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Functional inhibition of protein kinase C-mediated effects in myocardial tissue is due to the phosphatase 2A

BRACONI QUINTAJE, Silvia, et al.

Abstract

An endogenous protein which inhibits protein kinase C (PKC)-mediated effects has been detected in rat heart ventricular tissue. This functional PKC-inhibitory activity was completely abolished by okadaic acid, making it possible to measure PKC activity in non-purified cell fractions. This suggests that the PKC-inhibitory activity is a type 1 or 2A serine/threonine phosphatase. Confirming this, membrane and cytosolic PKC-inhibitory preparations were found to contain phosphatase activity which was suppressed by okadaic acid, exhibiting an IC50 (concn. required for 50% inhibition) of 1.5-2 nM. Furthermore, okadaic acid stimulated prostacyclin production in rat cardiomyocytes and aortic smooth-muscle cells and, like the PKC activator phorbol 12-myristate 13-acetate, it augmented the prostacyclin formation induced by the Ca2+ ionophore A23187. Our results strongly suggest that the endogenous PKC 'inhibitor' is the cellular phosphatase 2A, which plays an important role in regulating the phosphorylation level of PKC target proteins.

BRACONI QUINTAJE, Silvia, et al. Functional inhibition of protein kinase C-mediated effects in myocardial tissue is due to the phosphatase 2A. Biochemical Journal, 1992, vol. 286 (Pt 3), p.

851-855

PMID : 1329718

Available at:

http://archive-ouverte.unige.ch/unige:1167

Disclaimer: layout of this document may differ from the published version.

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Functional inhibition of protein kinase C-mediated effects in myocardial tissue is due to the phosphatase 2A

Silvia BRACONI, Dennis

J.

CHURCH, Michel B. VALLOTTON and Ursula LANG*

Division ofEndocrinology, University Hospital, CH-121 1 Geneva 4, Switzerland

Anendogenous proteinwhich inhibits protein kinase C (PKC)-mediated effects has been detected in rat heart ventricular tissue.Thisfunctional PKC-inhibitoryactivity wascompletelyabolished byokadaicacid, making itpossible to measure PKC activity in non-purified cell fractions. This suggests that the PKC-inhibitory activity is a type 1 or 2A serine/threonine phosphatase. Confirming this, membrane and cytosolic PKC-inhibitory preparations were found to contain phosphatase activity which was suppressed by okadaic acid, exhibiting an IC50 (concn. required for 50 % inhibition) of 1.5-2 nm. Furthermore, okadaic acid stimulated prostacyclin production in rat cardiomyocytes and aortic smooth-musclecellsand, like the PKCactivator phorbol 12-myristate 13-acetate, it augmented the prostacyclin formation induced by theCa2+ionophore A23187. Our results strongly suggest that the endogenous PKC 'inhibitor' is the cellular phosphatase2A, which plays an important role in regulating the phosphorylation level of PKC target proteins.

INTRODUCTION

Protein kinase C (PKC), a multifunctional serine/threonine protein kinase, is known to play an important role in cellular signal transduction [1,2]. Various physiological functions have been attributed to PKC, such as involvement in secretion and exocytosis, interaction and down-regulation of receptors, smooth-muscle contraction, gene expression and cell prolifer- ation [2].

The enzyme is Ca2+-sensitive and requires phospholipids, particularly phosphatidylserine, for its activation [1]. Agonist- stimulated phospholipid hydrolysis produces diacylglycerol, which activates PKCby promoting its associationwith the cell membrane. Tumour-promoting phorbolesterssuch as phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate cansubstitute fordiacylglycerol asactivators fortheenzyme.

Little is known about thephysiological inhibition of PKC, as there have been few studies concerning endogenous factors inhibiting PKC activity. Pearsonet al. [3] have characterized a heat-stabile13.7 kDainhibitorprotein from bovinebraincytosol.

