Bombesin, Vasopressin, Endothelin, Bradykinin, and Platelet-derived Growth Factor Rapidly Activate Protein Kinase D through a Protein Kinase C-dependent Signal Transduction Pathway*

Protein kinase D (PKD) is a serine/threonine protein kinase that is activated by phorbol esters via protein kinase C in intact cells. To assess the physiological sig- nificance of this putative pathway, we examined the regulation of PKD in living cells by mitogenic regulatory peptides and by platelet-derived growth factors (PDGF). Our results demonstrate that bombesin rapidly induces PKD activation in Swiss 3T3 cells, as shown by autophosphorylation and syntide-2 phosphorylation assays. Maximum PKD activation (14-fold above base-line levels) was obtained 90 s after bombesin stimulation. Bomb- esin also induced PKD activation in Rat-1 cells stably transfected with the bombesin/gastrin releasing peptide (GRP) receptor and in COS-7 cells transiently co-trans-fected with PKD and bombesin/GRP receptor expres- sion constructs. No inducible kinase activity was demonstrated when COS-7 cells were transfected with a kinase-deficient PKD mutant. Bombesin-mediated PKD activation was prevented by treatment of Swiss 3T3 cells with the protein kinase C inhibitors GF 1092030X and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro . Vasopressin, endothelin, and bradykinin also activated PKD in Swiss 3T3 cells through a PKC-dependent pathway. Platelet-derived growth factor-stimulated PKD activation in Swiss 3T3 cells and To determine activity were phosphorylation were further analyzed by and autoradiography. Quantifi- cation autophosphorylation was performed by scanning densitometry. The results are expressed as a of were treated for 10 in the presence or absence ( bar ) of 10 n M bombesin. After lysis and immunoprecipitation with PA-1 antiserum, PKD activity in immunocomplexes was determined by syntide-2 phosphorylation as described under “Experimental Proce- dures.”

Protein kinase C (PKC), 1 a major cellular target for the potent tumor-promoting phorbol esters (1,2), has been implicated in both short and long term regulation of cellular responses, including ion fluxes, gene expression, cell-cell commu-nication, cell morphology, differentiation, and proliferation (1,(3)(4)(5)(6)(7). However, despite the recognized importance of this key enzyme, events that occur downstream of PKC activation remain poorly defined.
The recently identified mouse serine protein kinase, named PKD, also consists of regulatory and catalytic domains (18). However, comparison of the deduced amino acid sequence of the catalytic domain of PKD with that of other protein kinases indicates that PKD is a distinct protein kinase that is distantly related to Ca 2ϩ -regulated kinases but does not belong to any of the protein kinase subfamilies (19). In particular, the kinase subdomains of PKD show little similarity to the highly conserved regions of the kinase subdomains of the PKC family. Consistent with this, PKD does not phosphorylate a variety of substrates utilized by PKCs, indicating that PKD is a protein kinase with distinct substrate specificity (18,20). The aminoterminal region of PKD contains a putative transmembrane domain, two cysteine-rich, zinc finger-like motifs, and a pleckstrin homology domain that is not found in any of the PKCs. A fusion protein containing the zinc finger-like domains of PKDbound [ 3 H]PDB with high affinity (18). Furthermore, immunopurified PKD was markedly stimulated by PDB or DAG in the presence of phosphatidylserine (20). A human protein kinase called atypical PKC (21,22) with 92% homology to PKD (extending to 98% homology in the catalytic domain) is also stimulated by phorbol esters and phospholipids (23). These in vitro results indicate that PKD/PKC is a novel phorbol ester/ DAG-stimulated protein kinase.
Recently we reported that exposure of intact cells to biologically active phorbol esters and membrane-permeant DAG induces PKD activation via a PKC-dependent pathway (24). PKD activity recovered from phorbol ester-stimulated cells can be measured by kinase assays in the absence of lipid activators (24). These results revealed an unsuspected connection between PKCs and PKD and implied that PKD can function downstream of PKCs in a novel signal transduction pathway. To assess the physiological significance of this putative pathway, it was important to determine whether growth promoting factors that elevate DAG, and thus activate PKC, also induce PKD activation in intact cells.
