1.3ml of buffer(15mMHepes/NaOH pH7.2,20mMKCI,

1mMEDTA,0.3mMEGTA, 0.3 mg/mlBSA,0.15mg/mlsoybean trypsininhibitor) containing

10 mMmagnesium acetate, 3 mM ATP and 30FMcAMP. Inoneexperiment(El)magnesium

acetatewasomitted. For theexperimentsshownin(b)the buffer contained30 nM(A),90 nM

(O)or no(0)added Csubunit. About2 nMC subunitwascarriedoverwith thepreincubation

mixture. Samples (150Ful) were spottedon to filter-paper discs todetermine 32P-RII, as

subunit depended on its concentration, rather than the ratio

between P-RII andCsubunit concentrations(tested from 1:1 to 1:200), indicating a catalytic effect of the Csubunit,as expected

whenexcess cAMP is present to prevent 'trapping' of C subunit incA-PKIIholoenzyme complex.

The dephosphorylation was dependent on continuous phosphorylation of kemptide, since the reaction rate declined as

the amount of kemptide became insufficient to support the

kinase activity (Figure la). This was demonstrated by starting

the reactionwith aconcentration of kemptide (50,uM) that, at

the level of C subunit present, became nearly completely

con-verted into phospho-kemptide during the reaction period. The conversion of kemptide intophospho-kemptidewasdetermined in aseparate experiment run under similar conditions to those

shown inFigure

1(a)

with 50,uM kemptide and using cA-PKII holoenzyme preincubated without labelledATP. Samples were

diluted4-fold inaprotein kinaseassaywith200,tM[y-32P]ATP and 100 nM C subunit for 15min, and 32P-kemptide was

determined.Only20

%

of theoriginally added 50

ptM

kemptide

wasstillphosphorylatable after30 sofincubation, and lessthan 5

%

was left after60 s(resultsnotshown).

The need for catalytic activity of the C subunitwassupported bythe lower rateofautodephosphorylation in the presence of kinaseinhibitors (Table 1), such as apeptidecorresponding to

the active sequence of the heat-stable protein kinase inhibitor,

and the ATP-directed inhibitors H-8 and KT-5720 (reviewed by Hidaka and Kobayashi, 1992). The abolition of

auto-dephosphorylation by polylysine (Table 1)was presumably due

to blocking ofthe interaction between RII and the C subunit, sincepolylysineprevented the reassociation ofRIIand Csubunit

(results notshown).

Evidence that

MgADP, formed

in

situ

on

the

C subunit from

MgATP,

is

the

phosphate acceptor

during

autodephosphorylation

of cA-PKII

The molecule accepting the 32P from

32P-RI(cAMP)2

during

autodephosphorylation

in the presence of kemptide, MgATP andan

ATP-regenerating

system wassoughtby h.p.l.c. analysis of the

HCl14-soluble

fraction from such anincubation. More than 90

%

of the

HCl14-soluble

32pwasidentified as[32P]ATP. About75

%

ofthis[32P]ATP could be incorporatedinto kemptide

underprotein kinaseassayconditions, where70-75

%

of authen-tic

[y-32P]ATP

was incorporated.

[y-32P]ATP

was therefore the main, and possibly the only, radioactive product formed from 32P-RII duringautodephosphorylation. The origin of the ADPrequiredtoacceptthelabelledphosphate from 32P-RIIto

produce

[y-32P]ATP

was not immediately evident, since no exogenous ADPhad been addedtotheincubation.Furthermore,

an efficient (seebelow and Figure 2) ATP-regenerating system was present, and any free ADP molecules would have had to compete (Table 1, Figure 2) with a vast excess ofATP in the reaction mixture. TheacceptorADP therefore hadtobepresent at asite inaccessible to theATP-regenerating system and not in

equilibrium with thegeneralATPpoolinthe incubation. Theexperimentsshown inFigure2 wereperformedtofind to what extent MgADP, added to the reaction medium, could substitute for the ADP produced in situ (see above). One would expect that onlya fraction

([C*(MgADP)]/[total

C]) of the C-subunit molecules carried MgADP during kemptide

phosphorylationinthe presence ofMgATP(seeright-handside

of Table2).Therefore, dependingonthesize ofthisfraction, the autodephosphorylation with MgATP and kemptide should be slower than in the presenceofmillimolarMgADP,which should

nearlysaturatetheCsubunitwith MgADP. Surprisingly, when

c1.0 X0.5 A d. N~~~~~~ .I4- uu0 o 0.25 fl0 3mMATP+Cr-P/cPK

0A3mM ATP +0.3mMADP 3

3mMATP+3mM ADP

_ A 3mM ADP

0.1 - 0 3 mM ADP+

Cr-P/CPK

1 2

Timeofincubation(min)