Another heat-stabile inhibitor of 19kDa, possibly

calmodulin,

hasbeenisolated from the sametissue [4],whereas in rat brain aheat-labile20kDa PKC inhibitor has been detected[5]. Toker etal. [6] have characterized threeheat-sensitive PKCinhibitors (29-33kDa) from sheepbrain containingsequencehomologies with lipocortins. An endogenous PKC inhibitor has also been found in humanneutrophils [7].

Themechanism(s) by whichthese PKC inhibitors interact with PKC remains to be elucidated. Studies using the supposed phosphatase inhibitorNaF[6,7]indicatethat thesePKCinhibi- tors are not associated with phosphatase activity. They also appear not to interact with PKC substrates or cofactors [6,8].

However, Sahyounetal. [9]observed that thecytosolicfraction ofratlivercontainsphosphataseactivitywhichacts onhistones phosphorylated byPKC.

In the present work we describe a functional

myocardial

PKC-inhibitory activity, which was found to be due to the cellularphosphatase 2A.

EXPERIMENTAL

Preparation ofcytosolic and particulate fractions of rat heart ventricular tissue

Heartventriculartissuefrom 50-day-oldrats waswashedand homogenized with 20mM-Tris/HCl buffer, pH 7.5, contain- ing 2mM-EDTA, 10mM-EGTA, 10mM-dithiothreitol, 0.25 M- sucrose and 30,M-leupeptin (buffer A; 1 ml/heart ventricle).

Thehomogenatewascentrifugedat100000g for 1 h at 4°C, and thesupernatantwasused as thecytosolic fraction. The pelletwas incubated for 30 min at 20°CwithTritonX-100(1% in buffer A),diluted to theoriginalvolumeofthehomogenatewithbuffer A (final Triton concn. 0.2%), and centrifuged at 100000g for 1h. The

supernatant

obtained was used as the solubilized particulate fraction.

SeparationofPKC from itsendogenous PKC 'inhibitor(s)' PKCactivitywasalmostundetectable in crude cell fractions, but was

expressed

after DEAE-cellulose chromatography, in- dicatingthatcrudecell fractionsprobably containaninhibitorof PKC and/or a

Ca2l-dependent

proteinase. The cell fractions were applied to a DEAE-cellulose column (2.5cmx0.9cm) equilibratedat4°CinbufferB(20mM-Tris/HCl,pH 7.5, 2 mm- EDTA, 2mM-EGTA and 10mM-dithiothreitol). The columns werewashed with 2x1 ml of bufferB. No enzymeactivitywas detected in these eluates. The PKC activity and the PKC- inhibitory activitywereelutedby using 14ml of a linear NaCl gradient (0-0.3M)inbuffer B. The flow rate was 350,cl/min,and

1.5ml fractions were collected.

Determination ofPKC activityandPKC'inhibitory

activity'

Asample(50

,d)

of each fraction from the columnwasassayed for PKC activity as previously described [10]. Protein kinase activitywasdeterminedby measuringthetransfer of32P from[y- 32P]ATPto histone III-S (Sigma, St. Louis, MO, U.S.A.).The finalreaction mixture(240

,1e)

contained(0.7-1.2)x106

c.p.m.

of

[y-_2P]ATP,

0.15

,tM-ATP,

0.46

mM-CaCl2,

6.9

mM-MgCl2

and 30

,tg

of histone III-S. Each fractionwas

assayed

in the absence

Abbreviations used:PKC,proteinkinaseC; PMA,phorbol 12-myristate 13-acetate;ANP,atrial natriureticpeptide.

* Towhomcorrespondence should beaddressed.

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S. Braconi and others

and presence ofphosphatidylserine (80 ,ug) and diolein (4gg).

The reaction was carried out at 30 °C for 10min and was stopped by adding2ml of 12 % (w/v) trichloroacetic acid in the presenceof 600 jig ofy-globulinadded as carrier. Aftercentrifug- ation(3000 g, 2 min), the pellet was dissolved in 0.5 ml of 1 M- NaOH and precipitated again with 12% trichloroacetic acid.

After a second centrifugation the protein precipitate was dis- solved in 0.5 ml of 1 M-NaOH, and 32P incorporation was measuredbyscintillationcounting in Ultima Gold (Packard).

