Thrombin Rapidly Induces Protein Kinase D Phosphorylation, and Protein Kinase C delta  Mediates the Activation*

Mingqi TanDagger , Xuemin XuDagger , Motoi Ohba§, Wataru Ogawa, and Mei-Zhen CuiDagger ||

From the Dagger  Department of Pathology, University of Tennessee, Knoxville, Tennessee 37996, the § Institute of Molecular Oncology, Showa University, Shinagawa-ku, Tokyo 142-8555, Japan, and the  Department of Clinical Molecular Medicine, Kobe University, Kobe 650-0017, Japan

Received for publication, November 4, 2002, and in revised form, November 12, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Thrombin plays a critical role in hemostasis, thrombosis, and inflammation. However, the responsible intracellular signaling pathways triggered by thrombin are still not well defined. We report here that thrombin rapidly and transiently induces activation of protein kinase D (PKD) in aortic smooth muscle cells. Our data demonstrate that protein kinase C (PKC) inhibitors completely block thrombin-induced PKD activation, suggesting that thrombin induces PKD activation via a PKC-dependent pathway. Furthermore, our results show that thrombin rapidly induces PKCdelta phosphorylation and that the PKCdelta -specific inhibitor rottlerin blocks thrombin-induced PKD activation, suggesting that PKCdelta mediates the thrombin-induced PKD activation. Using dominant negative approaches, we demonstrated that expression of a dominant negative PKCdelta inhibits the phosphorylation and activation of PKD induced by thrombin, whereas neither PKCepsilon nor PKCzeta affects thrombin-induced PKD activation. In addition, our results of co-immunoprecipitation assays showed that PKD forms a complex with PKCdelta in smooth muscle cells. Taken together, the findings of the present study demonstrate that thrombin induces activation of PKD and reveal a novel role of PKCdelta in mediating thrombin-induced PKD activation in vascular smooth muscle cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 NFkappa 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 PKCdelta -specific inhibitor inhibits thrombin-induced activation of PKD; 2) overexpression of a dominant negative PKCdelta abolishes PKD activation; and 3) PKCdelta 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 PKCdelta -dependent PKD activation reveals a novel thrombin-induced signaling pathway in living cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
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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 PKCepsilon and PKCdelta from BD Transduction Laboratories (San Diego, CA); an antibody against PKCzeta from Upstate Biotechnology (Waltham, MA); antibodies against PKD and phospho-PKCepsilon from Santa Cruz Biotechnology (Santa Cruz, CA); and antibodies against phospho-PKC isoforms (delta  and zeta ) 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 (delta , epsilon , or zeta ) 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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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 [gamma -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.

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.

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.

PKCdelta 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 PKCalpha , PKCbeta , PKCdelta , PKCepsilon , and PKCzeta are expressed in SMC (16-19), and among them, PKCdelta 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 PKCdelta was rapidly induced at 45 s upon thrombin treatment of the SMC; in contrast, no thrombin-induced phosphorylation of PKCalpha , PKCbeta , PKCepsilon , or PKCzeta was detected (Fig. 3A).


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Fig. 3.   Thrombin rapidly activates PKCdelta , and the PKCdelta -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 PKCalpha /beta (second panel), PKCepsilon (third panel), or PKCzeta /lambda (fourth panel) were detected through the time course of thrombin stimulation. B, effect of the PKCdelta -specific inhibitor rottlerin on PKD activation.

PKCdelta Inhibitor Rottlerin Blocks PKD Activation-- We next examined whether activation of PKCdelta contributed to thrombin-induced PKD activation by determining the effect of the PKCdelta -specific inhibitor rottlerin on thrombin-induced PKD activation. Rottlerin has been reported to inhibit selectively PKCdelta activation (IC50 = 3-6 µM) 5-10-fold more potently than PKCalpha and PKCbeta and 13-33-fold more potently than PKCepsilon , PKCzeta , and PKCeta (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 PKCdelta activity in SMC.

Dominant Negative PKCdelta Blocks Thrombin-induced PKD Activation-- To substantiate further the role of PKCdelta in mediating thrombin-induced PKD activation in living cells, we examined the effect of the dominant negative form of PKCdelta on thrombin-induced PKD activation. The dominant negative nature of the ATP-binding site mutant PKCdelta 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 PKCdelta 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 PKCdelta had no detectable effect on thrombin-induced PKD activation when compared with the effect of non-infected controls. In contrast, neither dominant negative PKCepsilon and PKCzeta nor wild type PKCepsilon and PKCzeta , at the same multiplicity of infection, affected PKD activation (Fig. 4, B and C). These data further indicate that PKCdelta mediates thrombin-induced PKD activation in SMC.


