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THE JOURNAL or BiOLOGICAL CHEMISTRY ' _ Vol. 263, No. 34, Issue of December 5, pp. 184S9-18465,1988
© 1988 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Pattern of Protein Phosphorylation in Aortic Endothelial Cells
MODULATION BY ADENINE NUCLEOTIDES AND BRADYKININ*
(Received for publication, May 11, 1988)
Dominique Démolie^, Marc Lecomte, and JeanMarie Boeynaems
From the Institute of Interdisciplinary Research, School of Medicine, Free University of Brussels, Campus Erasme, Building C,
Route de Lennik 808, 1070 Brussels, Belgium
In bovine aortic endothelial cells, ATP (10—100 ^m)
and bradykinin (0.11.0 ^M) enhanced the phospho
rylation of two major protein substrates with apparent
molecular masses of 95 and 28 kDa. The action of ATP
involved P2y purinoceptors. The kinetics were distinct
for the two phosphopeptides. The phosphorylation of
the 95kDa protein was rapid (within 30 s) but tran
sient (maintained for only 2 min). This time course
agrées with that observed for the increase of the cyto
solic Ca^* level induced by ATP in thèse cells. lono
phore A23187 (^ 100 nM) induced this phosphorylation
for a longer period (510 min), whereas phorbol 12
myristate 13acetate (PMA) was completely inactive.
The enhancement of the 28kDa protein phosphoryla
tion was détectable after a 5min lag and was main
tained for at least 20 min. PMA (50 nM) stimulated
weakly the phosphorylation of the 28kDa protein,
whereas A23187 (100300 nM) was even more effec
tive than ATP and bradykinin. The 95kDa phospho
protein seems to be related to a 100kDa substrate of
calmodulindependent protein kinase III recently iden
tified as elongation factor2. The 28kDa protein,
which was resolved as three variants in bidimensional
gel electrophoresis, appears very similar to a slightly
heavier phosphoprotein from thrombinstimulated hu
man platelets. In addition, bidimensional electropho
resis allowed the détection of at least 10 substrates
(from 18 to 46 kDa) whose phosphorylation was en
hanced equally well by ATP, bradykinin, and A23187
and only partially by PMA. In conclusion, protein phos
phorylation induced by ATP and bradykinin in aortic
endothelial cells seems to be catalyzed mostly by Ca^*-
dependent kinases, distinct from protein kinase C.
The thromboresistance of the vascular endothelium in
volves several mechanisms. One of them is the release of
prostacyclin (PGI2)' and nitric oxide: PGI2 and nitric oxide
* This work was performed under contract of the "Ministère de la
Politique Scientifique" (Action Concertée) and supported by a grant
from the "Fonds de la Recherche Scientifique Médicale." The costs
of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked "aduer-
tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
t Supported by the Association de Recherche Biomédicale et de
Diagnostic (ARBD).
' The abbreviations used are: PGI2, prostaglandin I2 (prostacyclin);
ADP;8S, adenosine 5'0(2thiodiphosphate); ATP7S, adenosine 5'
0(3thiotrisphosphate); APCPP, adenosine 5'(a,/3methylene)tris
phosphate; APPNP, adenosine 5'(/3,7imido)triphosphate; BAEC,
bovine aortic endothelial cells; Chaps, 3[(3cholamidopro
pyl)dimethylammonio]lpropanesulfonic acid; CaM, calmodulin;
IP3, inositol (l,4,5)trisphosphate: MEMDVal, minimal essential
médium with DvaHne; PAEC: porcine aortic endothelial cells; PGEi,
prostaglandin Ei; PMA, phorbol 12myristate 13acetate; SDS, so
dium dodecyl sulfate; HUVEC, human umbilical vein endothelial
cells.
synergize with each other to inhibit the aggregation of plate
lets (1), whereas nitric oxide decreases their adhésion to the
endothelium (2). Cultured endothelial cells release PGI2 in
response to multiple agonists. Human umbilical vein endo
thelial cells (HUVEC) are stimulated to synthesize PGI2 after
addition of thrombin (3) and histamine (4), whereas ATP and
ADP stimulate PGI2 production selectively in aortic endothe
lial cells from bovine (BAEC) (5) and porcine (PAEC) (6)
origin. Bradykinin, in addition to its stimulatory effect on
PGI2 release from BAEC and PAEC (7), stimulâtes the pro
duction of nitric oxide by PAEC (8).
The action of ADP and ATP might be physiologically
relevant. Thèse adenine nucleotides are indeed released dur
ing platelet activation, so that they might act as the mediators
of a négative feedback loop: they would stimulate the release
of PGI2 and nitric oxide from the endothelium adjacent to the
thrombus in formation, in order to limit the extent of platelet
aggregation. The responses of endothelial cells to ATP seem
to be mediated by P2y purinoceptors according to Burnstock's
classification (9, 10). One transduction mechanism linked to
thèse receptors has been recently identified: the hydrolysis of
phosphatidylinositol bisphosphate. ATP induces indeed a
rapid and transient increase of the level of inositol (1,4,5)
trisphosphate (IP3) (11, 12) and of cytosolic Ca^* (11, 13, 14)
in aortic endothelial cells. Bradykinin reproduces thèse effects
of ATP in aortic endothelial cells (1518), as do thrombin
and histamine in HUVEC (19, 20). In addition, several studies
have pointed out the rôle played by protein kinase C in the
control of endothelial cells functions. Phorbol 12myristate
13acetate (PMA), a potent activator of protein kinase C
which mimics the effect of endogenous diacylglycerol, induces
the production of plasminogen activator (tPA) (21), the
sécrétion of von Willebrand protein (22), and angiogenesis
(23). Our démonstration that PMA and the Ca^* ionophore
A23187 act synergistically to stimulate PGI2 production by
BAEC (24) seems to indicate a coopération between cytosolic
Ca^"^ and diacylglycerol as second messengers.
In many Systems, the effects of second messengers appear
to be mediated by the activation of spécifie protein kinases
that phosphorylate a variety of substrate proteins. Studies of
protein kinases have already been performed in extracts of
BAEC where cAMPdependent kinase, calmodulin (CaM)
dependent protein kinase (CaM kinase II, CaM kinase III)
and protein kinase C activities were detected (25); in addition,
a kinase activity distinct from those mentioned above was
measured in membranes of proliferating BAEC (26). However,
except for a few preliminary (27, 28) or partial (29) studies,
the patterns of protein phosphorylation in intact endothelial
cells have not yet been fully characterized. In this paper, we
show that ATP and bradykinin induce or enhance the phos
phorylation of several proteins in intact BAEC, mostly via
Ca^*dependent kinases.
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