Asample (50,dl) ofeachfraction resulting from the DEAE- cellulose chromatography of the ventricular cytosolic ormem- branefraction was assayed for its inhibitory activity on the PKC- induced incorporation of 32P into histone III-S. The pooled PKC-containing fractions depicted in Fig. 1 were used for monitoringPKC-inhibitory effects.

Determination of phosphatase activity

HistoneIII-S wasphosphorylatedwithpartiallypurified PKC inthe presenceof[y-32P]ATPat 30 °C for 10 min asdescribed for the PKC assay.Phosphorylationwasstoppedbyincubation for 5min at 50'C. Then a sample (50-200,1) of the inhibitor, partially purified by DEAE-cellulose chromatography, was addedinthepresence andin theabsence of1,lM-okadaicacid. The reaction(dephosphorylation)wascarried out at 37 'C for various times and was stopped by adding 2ml of 12% trichloroacetic acid in thepresenceof 600,tgofy-globulin.Theamountof 32P incorporatedintohistone III-S wasdeterminedasdescribed for thePKC assay.

Cellcultures

Neonatal-rat ventricular cardiomyocytes wereobtained from 1-2-day-oldWistar ratsbythemethod ofKemet al.[11].After sequential digestions with trypsin and DNAase, cells were cultured in 5 ml ofMcCoy's modified SA medium containing insulin/transferrin/sodium selenite medium supplement (5 ,ug/

ml, 5

,gg/ml

and 5ng/ml respectively) and 10 % (v/v) fetal-calf serum. Cultured cardiomyocytes began to contract spontan- eously within 24 h ofplating (30-60 beats/min), released atrial natriureticpeptide (ANP; 5ng/hper 106 cells) and exhibited positive staining for pro-ANP,aprecursorform of ANP and a specific cardiomyocyte marker [12]. Although the presence of contaminating cell types (e.g. endothelial cells) cannot be excluded, prostacyclin production was found to be strongly correlatedwith ANP release, suggesting thatprostacyclin pro- ductionis an indicatorofcardiomyocytesstimulation.Confluent cells were used on the third day of culture for all experiments described herein.

Vascular smooth-muscle cells were isolated from thoracic aortas of female Wistar rats (40-50 days old) as

previously

described [10]. After enzymic dispersion with collagenase and elastase,cells were

plated

in 90mm

plastic

Petridishesorsix-well culture

plates

and cultured in Dulbecco's modified

Eagle's

mediumcontaining10%fetal-calfserum.Confluent

monolayers

were obtained after 6-7

days

of

plating.

Aorticsmooth-muscle cells ofpassages 2-5 were used for theexperiments described herein.

Functional tests

Six-welltissue-cultureplates containingaorticsmooth-muscle cellsor

rhythmically beating cardiomyocytes

were washed and incubated for 20 minat37'C with1 mlofa

Krebs-Ringer

buffer [10],containing0.2% BSA and 0.2

% glucose.

The

supernatant

wasthen

replaced by

fresh

buffer,

and the cellswereincubated at 37°Cwith

air/CO2 (19: 1),

in thepresenceof various

stimulating

agents. At the end of the incubation

period,

the

prostacyclin

content of the media was determined

by

a

specific

radio-

immunoassayof its stablemetabolite6-oxo-prostaglandin

Fl

as described previously

[10,13],

by using aspecific rabbit antiserum which was kindly provided by Dr M. J. Dunn (Division of Nephrology,CaseWestern Reserve University, Cleveland, OH, U.S.A.).

Statisticalanalysis

ANOVA using the Scheffe F-test criterion for unbalanced groups was applied forstatistical comparison.

RESULTS AND DISCUSSION

Fig. 1 showsthe elutionpatternsof PKC activityand PKC- inhibitoryactivitiesfromDEAE-cellulose columnswith alinear gradientof

0-0.3 M-NaCl.

OnepeakofPKC-inhibitory activity wasfoundinthe membrane fractions ofheartventricular tissue, eluted after PKC, between 110mm- and 240mM-NaCl. In contrast, twopeaksofPKC-inhibitoryactivity were eluted from thecytosol; afirst, smaller one, eluted before PKC atapprox.