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Fig. 4.   Overexpression of dominant negative PKCdelta but not of dominant negative PKCepsilon or PKCzeta 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 PKCdelta (A), PKCepsilon (B), and PKCzeta (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 PKCdelta on thrombin-induced PKD activation also was determined by an IVK assay (fifth row). MOI, multiplicity of infection.

PKCdelta Interacts with PKD in Intact Cells-- The above results indicated that PKCdelta functionally mediates PKD activation in response to thrombin. We further asked whether PKCdelta physically interacts with PKD in SMC. To address this question, we infected SMC with an adenovirus vector encoding PKCdelta , PKCepsilon , or PKCzeta 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 PKCdelta -, PKCepsilon -, or PKCzeta -specific antibodies. As shown in Fig. 5A, PKCdelta was co-immunoprecipitated with PKD in living cells. In addition, we also observed that, consistent with the observation reported previously (15), PKCepsilon , but not PKCzeta , was co-immunoprecipitated with PKD (Fig. 5, B and C). It should be noted that although both PKCdelta and PKCepsilon physically interact with PKD in SMC, only PKCdelta functionally mediates thrombin-induced PKD activation in aortic SMC.


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Fig. 5.   PKD interacts with PKCdelta and PKCepsilon but not with PKCzeta . SMC were infected (infec) with adenoviruses expressing wild type PKCdelta , PKCepsilon , and PKCzeta 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 PKCdelta (A), PKCepsilon (B), or PKCzeta (C). Cell lysates were used as controls (third lane of each panel).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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), NFkappa B (24), and nuclear diacylglycerol kinase theta  (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 PKCdelta mediates thrombin-induced PKD activation. To date, the role of PKCdelta in PKD activation is totally unknown, and although thrombin has been shown to activate PKCdelta in several types of cells (9, 26, 27), the downstream target of PKCdelta is still unclear. Our data established for the first time that thrombin-induced PKD activation is mediated by PKCdelta in vascular SMC.

We employed multiple approaches to address the specificity of the PKCdelta 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 PKCdelta and that the PKCdelta inhibitor rottlerin blocked thrombin-induced PKD activation in a concentration-dependent manner strongly suggests the functional involvement of PKCdelta 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 PKCdelta , PKCepsilon , and PKCzeta in SMC. Our results revealed that overexpression of the dominant negative PKCdelta almost completely blocks thrombin-induced PKD activation. In contrast, neither PKCepsilon nor PKCzeta affects thrombin-induced PKD activation (Fig. 4). Together, these data revealed a novel role of PKCdelta in mediating thrombin-induced PKD activation in living cells.

In addition, our results also demonstrate the formation of a complex between PKCdelta and PKD in SMC, suggesting that PKCdelta mediates thrombin-induced PKD activation through its direct interaction with PKD. Notably, PKCepsilon was also found to form a complex with PKD (Fig. 5) (15). However, PKCepsilon is not functionally involved in thrombin-induced PKD activation. This finding provides further support of the specific role of PKCdelta in mediating thrombin-induced PKD activation in SMC.

Based on the observations that PKD kinase activity was enhanced upon transient coexpression with constitutively active PKCeta , PKCepsilon , and PKCtheta , each of which is a novel PKC, recent studies have suggested that PKCeta , PKCepsilon , and PKCtheta 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 (PKCeta , PKCepsilon , and PKCtheta ) and PKD in intact cells responding to extracellular stimuli has not yet been established. Moreover, there is no information about the potential role of PKCdelta , 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 PKCdelta -dependent pathway in SMC open a new avenue to study the biological function of PKD and PKCdelta in living cells.

In summary, our study demonstrated that thrombin activates PKD in SMC. Furthermore, our results revealed a novel function of PKCdelta in mediating PKD activation induced by thrombin. In addition, our experiments also provide for the first time evidence of a complex formation between PKCdelta 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.

    ACKNOWLEDGEMENTS

We thank Drs. Donald McGavin, Jack Oliver, and Kellie Fecteau for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by American Heart Association Scientist Development Grant 9730039N (to M.-Z. C.) and National Institutes of Health Grant NS42314 (to X. X.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Pathology, University of Tennessee, College of Veterinary Medicine, 2407 River Dr., Knoxville, TN 37996. Tel.: 865-974-8212; Fax: 865-974-5616; E-mail: cuim@utk.edu.

Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M211523200

    ABBREVIATIONS

The abbreviations used are: SMC, smooth muscle cell(s); PAR, protease-activated receptor; PKD, protein kinase D; PKC, protein kinase C; IVK, in vitro kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.

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REFERENCES

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