Quiescent Swiss 3T3 fibroblasts have proved to be a useful model system for elucidating signal transduction pathways involved in cell proliferation (4), and regulatory peptides of the bombesin family have been identified as potent mitogens for these cells (25,26). Binding of bombesin to its specific seven transmembrane domain receptor activates heterotrimeric G proteins of the G␣ q subfamily (27,28) that stimulate the ␤ isoforms of phospholipase C to induce the rapid formation of the intracellular second messengers DAG and inositol 1,4,5trisphosphate, which activate classic and novel PKCs and mobilize Ca 2ϩ , respectively (29 -34). PDGF, which stimulates tyrosine phosphorylation and subsequent activation of phospholipase C␥1 (reviewed in Ref. 35), leads to a similar sequence of events. The experiments presented here were designed to determine whether bombesin and other growth factors can induce PKD activation in intact cells. Our studies demonstrate that PKD activation is a novel early event in the action of multiple signaling peptides.

EXPERIMENTAL PROCEDURES
Cell Culture-Stock cultures of Swiss 3T3 cells were maintained in DMEM supplemented with 10% FBS in a humidified atmosphere containing 10% CO 2 at 37°C. For experimental purposes, cells were plated in 90-mm dishes at 6 ϫ 10 5 cells/dish in DMEM containing 10% FBS and used after 6 -8 days, when the cells were confluent and quiescent. COS-7 cells were plated in 90-mm dishes at 9 ϫ 10 5 cells/dish in DMEM containing 10% FBS.
cDNA Expression Vectors and Transfection of COS-7 Cells-The PKD cDNA fragment spanning bases Ϫ125 to 3179 was inserted into the mammalian expression vector pcDNA3, as described (20). A kinasedeficient mutant (PKDK618M) was generated by site-directed mutagenesis using the Altered Sites II in vitro mutagenesis kit (Promega) and subcloned into pcDNA3 (pcDNA3-PKDK618M).
Exponentially growing COS-7 cells, 40 -60% confluent, were transfected with the various plasmids using Lipofectin (Life Technologies, Inc.). Briefly, 12 g of DNA were used for 90-mm dishes. The DNA was diluted to 100 l with Opti-MEM I medium (Life Technologies, Inc.) and then mixed with Lipofectin (36 l) diluted to 100 l with Opti-MEM I medium. After 15 min, the DNA-Lipofectin complex was diluted to 10 ml with Opti-MEM I medium, mixed gently, and overlaid onto rinsed (1 ϫ with Opti-MEM I) COS-7 cells. The cultures were incubated at 37°C for 6 h, and the medium was then replaced with fresh DMEM containing 10% FBS. The cells were used for experimental purposes 72 h later. In the co-transfection experiments, 6 g of pcDNA3-PKD or pcDNA3-PKDK618M and 6 g of BNR-pCD2 containing the cDNA encoding the bombesin/GRP receptor (38) as indicated were mixed prior to dilution with Opti-MEM I medium.
Kinase Assay of PKD-PKD autophosphorylation was determined in an in vitro kinase assay by mixing 20 l of immunocomplexes with 20 l of a phosphorylation mixture containing (final concentrations) 100 M [␥-32 P]ATP (specific activity, 400 -600 cpm/pmol), 30 mM Tris/HCl, pH 7.4, 10 mM MgCl 2 , and 1 mM dithiothreitol. After 10 min of incubation at 30°C, the reactions were terminated by adding an equal volume of 2 ϫ SDS-PAGE sample buffer (1 M Tris/HCl, pH 6.8, 0.1 mM Na 3 V0 4 , 6% SDS, 0.5 M EDTA, 4% 2-mercaptoethanol, 10% glycerol), and analyzed by SDS-PAGE. The gels were dried and the 110-kDa radioactive band corresponding to autophosphorylated PKD was visualized by autoradiography. Autoradiograms were scanned in an LKB Ultrascan XL densitometer, and the labeled band was quantified using Ultrascan XL internal integrator.
Exogenous substrate phosphorylation was assayed using PKD immunoprecipitates and syntide-2 (PLARTLSVAGLPGKK), a peptide based on the phosphorylation site two of glycogen synthase (39,40). The immunocomplexes were washed twice with buffer A and twice with a buffer containing 30 mM Tris/HCl, pH 7.4, 10 mM MgCl 2 , and 1 mM dithiothreitol. Syntide-2 (2.5 mg/ml) and 100 M [␥-32 P]ATP (400 -600 cpm/pmol) were then added to the incubation mixture. The final total volume was 30 l. After incubation for 10 min at 37°C the reaction was terminated by adding 100 l of 75 mM H 3 PO 4 and spotting 75 l of the reaction mix onto P-81 phosphocellulose paper. Free [␥-32 P]ATP was separated from the labeled substrate by washing the P-81 paper four times for 5 min each in 75 mM H 3 PO 4 . The papers were dried, and the radioactivity incorporated into syntide-2 was determined by Cerenkov counting.