14

mM-NaCl,

anda second, muchlarger one, elutedafter PKC between

110

mM- and 250

mM-NaCl

(Fig. 1). In this study we examined the PKC-inhibitory activity eluted at the higher

80

60

40

._

0 E 0i

x 0

20

0 120 100 80 60 40 20 0

Membranes

1 2 3 4 5 6 7 8 9 10 11

120

80

40

m-q

0

St

C

U.2.

oi 120 .~.0

C

C.)

80

40

1 2 3 4 5 6 7 8 9 10 11

Fractionno.

Fig.1.Elution of PKC andPKC-inhibitoryactivitiesfromDEAE-cellulose chromatography

Cytosolic and membrane fractions wereprepared fromrat heart ventricular tissue and applied to a DEAE-cellulose column as describedin theExperimentalsection. Elutionwascarriedoutwith a linear(0-0.3M) NaCl gradient. Samples of each fraction were

assayed for PKCactivityand forPKC-inhibitory activity (seethe Experimental section). PKCactivity

(EO)

is expressedasc.p.m. of 32Pincorporatedinto histoneIII-S/10minper100,1 sample.PKC- inhibitory activity(El)wastestedby adding50,ul of each fraction to PKC samples of previously determined activity (defined as 0%

inhibition). NaCl concentration (iii i), determined byconductivity measurements,wasfoundtoincreasefrom0.01M(fractions1-3)to

0.25M(fraction 1).TheFigurerepresentsatypicalexamplefroma

series ofsevenexperimentsthatconsistently gave thesametypeof results.

852

(4)

Table 1. Influence ofcytosolic and membrane PKC-inhibitor preparations onPKC activity

Cytosolicand membrane PKC activity eluted fromDEAE-cellulose columns was measured without (control) and with membranous (Memb) or cytosolic (Cyt) PKC-inhibitor preparations (IP) (18,g of protein/50,l), in the presence and in the absence of1UMokadaic acid (OA). PKC activity is expressed as c.p.m. of32pincorporated into histone III-S/10min per 50,ul sample. Each values is the mean +S.E.M. of four determinations.

PKC activity (c.p.m.)

Treatment Cytosol Membrane

Control Control +OA CytIP Memb IP CytIP+OA Memb IP+OA

3094+75 3496+95 876+104 814+89 3214+ 166 3312 +281

1951 +47 2127+55 430+62 397 + 59 2012+ 114 2106+ 161

Table 2. Influence of okadaic acid on kinase activity in non-purified subceliularfractions of heart ventriculartissue

Kinase activity was measured in non-purified cytosolic and mem- brane fractions with or without 1

IsM-okadaic

acid (OA). The difference between kinase activities in the presence and in the absence of 80,g ofphosphatidylserine (PS) and4,tgof diolein (D) represents PKC activity. Protein kinase activity is expressed as c.p.m.of32pincorporated into histone

III-S/IO

min per 5,lsample.

Values are means + S.E.M. of six determinations.

Kinase activity (c.p.m.) Cell fraction WithoutPS/D WithPS/D Cytosol

Without OA With OA Membranes

Without OA WithOA

3884+613 3641 +496 4240+384 10824+129 2892+461 2165+320 2803 +285 6768+488 concentrations ofNaCl. Thecharacterization ofthefirst small

peak of PKC-inhibitory activity eluted from cytosolic prepar- ations and its relationship to the second large peak require further study.

The fractionscontaining the cytosolic and the membrane PKC 'inhibitor', eluted afterthe PKC, were pooled and were compared for their inhibitory action on cytosolic and membrane PKC activity. As showninTable 1, these two 'inhibitors' decreased both cytosolicandmembrane PKC activity to the same extent.

Both inhibitorswereheat-sensitive; inhibitory activitywasfully retainedafter5min at 50 °C,butwascompletelyabolished after heating for 5 min at

700

or 100'C.