Figure2 MgADP, but not MgATP, can promote

autodephosphorylatlon

in

theabsenceof

kempfide

The timecourseof32P-RIIdephosphorylation wasstudied in the presence of 10 mM Mg2+, 30uM cAMP and 75 nM Csubunit. The rateofdephosphorylation was low without added nucleotide (0) as well as with 3 mM ATP (E). With 3 mM ADP (A) alone the

dephosphorylation was about twice as fastas when equimolar (3 mM ADP/3 mM ATP)

amountsof ADP and ATP (R)were present. Theeffect of ADP (0.3mM) was efficiently counteractedby3 mM ATP(A),and eventheeffect of 3 mM ADPwasabolished(i) by

an ATP-regeneratingsystem(Cr-P/CPK,asin Figure 1).

the

dephosphorylation

ratewas

compared

inincubationsrunin

parallel

with

MgADP

andwith

MgATP-kemptide,

itwas

slightly

(20

%),

but

significantly,

faster under the latter conditions

(Table

1). The effect of ADP

(kemptide

absent)

was

completely

blocked

by

an

ATP-regenerating

system

(phosphocreatine/

creatine

kinase),

andwas

competitively

inhibited

by

ATP

(Figure

2, Table

1).

The apparent Km

(11 ,uM)

for

MgADP

in the

auto-dephosphorylation

reaction

(Table 1)

was close to the K1 of

MgADP

estimated from the

ability

of

MgADP

to inhibit

competitively cA-PK-catalysed

phosphorylation

of substrates

(Whitehouse

et

al., 1983;

results not

shown).

A low

Km

for

MgADP

wasalsonoted in the

early

study

ofreversal of 32P-R11

phosphorylation by

Rosenand Erlichmann

(1975).

On theother

hand,

a Km value for

MgADP

of 3.3mM was found for the

reversal of 32P-casein

phosphorylation

by

the C subunit in an

important

early

study

(Shizuta

et

al., 1975).

This raises the

interesting

possibility

that

dephosphorylation

of standard

phosphorylated substrates,

unlike

autodephosphorylation,

hasa

MgADP-dependent

step

(possibly

a

ternary

complex

between

C,

P-casein and

MgADP),

with a low

affinity

for

MgADP.

An

alternative

explanation

is that the

high

32P-casein concentration used

(four

ordersof

magnitude higher

than thatof32P-RIIinthe

present

study)

may have led to the accumulation of

enough

[y-32P]ATP

to

give

significant

rephosphorylation

of substrate

at moderate concentrations of ADP. A very recent

study

of

C-subunit-catalysed

dephosphorylation

of

P-kemptide

reported

apparentKmvalues for

MgADP

in the

,uM

range

(Quamar

et

al.,

1992).

The

autodephosphorylated

RII could be

rephosphorylated

with retained

mobility

on

SDS/PAGE,

and retained its

[3H]cAMP-binding

characteristics,

indicating

that the loss of 32P from the

autophosphorylation

site was not caused

by

or

ac-companied

by

any

degradation

ordenaturation ofRII.

Effect of cAMP and

kemptide

on the

autophosphorylation

and

autodephosphorylation

of cA-PKII

Table 2 Scheme explaining preferential dephosphorylation of P-RII In thepresence of kiasepeptide substrate

The scheme shows the possible complexes involving C subunit, MgADPor MgATP, RII(cA)2and P-RII(cA)2in the absence (left;solid arrows) and the presence (right; broken arrows)of exogenouspeptide substrate [which, unlike RII(cA)2, is presentat aconcentration well above its Km value for phosphorylation]. In the absence of exogenous substrate RII(cA)2is readily

phosphorylated. In the presence of substrate, dephosphorylation of P-RII(cA)2 prevails relative tophosphorylation of RII(cA)2, because: (1) peptide substrate occupies C(MgATP), which is thereby made unavailable forphosphorylation of RII(cA)2; (2) peptide phosphorylation generates C(MgADP), which catalyses dephosphorylation ofP-RII(cA)2. The concentration ofATP is taken to be high, and well above that ofADP (as in the intactcell). Compounds of low abundance are shown in italics and others in bold. Furtherexplanations aregiven in thetext.

