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INTRODUCTION |
Thrombin belongs to the multifunctional serine protease family and
plays an important role in the blood coagulation cascade through the
cleavage of fibrinogen to fibrin (1, 2). Thrombin also exerts direct
effects on cells to regulate platelet aggregation, endothelial cell
activation, and smooth muscle cell
(SMC)1 proliferation via
interactions with members of the protease-activated receptor (PAR)
family, such as PAR1, PAR2, PAR3, and PAR4, known as G-protein-coupled
receptors (2, 3). However, the intracellular signaling cascades
downstream from the thrombin receptors are surprisingly complex and are
still not well understood.
Protein kinase D (PKD), also known as protein kinase Cµ (4, 5), is a
newly described serine/threonine protein kinase with unique structural,
enzymological, and regulatory properties that are different from those
of the PKC family members. The most distinct characteristics of PKD are
the presence of a catalytic domain distantly related to
Ca2+-regulated kinases, a pleckstrin homology domain within
the regulatory region, and a highly hydrophobic stretch of amino acids
in its N-terminal region (6, 7).
PKD can be activated by a variety of stimuli including biologically
active phorbol esters, growth factors, and T- and B-cell receptor
agonists via PKC-dependent pathways (6, 7). PKD activation
appears to involve the phosphorylation of Ser-744 and Ser-748 within
the activation loop of the catalytic domain as well as the
autophosphorylation of Ser-916 (6). PKD has been implicated in the
regulation of a variety of cellular functions including NF
B-mediated
gene expression, Na+/H+ antiport activity,
Golgi organization and function, and protein transport (7, 8). The aim
of the present study is to determine whether and how thrombin activates
PKD in living cells. Our results demonstrate that thrombin rapidly and
markedly induces PKD activation in SMC. Furthermore, our results
demonstrate the following: 1) a PKC
-specific inhibitor inhibits
thrombin-induced activation of PKD; 2) overexpression of a dominant
negative PKC
abolishes PKD activation; and 3) PKC
interacts with
PKD. PKC has been implicated in many cellular responses to thrombin
(9-11). Despite the importance of PKC in thrombin-induced signal
transduction, the downstream targets of PKC in the signaling cascades
remain largely undefined. Thus, our finding that thrombin induces
PKC
-dependent PKD activation reveals a novel
thrombin-induced signaling pathway in living cells.
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EXPERIMENTAL PROCEDURES |
Materials--
Reagents were obtained as follows:
thrombin from Sigma; protein kinase inhibitors Ro 31-8220, U-0126, GF
109203X, SB-203580, LY294002, and rottlerin from Biomol (Plymouth
Meeting, PA); antibodies against PKC
and PKC
from BD Transduction
Laboratories (San Diego, CA); an antibody against PKC
from Upstate
Biotechnology (Waltham, MA); antibodies against PKD and phospho-PKC
from Santa Cruz Biotechnology (Santa Cruz, CA); and antibodies against
phospho-PKC isoforms (
and
) and phospho-PKD (phosphorylated
Ser-744/Ser-748 and phosphorylated Ser-916) from Cell Signaling
Technology (Beverly, MA).
Cell Culture--
Rat aortic smooth muscle cells were isolated
from explants of excised aortas of rats and were maintained in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
as described previously (12). The SMC between passages 6 and 17 were
used in this study.
Adenovirus Constructs and Adenoviral Infection of
SMC--
Adenoviruses encoding mouse PKC isotypes (
,
, or
)
were constructed as previously described (13, 14). SMC were infected for 24 h with either wild type or dominant negative PKC isotypes.
Immunoprecipitation and Western Blotting Analysis--
SMC or
SMC infected with virus expression vectors were serum-starved in a
serum-free medium for 24 h prior to treatment with thrombin. After
treatment with thrombin, the cells were lysed and were subjected to
immunoprecipitation and Western blot analysis as described previously
(15).
In Vitro Kinase (IVK) Assay--
PKD autophosphorylation was
determined as described previously (15).
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RESULTS |
Thrombin Induces PKD Activation in Living Cells--
To examine
whether thrombin induces PKD activation in living cells, we first
performed an IVK assay to determine the autophosphorylation activity of
PKD. Serum-starved rat aortic SMC were exposed to 0.1 unit/ml thrombin
for various periods of time, the cells were lysed, and PKD was
immunoprecipitated with a PKD-specific antibody. The resulting
immunocomplexes were incubated with [
-32P]ATP, and the
incorporation of 32P into PKD was analyzed by SDS-PAGE and
autoradiography. As shown in Fig.