Exposureofcytosolic and membrane PKC inhibitor to trypsin (0.2mg/ml) for2h at 37 'C also completely suppressed PKC- inhibitory activity, suggesting that the PKC inhibitor is a polypeptide. Inthe presenceofthealleged phosphatase inhibitor NaF (10

mM),

the heart ventricular PKC inhibitor retained its fullactivity.These results are in agreement with thefindings by Toker et al. [6], who observed that sheep brain contains PKC- inhibitory activity which is eluted after PKC from DEAE- cellulose chromatography and which is also insensitive to the presence of NaF. Toker et al. [6] concluded that the PKC- inhibitory activityisolated frombrainhomogenatecontains no phosphatase activity.

In the present study we have taken advantage of the recent availability of okadaic acid, a potent and specific inhibitor of serine/threonine protein phosphatases [14,15], to characterize the nature ofthe endogenous PKC-inhibitory activityin heart ventricular tissue. Okadaic acid, a polyetherderivative of

C3.

fatty acid [16], has ahighly specific inhibitory action of type 1 and type 2Aphosphatases [14,15].Ithas been shownto cause a general stimulation of

protein phosphorylation

in intact cells withoutaffectingany of the relevantproteinkinases [17],and it has also been reported to be a potent tumour promoter that is not anactivatorof PKC[18].

Addition of 1,lM-okadaic acid abolished all PKC-inhibitory activityeluted from DEAE-cellulosechromatographyfrom cyto- solic and membrane fractions of heart ventricular tissue, as shown in Table 1. This observation indicates that the heart ventricular PKC 'inhibitor' is a serine/threonine phosphatase

(phosphatase-l

and/or -2A).

Asmentionedpreviously,PKCactivitywas notmeasurable in non-purified cell fractions. However, addition of 1

/nM-okadaic

acid made itpossibleto measurePKCactivityinsmallsamples of non-purified cytosolic and membrane fractions from heartven-

triculartissue,asshownin Table 2. The valuesobtainedby this method were slightly but not significantly higher than those obtained by using DEAE-cellulose chromatography. In four different preparations the values of cytosolic and membrane PKC activity were found to be 116+12% and 121+16%, respectively,of thePKCactivitiesmeasured inDEAE-cellulose- purified subcellular fractions. Similarly, addition of0.1-11M- okadaic acidmade italso possibleto measure PKCactivity in smallsamples of crude cell fractions from cultured neonatal rat cardiomyocytes and aortic smooth-muscle cells (results not shown). Thus, in the presence of an adequate okadaic acid concentration for the amount of phosphatase(s) present in the sample, it is possible to measure PKC activity without prior chromatographicpurification.

The elution pattern of PKC activity after DEAE-cellulose chromatography (Fig. 1) was not significantly changed when PKCactivity was measured in the presence of 1

,tM-okadaic

acid (results not shown), whereas all PKC-inhibitory activity was abolished, suggesting that DEAE-cellulose chromatography efficientlyseparates PKCfrom cellularphosphatase(s).

The conclusion that endogenous PKC-inhibitory activity is due to the presence of cellular phosphatase(s) was further confirmedbythefactthatPKC-inhibitory preparationsshowed a pronounced phosphatase activity which was concentration- andtime-dependent (Fig. 2). However,thisphosphatase didnot dephosphorylateentirelyhistone III-S that had beenphosphoryl- atedwithpartially purifiedPKC and[y-32P]ATPinthe presence ofphospholipids. Interestingly,the remaining8410+644c.p.m.

of32P-labelledhistoneIII-Scorrespondedtothe8559+591 c.p.m.

of 32P incorporated in the absence of phospholipids. Thus the phosphatase activity of the PKC-inhibitory preparation appears to affect mainly

PKC-specific

phosphorylation. The samephosphatase activitywascompletelyabolishedbyaddition of 1

4aM-okadaic

acid, asillustrated in

Fig.

2.