Exogenous peptide substrate absent ( - )

C(MgATP) Rll(cA)2

7

C(MgATP)RII(cA)2

_--

C(MgATP)

Peptide substrate present (- -l-) peptide C(MgA TP)peptide

4

C(MgADP)P-RII(cA)2 C*(MgADP)

P-RIIc42

C(MgADP) C CP-RII(cA)2

4

- -C*(MgADP)P-peptide

I

C*(MgADP)

A~\k

P-peptide

C CP-peptide

[RII(cA)2] < Km forRII(cA)2; [peptide] >Km forpeptide

1.0 E 0.5 0. 0 0.25 0 U-0.125 0 1 2

Timeofincubation(min)

Figure 3 cAMP-dependenceofthecA-PKIIautodephosphorylationreaction The timecourseof32P-RII dephosphorylationwasstudied under the conditions described in

thelegendtoFigure1 (10mMMg2+,2 mMADP and 60 nM C subunitwerepresentin all

incubations).,Thedephosphorylationwasstrongly dependentontheconcentrationofcAMP in the medium,whichwas0(0),0.1,M (/\),1,uM ([1),10,uM (@),or0.1 mM(A).

the presence of added MgADP was enhanced several-fold by cAMP(Figure3). The simplest explanation is that only free32p_

RII(cAMP)2 (not 32P-RII in the cA-PKII holoenzyme) was proneto significant dephosphorylation. This was supported by the closely corresponding cAMP-saturation curves (Figure 4) basedondatacalculated from theconcentration-dependence of

the cAMP effect on autodephosphorylation and actual

[3H]cAMP-binding

data (determined in parallel assays). In another set ofexperiments isolated cA-PKII holoenzyme was incubated with

Mg[y-32P]ATP

in the absence and presence of

0 1.00 a,; D .0 X 0.75 a, .E

~0

m 0.50 cc ,0.25 in -0 0 .r-m cu LL 0 10-7 10-6 i-Concn. of cAMP(M) 10-4 0) cm E-'na C) 0 0 cu"

Figure4 Correlation between cAMP binding and cAMP promotion of autodephosphorylation

The rate constantfor thepseudo-first-order autodephosphorylationreactionwascalculatedfrom data(obtainedwith different cAMPconcentrations) asshown in Figure 3,and thefractional

occupancyof theputativecAMPbinding site(s) responsiblefor the cAMP effectwascalculated

(see the Experimental section for details) and plotted (0) as a function of the cAMP concentrationduring autodephosphorylation.TheFigurealsoshows datafor[3H]cAMPbinding toRll (0)under similarconditions(presence of MgADP,60 nM Csubunit,37OC)to those usedfortheautodephosphorylation experiment.Note the close correlation betweenthe increase inthe twoparametersas afunctionof risingconcentrations(abscissa) ofcAMP(labelledor

unlabelled). Other conditions: [ATP]>[ADP];

C(MgA TP)

0

. ~ ~~~~

+kemptide No kemptide

NocAMP+cAMP NocAMP +cAMP 20s 60s 20s 60s 20s 60s 20s 60s 67k

-44k

-2 3 4 5 6 7 8

Figure 5 Kemptide blocks the intermolecular, butnotthelntra-holoenzyme, autophosphorylation of cA-PKII

cA-PKIIholoenzyme(0.1 uMwithrespect tosubunitconcentration)wasincubatedat0 °C for

20s(lanes 1, 3, 5, 7)or60s(lanes 2, 4, 6, 8)inthebufterdescribed in thelegendtoFigure 1,but with 2.5 mM Mg2+and4,uM [y-32P]ATP.Insomeincubations (lanes 3, 4 and 7, 8)

cAMP(30 ,uM)waspresent. Inothers(1-4) 300 ,uMof the kinase substratekemptidewas present. Theautoradiographic intensity of the RII bands show that kemptide canblock the

intermolecularphosphorylation of RII (lanes 3, 4), butnotthe intramolecularphosphorylation occurringin the absenceofdissociatingcAMP(lanes 1, 2). The incubation volumewas100,ul,

and 20#l samples were removed for SDS/PAGE and autoradiography as detailed in the

Experimental section.The arrowheads at the left showthe positions (determinedfromthe protein staining pattern)ofstandardproteins (BSAandovalbumin respectively)ofMr67000 (67 k)and 44000(44 k).

cAMP. In either case a rapid phosphorylation of RII was

observed in the absence of kemptide. Kemptide selectively prevented phosphorylation of RII in the presence of cAMP

(Figure 5), i.e. when RII was dissociated from the cA-PKII

holoenzyme. It appeared therefore that kinase substrates suchas

kemptide counteract the accumulation of autophosphorylated RIIby both inhibitingphosphorylation (Figure 5; Table 2) and promotingdephosphorylation (Figure 1; Table 2).