Phosphopeptide Mapping-Two-dimensional tryptic phosphopeptide mapping on thin layer cellulose plates was performed according to Boyle et al. (41). Briefly, confluent and quiescent Swiss 3T3 cells (1 ϫ 10 7 cells/condition) were either unstimulated or stimulated with serumfree culture medium containing either 200 nM PDB or 10 nM bombesin for 10 min. Cells were lysed, and PKD was immunoprecipitated from cell lysates using PA-1 antiserum according to the procedure described above. PKD immunoprecipitates were subjected to in vitro kinase reactions as described above but containing 1 M [␥-32 P]ATP (800 -1200 cpm/pmol) in the assay mixture. Autophosphorylated PKD was resolved by SDS-PAGE on 1.5-mm thick, 8% polyacrylamide gels. Gels were briefly fixed, dried, and subjected to autoradiography. The 110-kDa bands corresponding to intact PKD protein were excised from gels and further processed for two-dimensional maps as described (41). Electrophoresis in pH 1.9 buffer (2.2% formic acid and 7.8% acetic acid in water) was performed for 30 min at a constant current of 21 mA. The solvent system for the ascending chromatography consisted of n-butyl alcohol/pyridine/acetic acid/water in the ratio 75:50:15:60. Thin layer plates were air-dried and subjected to autoradiography to allow visualization of radiolabeled phosphopeptides. 32 P i Labeling of Cells and Analysis of PKD Phosphorylation-Quiescent and confluent cultures of Swiss 3T3 cells were washed twice in phosphate-free DMEM and incubated at 37°C with this medium containing 200 Ci/ml carrier-free 32 P i for 12 h. Cells were stimulated with different concentrations of bombesin for 10 min. Parallel cultures of 32 P i -labeled cells were stimulated with 10 nM bombesin for different times. The cells were subsequently lysed with buffer A, and the lysates were immunoprecipitated with PA-1 antibody and analyzed by SDS-PAGE prior to autoradiography.
Western Blot Analysis-For Western blot analysis of PKD, immunoprecipitates were washed three times with lysis buffer A and extracted for 10 min at 95°C in 2 ϫ SDS-PAGE sample buffer and analyzed by SDS-PAGE followed by transfer to Immobilon membranes. The transfer was carried out at 100 V, 0.4 A at 4°C for 4 h using a Bio-Rad transfer apparatus. The transfer buffer consisted of 200 mM glycine, 25 mM Tris, 0.01% SDS, 20% methanol. Membranes were blocked using 5% non-fat dried milk in phosphate-buffered saline, pH 7.2, and incubated with PA-1 antiserum (1:500) at room temperature for 4 h in phosphatebuffered saline containing 3% non-fat dried milk. Immunoreactive bands were visualized using horseradish peroxidase-conjugated antirabbit IgG and subsequent enhanced chemiluminescence detection.

RESULTS
Bombesin Induces PKD Activation in Swiss 3T3 Cells-To examine whether bombesin induces PKD activation, confluent and quiescent Swiss 3T3 cells were exposed to a saturating concentration of this peptide (10 nM) for various periods and lysed. The extracts were immunoprecipitated with the PA-1 antiserum raised against a peptide composed of the 15 carboxyl-terminal amino acids of PKD. The immunocomplexes were incubated with [␥-32 P]ATP and then analyzed by SDS-PAGE and autoradiography to examine the level of autophosphorylation. Stimulation of the cells with bombesin induced a striking increase in PKD activity that was maintained during cell disruption and immunoprecipitation ( Fig. 1). A marked increase of PKD activity (8-fold) was obtained after 0.5 min and reached a maximum (14-fold over base-line levels) after 1.5 min of bombesin stimulation (Fig. 1A).
We also determined whether bombesin-mediated PKD activation could be demonstrated using an exogenous substrate. The synthetic peptide syntide-2 (39, 40) has been identified as an efficient substrate for the catalytic domain of PKD (18) and for the full-length PKD (20). Therefore, we have chosen syntide-2 as a model exogenous substrate to assay PKD activity immunoprecipitated from lysates of Swiss 3T3 cells treated with or without bombesin. As illustrated in Fig. 1B, bombesin stimulated rapid and robust PKD activation as shown by the syntide-2 phosphorylation assay. These results demonstrate that bombesin stimulation of intact Swiss 3T3 cells induces an activated state of PKD that is maintained during cell lysis and protein isolation.