Fig. 3 shows that okadaic acid inhibited this phosphatase activityinaconcentration-dependentmanner,

exhibiting

for the membrane and thecytosolic phosphataseasimilar

IC50

of1.5nm and 2nm respectively. These results suggest that in both cell fractions theendogenousPKC 'inhibitor' is thephosphatase

2A,

since it has been reported that the

IC50

for

phosphatase

2A ranges from 1 to 2 nm[14,15],whereasthosefor

phosphatases

1 and 2B are in the ranges 270-500 nm and 3-10

/tM respectively,

andphosphatase 2C isnotaffected byokadaic acid

[14,15].

In order to study the cellular influence of

serine/threonine

(5)

S.Braconi and others

25 E

Q

0-

C)

0

CL

0~

0

15

0)

F .D

10 lo

x

5 0

Fig.2. Determination aration

30 60

Time (min)

*i 1.5-

0

0.

o 1.0-

0

0m 0.5- aC') a

(D 0

= 8 - 'a

Q 4-

m

x

O 2- Co 90

0 of phosphatase activity in PKC-inhibitory prep-

HistoneIII-S wasphosphorylatedwith partially purified PKC in the presenceof[y-3CP]ATPandphospholipidsat 30'Cfor 10min(see theExperimental section). Dephosphorylation was carried out by adding200,ul of20mM-Tris/HClbuffer, pH 7.4, containing PKC- inhibitory preparation from DEAE-cellulose chromatography, in the absence and in the presence of 1,uM-okadaic acid. Control, I---LE;addition ofPKC-inhibitory activity (62 and 124 ug of protein, O---O and 0 O; addition of okadaic acid and PKC-inhibitory activity (124,mgof protein),0 0.The reaction mixture was incubated at 37°C for the indicated times, and the phosphorylation level of histone III-Swasdetermined as described in theExperimental section. Each value is themean +S.E.M.of3-5 determinations.

(a)Cardiomyocytes r P < 0.01 -

(b) Aortic smooth-muscle cells

2r

r P<0.01 -,

X1 71 -z

C A23187 PMA A23187

+PMA

Fig. 4.Effect of okadaic acid on prostacyclin production in rat cardio- myocytes and aortic smooth-musclecells

Neonatal-ratventricular cardiomyocytes and aortic smooth-muscle cells were isolated and cultured as described in the Experimental section. Confluent cultured cardiomyocytes (a)and aortic smooth- muscle cells (b) were stimulated with 0.1 zM-PMA and/or 1,M- A23187for 30 min in the absence (O) and in the presence 0)of 1

/LM-okadaic

acid. The incubation medium was removed and assayed for 6-oxo-prostaglandin

F,,

release.Valuesrepresent means (± S.E.M) of3-4 separateexperiments performed in triplicatedeter- minations.

150-

.220

"o 0.

o 0100 1

0-,) If

C.)

50-

(D4..

a4..r

00 Ln.-...

0 lo-lo 10~q 10 -7i0

[Okadaicacid](M)

Fig.3.Concentration-dependenceof theinhibitory effectof okadaic acid onphosphataseactivity

HistoneIII-Swasphosphorylatedwithpartially purifiedPKC and

[y-3CP]ATPin thepresence and absenceofphospholipids at30°C for 10 min (see the Experimental section). PKC-specific 32P in- corporation into histone III-S was calculated by subtracting the phospholipid-independent32Pincorporationfrom thephospholipid- dependent32Pincorporation.Phosphataseactivitywasinvestigated by adding200,1 of20mM-Tris/HCl buffer, pH7.4(control), or 200,1 ofTris/HCl buffercontaining membrane (0) orcytosolic

(0) PKC-inhibitory preparation

from DEAE-cellulose chromat- ography, in thepresenceof theindicated concentration of okadaic acid. Thereactionmixturewasincubatedat37'Cfor 40 min. The amount of32Pincorporated intohistone III-S wasdetermined as described in the Experimental section and is expressed as % of control.Each valueisthe mean+S.E.M.of3-6determinations.