DISCUSSION

Coupling

of

autodephosphorylation

of P-RII and

phosphorylation

of standard

peptide

substrate:

Implications

for the kinetic

mechanism

of the

protein kinase reaction

Thepresentstudy has shown that autodephosphorylation of 32p_ RII(cAMP)2 canbe coupled to phosphorylation ofa standard

protein kinase substrate with theproduction of [y-32P]ATP. This implies that MgADP producedinsituonthe C subunit of cA-PK canacceptdirectly the 32p from the autophosphorylated siteon

RII, i.e. that the kinase reactioncanproceed from theternary catalytic complex [C(MgATP)peptide] accordingto:

C(MgATP)peptide-+C*(MgADP)P-peptide

C*(MgADP)+P-peptide (2)

Furthermore, the intermediary C*(MgADP) must be able to reactwithP-RII accordingto:

C*(MgADP)+P-RII(cAMP)2--

C*(MgADP)P-RII(cAMP2-+C(MgATP)+RII(cAMP)2 (3)

[that only one reaction direction is shown does not imply

irreversibility; C*(MgADP) denotes C subunit with bound

MgADP formed in situ through transfer of phosphate from

MgATPto peptide substrate].

Several detailed studies (containing references to important earlierstudies on thesubject)deal with the kinetic mechanism of

the kinase reactionofcatalysed by cA-PK (Whitehouse et al., 1983; Kong and Cook, 1988; Quamar et al., 1992) and the multifunctional Ca2+/calmodulin-dependent protein kinase II

(Kwiatkowski et al., 1990; Katoh and Fujisawa, 1991a). Data have beenpresented supporting sequential binding of MgATP

and peptide, followed by ordered release of P-peptide and MgADP(Whitehouse etal., 1983;Kwiatkowskietal., 1990)as

well as random binding (Kong and Cook, 1988; Katoh and

Fujisawa, 1991a; Quamar et al., 1992). The present study probablyprovides the mostexplicit evidencesofar for releaseof

phosphopeptide before MgADP. Furthermore, the C*(MgADP) complexsoformedis able tobind andcatalysedephosphorylation ofP-RII(cAMP)2.This means that the kinase reaction can also be

reversed in anorderedmanner,with P-RII(cAMP)2 joiningthe

preformed C*(MgADP).

Inapparentcontradictiontothis,acarefulisotope-partitioning study failedtodetectphosphotransfer from phosphokemptideto

C(MgADP), indicating that ADP dissociated faster than

P-kemptide from the complex C(MgADP)P-peptide (Kong and Cook, 1988). Thisdiscrepancycanberesolvedby assumingthat

C*(MgADP) differs from C(MgADP) by promoting more

efficiently thereversal of the kinasereaction. This idea receives

somesupportfromthe presentstudy (Table 1): in the presence

of MgATP and kemptide [when C*(MgADP) is formed] the

autodephosphorylationwasslightly fasterthan inthepresenceof

MgADP [when C(MgADP) dominates]. Unfortunately, the prevalence of C*(MgADP) relativetoothercomplexesof the C subunit(Table 2)is unknown. If 10

%

of totalC subunit is in the

C*(MgADP) state in the presence of MgATP and kemptide, C*(MgADP)isabout 12-foldmoreefficient thanC(MgADP)in

catalysing P-RII(cAMP)2 dephosphorylation. If

900%

is in the

C*(MgADP) state, the difference in catalytic activity must be slight.

Whereas the

autophosphorylation

ofRII canproceedasboth

anintramolecular andanintermolecularreaction(Rangel-Aldao and Rosen, 1976; Figure 5), autodephosphorylation was

detectedonly forP-RIIcomplexed with cAMP (Figures3and4), implyingthat it occurredonlyas anintermolecularreaction.

Possible

physiological

significance

of

autodephosphorylation of

Rul

In standard low-salt buffer,

500%

dephosphorylation of 32p_ RII(cAMP)2 was achieved in about 30 s by 90 nM C subunit (Figure 2). The concentration ofC subunit (0.78,umol/kg) in brain(Hofmannet al., 1977) translates to about 1.6,sM in the cell waterphase, assumingthat the latter occupies 50

%

of the

brain volume. Extrapolation would indicate an

auto-dephosphorylation rate of 0.4

s-Q.

This compares favourably

with the value (0.45

s-1)

recently estimated from data on

phosphataseaction onautophosphorylatedP-RII inDrosophila head extract(Aszodi etal., 1991).