1A, stimulation of the SMC
with thrombin resulted in a striking activation of PKD, which was
detected after 45 s of thrombin stimulation and reached a peak at
2-4 min.

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Fig. 1.
Thrombin induces PKD activation in SMC.
A, IVK assay. B, PKD phosphorylation was detected
by using phosphospecific antibodies: anti-p-PKD
(S744/748) (middle panel) and
anti-p-PKD (S916) (top panel). PKD expression
levels were determined using a PKD antibody (bottom panel).
All data presented in this study are representative of at least three
independent experiments.
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Thrombin-induced PKD activation also was determined by using two
recently available phospho-PKD-specific antibodies that recognize phosphorylated Ser-916 as well as phosphorylated Ser-744 and Ser-748 of
PKD. The residues Ser-744 and Ser-748 in the activation loop of PKD
have been identified as critical phosphorylation sites in PKD
activation induced by phorbol esters, and Ser-916 is autophosphorylated when PKD is activated (6). By using these antibodies, we observed that
thrombin rapidly and transiently induced PKD phosphorylation (Fig.
1B).
Thrombin Stimulates PKD Activation through a
PKC-dependent Pathway--
Reportedly, PKD is activated in
PKC-dependent fashion (6). To determine whether PKC
activation is involved in thrombin-induced PKD activation in SMC, we
examined the effect of two PKC inhibitors, GF 109203X and Ro 31-8220, on PKD activation stimulated by thrombin. Serum-starved SMC were
treated with GF 109203X and Ro 31-8220 for 40 min prior to a 3-min
exposure to thrombin (0.1 unit/ml). As shown in Fig.
2, GF 109203X at a concentration as low
as 0.5 µM completely blocked PKD activation (left
panels). Thrombin-induced PKD phosphorylation also was blocked by
Ro 31-8220 in a concentration-dependent fashion
(middle panels). These data suggest that PKC is involved in
the thrombin-stimulated PKD activation.

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Fig. 2.
Thrombin induces PKD activation through a
PKC-dependent pathway. General PKC inhibitors GF
109203X (left panels) and Ro 31-8220 (middle panels) but not the MEK inhibitor (U0, 10 µM), the phosphoinositide 3-kinase inhibitor (LY, 50 µM), or the p38 MAPK inhibitor (SB, 10 µM)
(right panels) inhibit thrombin-induced PKD activation. PKD
activation was determined by Western blot analysis using the
phospho-PKD antibodies anti-p-PKD (S744/748) and
anti-p-PKD (S916). PKD expression levels were determined
using the PKD antibody. p, phospho; U0, U-0126;
LY, LY 294002; SB, SB-203580.
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We also examined whether MEK inhibitor U-0126,
phosphoinositide 3-kinase inhibitor LY 294002, or p38 mitogen-activated
protein kinase inhibitor SB-203580 affects the activation of PKD. As
shown, none of these inhibitors had any effect on thrombin-induced PKD activation (right panels of Fig. 2). These results suggest
that PKC but not MEK, phosphoinositide 3-kinase, or p38
mitogen-activated protein kinase is required for thrombin-induced PKD
activation in SMC.
PKC
Is Rapidly Activated by Thrombin in SMC--
The finding
that PKC activation is involved in thrombin-induced PKD activation
prompted us to determine which isotype of PKC is required for PKD
activation. Previous studies of SMC have shown that PKC
, PKC
,
PKC
, PKC
, and PKC
are expressed in SMC (16-19), and among
them, PKC
is most abundantly expressed in rat aortic SMC (20). We
first determined which PKC was activated by thrombin in SMC. As shown
in Fig. 3A, phosphorylation of
PKC
was rapidly induced at 45 s upon thrombin treatment of the
SMC; in contrast, no thrombin-induced phosphorylation of PKC
,
PKC
, PKC
, or PKC
was detected (Fig. 3A).

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Fig. 3.
Thrombin rapidly activates
PKC , and the
PKC -specific inhibitor rottlerin blocks
thrombin-induced PKD activation in a dose-dependent
manner. A, time course of thrombin-induced
phosphorylation of PKC isotypes in SMC. No changes in the basal
phosphorylation levels of PKC / (second panel), PKC
(third panel), or PKC / (fourth panel) were
detected through the time course of thrombin stimulation. B,
effect of the PKC -specific inhibitor rottlerin on PKD
activation.