phosphatase(s), we examined the effect of okadaic acid on cardiomyocytes and aortic smooth-muscle cells. In both cell typesPMA has beenshowntoinducePKCactivation,leading to prostacyclin production [10,19]. PMA has also been found to augmenttheprostacyclinresponseinduced by increasedcytosolic freeCa2l concentrations[13,19]. AsshowninFig. 4, exposure to 1,tM-okadaic acid for30 minincreasedprostacyclin production from0.15+0.02 to0.47+0.04ng/mgofcellprotein in cardio- myocytes and from 0.14 +0.02 to 2.21 +0.29 ng/mg of cell protein inaorticsmooth-musclecells. Like PMA, okadaicacid augmented the A23187-induced prostacyclin response in both cell types. Sinceprotein

phosphatase-1

and-2Aare likelyto be themain enzymes thatreversetheeffects of PKC[20], it appears that a constant serine/threonine phosphatase activity is re- sponsible for maintaining low levels of PKC-dependent phosphorylation in resting cells. Our results obtained with okadaicacidon

prostacyclin

productionincardiomyocytesand aortic smooth-musclecells indeed resemble thoseobserved with PMA.Similarobservations havebeen madebyTantietal.[21], studyingthe influence of PMA and okadaic acidon theuptake of 2-deoxyglucose by skeletal muscle. However, inhibition of phosphatase 2A by okadaic acid does not appear to mimic entirely the activation of

PKC-dependent protein phosphoryl-

ation, since in aortic smooth-muscle cells stimulation of pro- stacyclinformation is greater withokadaic acid than with PMA (P<0.01), whereas incardiomyocytestheokadaic acid-induced response is weaker than that stimulated by PMA (P<0.01)

(Fig. 4).

In summary, ourresults suggest that the endogenous PKC- inhibitory activity detected in the

cytosolic

and membrane fractions of heart ventricular tissue is due to the cellular 854

(6)

phosphatase 2A playing an important role in regulating the phosphorylation level ofPKC targetproteins.

Wethank Ms. C. Gerber-Wicht, Mrs. M. Rey and Mrs. M. Klein for their excellent technical assistance, Mrs. C. Gerber-Wicht and Mrs. M. Rey for skilful secretarial help andDr.M. F. Rossier andDr. A. M. Capponi for helpful discussions. This study was supported by Grants from the Swiss National Science Foundation (31.27727.89), from the Swiss Foundation of Cardiology, from the Geneva Cancer League and from the Sandoz Foundation for Advancement of Medical and Biological Sciences.D. J.C.waspartially supported by a post-graduatescholarship from the Glaxo Institute of Molecular Biology, Geneva, Switzerland.

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18. Suganuma, M.,Fujiki, H., Suguri, H., Yoshizawa, S., Hirota, M., Nakayasu, M., Ojika, M., Wakamatsu, K.,Yamada,K.&Sugimura, T.(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1768-1771

19. Church,D.J., Lang, U., Erbrich, A. C. & Vallotton,M. B. (1991) in Prostaglandin, Leukotrienes and PAF (Bailey, J.M., ed.), pp. 187-197,PlenumPublishing Corp.,NewYork

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Received29January1992/23 March 1992; accepted 1 April 1992

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M. Bonnet, Jean-François Hocquette, Yannick Faulconnier, J. Fléchet, Francois Bocquier, et al.. Nu- tritional regulation of lipoprotein lipase activity and its messenger RNAs in

They first shown with two PknG substrate GarA, a FHA containing domain protein, and a peptide derived from the phosphorylation site (17-mer) that both Nter/Rdx

In order to identify epigenetic mechanisms through which hyperglycemia can affect gene expression durably in b cells, we screened DNA methylation changes induced by high

Site-directed mutagenesis experiments were performed on the RVXF binding motif of PfPP1 ( 255 FF 256 /AA) as well as on the RVXF motif of the PIP region of PfRCC-PIP ( 980 KSASA 984

The orientation of the WNW-oriented folds of stage 2, such as the Chelopech syncline and affecting the Late Cretaceous magmatic and sedimentary rocks in the Panagyurishte district,

Les salariés qui ne bénéficient pas de jours de RTT, ne rapportent jamais de travail à leur domicile et ne font jamais d’heures complémentaires ou supplémentaires ont une durée