We have shown that the rate of the C-subunit-mediated phosphorylationof a substrate in the intact cell was very close to thatcalculated fromexperimentswith isolatedenzymesusing a buffer of near physiological ionic strength (D0skeland et al., 1992). Using that same buffer we found the rate of

auto-dephosphorylation of P-RII(cAMP)2 to beabout 8-fold slower (resultsnotshown)than inthe standard low-salt buffer(Figure 2),suggestingthat underphysiologically relevant conditionsthe maximal rate of autodephosphorylation may be somewhat

slower than the maximal rate ofphosphoprotein phosphatase dephosphorylation of P-RII(cAMP)2. It should be stressed that, in the absence ofasuitablequantitative experimental approach

to study therateof dephosphorylation in intact cells, estimates of such rates must be considered hypothetical. A particular experimental problem applies to P-RII(cAMP)2, because enhanceddephosphorylation will leadtoenhancedre-association with the C subunit, formation of holoenzyme, and

re-autophosphorylation by an intramolecular mechanism

(Rangel-Aldao and Rosen, 1976; Figure 5). Therefore the overall phosphorylation of RII may not decrease even if

dephosphorylation is activated.

Buxbaum and Dudai (1989) have suggested that, owing to

autophosphorylation, cA-PKII can remain partially activated

formorethan 10min after cessation ofacAMPsignal,implying

long-term activation of cA-PKII as relevant for learning and memory; seealso Schwartz and Greenberg (1987) forareview of

thistopic. Thepresentdatasuggestthat, ifthe kinase is actively phosphorylating substrates giving risetoC*(MgADP), thebulk ofautophosphorylated P-RIImaybedephosphorylated in1min

evenin the absence ofphosphoprotein phosphatase activity.

Theoretical considerations

The equilibrium constants (in the forward direction) for the phosphorylation of casein by cA-PK(Shizutaetal., 1975) and of myosin light chain by myosin light-chain kinase (Geuss et al., 1985) have been reported tobe 24 and 60-70respectively. This

meansthat itrequiresoneto twoordersofmagnitudemoreADP

than of ATP to achieveequal rates of dephosphorylation and phosphorylation. Since cell ADP is one to two orders of magnitude lower than ATP,onewillexpectthe forwardreaction todominatecompletely in the intact cell. This consideration has led investigators to ignore the reversal of the kinase reaction underphysiological conditions.

The novel observation of the present study is that de-phosphorylation ofautophosphorylated P-RIIcanbe linked to phosphorylation ofa peptide substrate in sucha way that the

relative concentrations of bulk free cellularMgADP and MgATP become lessimportant (Table 2). In fact,evenwhen ATPwasin

extreme excess over ADP, the peptide substrate kemptide

en-hanced the dephosphorylation of P-RII(cAMP)2 (Figure 1). Kemptide also efficiently blocked the phosphorylation of RII (Figure 5), thus shifting the equilibrium between RII and P-RII in favour ofdephosphorylation. The blocking of phosphorylation ofRII(cAMP)2 by kemptide is easily explained bythelatter out-competing the former for complex formation with C(MgATP) (illustrated in Table 2), since kemptide was present at

concen-trations well above its apparent Km value of about 5,uM (Whitehouse et al., 1983). The enhanced dephosphorylation in the presenceofkemptide is explained (1) by the production of

C*(MgADP) from C(MgATP) during kemptidephosphorylation and (2) by the fact that P-RII competes successfully with P-kemptide for C*(MgADP). This ispossible because P-kemptide bindsto C withabout 1000times loweraffinity thankemptide, theapparentKdofP-kemptide bindingtoCbeing about2-4mM (Whitehouseetal., 1983; Quamaretal., 1992). In contrast, the C subunit binds P-RII(cAMP)2 with only moderately lower affinity than it binds

RII(cAMP)2

(Granotetal., 1980;D0skeland

etal., 1993). Thismeansthatattheconcentrationsofkemptide

used in thepresentstudy (andpresumablyattheconcentrations of most exogenous substrates encountered in the intact cell)

phosphorylated substrates do not compete efficiently with P-RII(cAMP)2 for binding toC*(MgADP).

The mechanism described in the present study may be

im-portant for other kinases releasing phosphorylated product before MgADP. One possible candidate reaction is auto-dephosphorylation of the Ca2+/calmodulin-dependent multi-functional protein kinase II. A sequential release of phosphoproduct and MgADP has been suggested for thisenzyme

(Kwiatkowskietal., 1990), but isnotgenerally accepted (Hanson and Schulman, 1992). It is of interest that the presence of exogenoussubstratewasshowntodecrease therateaswellasthe

steady-state level ofautophosphorylation of thisenzyme(Katoh

andFujisawa, 1991b).

This workwas supported by the Medical Research Council of Norway (NAVF) and the Committee of the Nordic Insulin Foundation.

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