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PKC
Inhibitor Rottlerin Blocks PKD
Activation--
We next examined whether activation of PKC
contributed to thrombin-induced PKD activation by determining the
effect of the PKC
-specific inhibitor rottlerin on thrombin-induced
PKD activation. Rottlerin has been reported to inhibit selectively
PKC
activation (IC50 = 3-6 µM) 5-10-fold
more potently than PKC
and PKC
and 13-33-fold more potently than
PKC
, PKC
, and PKC
(21). The SMC were pretreated with rottlerin
for 40 min followed by stimulation with thrombin for 3 min. As shown in
Fig. 3B, rottlerin inhibited thrombin-triggered PKD
activation in a concentration-dependent fashion. These results
suggest that thrombin-induced PKD activation is dependent on PKC
activity in SMC.
Dominant Negative PKC
Blocks Thrombin-induced PKD
Activation--
To substantiate further the role of PKC
in
mediating thrombin-induced PKD activation in living cells, we examined
the effect of the dominant negative form of PKC
on thrombin-induced
PKD activation. The dominant negative nature of the ATP-binding site mutant PKC
has been previously characterized (22). We used recombinant adenovirus constructs to overexpress specific PKC isoforms
in SMC and to determine the effects of these dominant negative isoforms
of PKC on thrombin-induced cellular PKD activation. As shown in Fig.
4, A-C, infection of the SMC
with adenovirus constructs containing cDNAs for wild type or
dominant negative PKCs resulted in robust expression of these PKC
isoforms. As shown in Fig. 4A, at a multiplicity of
infection of 30, infection of the SMC with an adenovirus construct that
encodes the dominant negative PKC
almost completely blocked
thrombin-induced PKD activation as determined by an IVK assay and by
measuring PKD phosphorylation at Ser-744/Ser-748 and Ser-916, whereas
wild type PKC
had no detectable effect on thrombin-induced PKD
activation when compared with the effect of non-infected controls. In
contrast, neither dominant negative PKC
and PKC
nor wild type
PKC
and PKC
, at the same multiplicity of infection, affected PKD
activation (Fig. 4, B and C). These data further
indicate that PKC
mediates thrombin-induced PKD activation in
SMC.

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Fig. 4.
Overexpression of dominant
negative PKC but not of dominant negative
PKC or PKC blocks
thrombin-induced PKD activation. Cells infected with the
adenovirus expressing wild type (PKC(wt)) and dominant
negative (PKC(D/N)) PKC isotypes are indicated at
the top. The expression levels of each recombinant protein
are shown in the top row of each panel. Effects
of wild type and dominant negative mutants of PKC (A),
PKC (B), and PKC (C) on thrombin-induced
PKD activation were examined using phospho-PKD-specific antibodies. The
fourth row in A, B, and C
is the PKD protein level determined using the anti-PKD antibody. As
shown in A, the effect of PKC on thrombin-induced PKD
activation also was determined by an IVK assay (fifth row).
MOI, multiplicity of infection.
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PKC
Interacts with PKD in Intact Cells--
The
above results indicated that PKC
functionally mediates PKD
activation in response to thrombin. We further asked whether PKC
physically interacts with PKD in SMC. To address this question, we
infected SMC with an adenovirus vector encoding PKC
, PKC
, or
PKC
at the same multiplicity of infection. The cell lysates were
immunoprecipitated with anti-PKD antibody. The resulting immunocomplexes were subjected to SDS-PAGE and were probed with PKC
-, PKC
-, or PKC
-specific antibodies. As shown in Fig.
5A, PKC
was
co-immunoprecipitated with PKD in living cells. In addition, we also
observed that, consistent with the observation reported previously
(15), PKC
, but not PKC
, was co-immunoprecipitated with PKD (Fig.
5, B and C). It should be noted that although
both PKC
and PKC
physically interact with PKD in SMC, only PKC
functionally mediates thrombin-induced PKD activation in aortic
SMC.

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Fig. 5.
PKD interacts with PKC
and PKC but not with
PKC . SMC were infected (infec)
with adenoviruses expressing wild type PKC , PKC , and PKC at a
multiplicity of infection of 30 for 24 h. Cell lysates were
immunoprecipitated (IP) with (+) or without ( ) a PKD
antibody in the presence of protein-A beads. The immunoprecipitates
were probed with isoform-specific antibodies against PKC
(A), PKC (B), or PKC (C). Cell
lysates were used as controls (third lane of each
panel).
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DISCUSSION |
Thrombin has many important biological effects on the vascular
wall. Though much has been learned during the last decade about the
cell-surface receptors of thrombin, the intracellular signaling cascades, especially the early signal transduction mediators
responsible for the action of thrombin on cells, are still not well understood.
The results presented here have demonstrated that thrombin induces a
remarkable activation of PKD in living cells. Stimulation of
aortic SMC with thrombin leads to a rapid and transient activation of
PKD, occurring within seconds of thrombin stimulation of aortic SMC.
Interestingly, PKD is activated more rapidly than other kinase cascades
induced by thrombin, including Elk1 (23), NF
B (24), and nuclear
diacylglycerol kinase
(25). Thus, PKD activation is one of the
earliest events induced by thrombin in living cells.
Our results revealed a novel signaling pathway in which
PKC
mediates thrombin-induced PKD activation. To date, the role of PKC
in PKD activation is totally unknown, and although thrombin has
been shown to activate PKC
in several types of cells (9, 26, 27),
the downstream target of PKC
is still unclear. Our data established
for the first time that thrombin-induced PKD activation is mediated by
PKC
in vascular SMC.
We employed multiple approaches to address the specificity of the
PKC
function in mediating thrombin-induced PKD activation. The
general PKC inhibitors GF 109203X and Ro 31-8220 blocked
thrombin-induced PKD activation in a concentration-dependent
manner (Fig. 2), suggesting that thrombin induces PKD activation
through a PKC-dependent pathway. The fact that thrombin
induced the activation of PKC
and that the PKC
inhibitor
rottlerin blocked thrombin-induced PKD activation in a
concentration-dependent manner strongly suggests the
functional involvement of PKC
in thrombin-induced PKD activation in
SMC (Fig. 3). To substantiate further the role of PKC, we employed the
dominant negative approach by using the adenovirus expression system to
express the wild type and dominant negative forms of PKC
, PKC
,
and PKC
in SMC. Our results revealed that overexpression of the
dominant negative PKC
almost completely blocks thrombin-induced PKD
activation. In contrast, neither PKC
nor PKC
affects
thrombin-induced PKD activation (Fig. 4). Together, these data revealed
a novel role of PKC
in mediating thrombin-induced PKD activation in
living cells.
In addition, our results also demonstrate the formation of
a complex between PKC
and PKD in SMC, suggesting that PKC
mediates thrombin-induced PKD activation through its direct interaction with PKD. Notably, PKC
was also found to form a complex with PKD
(Fig. 5) (15). However, PKC
is not functionally involved in
thrombin-induced PKD activation. This finding provides further support
of the specific role of PKC
in mediating thrombin-induced PKD
activation in SMC.
Based on the observations that PKD kinase activity was enhanced upon
transient coexpression with constitutively active PKC
, PKC
, and
PKC
, each of which is a novel PKC, recent studies have suggested that PKC
, PKC
, and PKC
may function as potential upstream kinases and may account for the PKC-dependent
activation of PKD (28, 29). However, to our knowledge, the functional relationship between endogenous novel PKCs (PKC
, PKC
, and PKC
) and PKD in intact cells responding to extracellular stimuli has not yet
been established. Moreover, there is no information about the potential
role of PKC
, another member of the novel PKC family, in PKD
activation. Very recently, Stafford et al. (30) reported that thrombin induces PKD activation in platelets in a
PKC-dependent pathway. However, the specific PKC isotype,
which mediates thrombin-induced PKD activation in platelets, has not
been identified. Therefore, our findings that thrombin induces PKD
activation via a PKC
-dependent pathway in SMC open a new
avenue to study the biological function of PKD and PKC
in living cells.
In summary, our study demonstrated that thrombin activates
PKD in SMC. Furthermore, our results revealed a novel function of
PKC
in mediating PKD activation induced by thrombin. In addition, our experiments also provide for the first time evidence of a complex
formation between PKC
and PKD. The present findings identified PKD
as a new component in the thrombin-induced intracellular signaling pathway in SMC, and this discovery may implicate PKD in mediating the
biological responses induced by thrombin in SMC.