Bombesin induced PKD activation in a concentration-dependent fashion with half-maximal stimulation occurring at 0.5 nM as seen by autophosphorylation and syntide-2 phosphorylation assays ( Fig. 2A). The bombesin/GRP receptor antagonist [D-F 5 -Phe 6 ,D-Ala 11 ]bombesin-(6 -13)-OMe (42) prevented the increase in PKD activity induced by bombesin but did not interfere with the activation of PKD promoted by PDB in parallel cultures (Fig. 2B). We determined whether the phosphopeptide maps of PKD autophosphorylation obtained from cells stimulated either with bombesin or with PDB were similar. Two-dimensional tryptic peptide mapping of PKD activated in response to either bombesin or PDB showed identical patterns of phosphopeptide fragments (Fig. 3).
Bombesin Stimulates PKD Activation in Rat-1 and COS-7 Cells Expressing Bombesin/GRP Receptors-As bombesin activates PKD in Swiss 3T3 cells, we also examined whether bombesin stimulates PKD activation in other cell types. As shown in Fig. 4, bombesin stimulated a rapid and concentration-dependent activation of PKD in Rat-1 cells stably transfected with the bombesin/GRP receptor (36). Half-maximal activation occurred at 0.5 nM. The antagonist [D-F 5 -Phe 6 ,D-Ala 11 ]bombesin-(6 -13)-OMe also prevented PKD activation induced by bombesin in these cells.
To confirm that the kinase activity induced by bombesin was due to activation of PKD rather than to the presence of a co-precipitating protein kinase, we examined bombesin-induced PKD autophosphorylation and syntide-2 phosphorylation in COS-7 cells co-transfected with a bombesin/GRP receptor construct (BNR-pCD2) and with either a wild-type PKD expression vector (pcDNA3-PKD) or a kinase-defective PKD mutant (pcDNA3-PKDK618 M) in which lysine 618 in the ATPbinding site is substituted by a methionine. Bombesin treatment of COS-7 cells, co-transfected with pcDNA3-PKD and BNR-pCD2, resulted in activation of wild-type PKD (Fig. 5). In contrast, no inducible kinase activity was seen in COS-7 cells transfected with pcDNA3K618M despite similar PKD and PKDK618M expression levels (Fig. 5). These results demonstrate that the bombesin-induced kinase activity measured in PKD immunoprecipitates was due to the activation of PKD.
Peptide mapping with S. aureus V8 protease of PKD labeled with 32 P i in COS-7 cells either transfected with a wild-type PKD expression construct and stimulated with PDB or cotransfected with the wild-type PKD expression construct and the bombesin/GRP receptor expression construct and stimulated with bombesin showed identical phosphopeptide fragments (results not shown).
Bombesin Induces PKD Phosphorylation in Intact Swiss 3T3 Cells-The preceding experiments demonstrated that treatment with bombesin markedly increased the level of PKD autophosphorylation in in vitro kinase assays. We next examined whether bombesin induces PKD phosphorylation in intact cells. Confluent and quiescent cultures of Swiss 3T3 cells metabolically labeled with 32 P i were stimulated with different concentrations of bombesin for 10 min or with 10 nM bombesin for different times. Cells were lysed, immunoprecipitated with PA-1 antiserum, and analyzed by SDS-PAGE and autoradiography. As shown in Fig. 6 (upper panel), bombesin induced PKD phosphorylation in a concentration-dependent manner with half-maximal phosphorylation occurring at 0.6 nM. Stimulation of the cells with bombesin induced a 2-fold increase (over base-line levels) in the incorporation of 32 P i after 1 min and reached a maximum (3.5-fold over base-line levels) after 2 min of bombesin stimulation (Fig. 6).
Inhibitors of PKC Prevent PKD Activation Induced by Bombesin-Recent results indicated that phorbol esters induce PKD activation via a PKC-dependent pathway (24). Consequently, we examined whether PKC was required for PKD activation in response to bombesin. As shown in Fig. 7, treatment of intact Swiss 3T3 cells with various concentrations of the PKC inhibitors GF I (43) and Ro 31-8220 (44) inhibited PKD activation induced by subsequent addition of bombesin in a concentrationdependent manner. In striking contrast, GF I or Ro 31-8220 added directly to the in vitro kinase assay at identical concentrations to those used previously in intact cells did not inhibit PKD activity stimulated by bombesin in intact cells (Fig. 7). Similar results were obtained when the effects of GF I and Ro 31-8220 on bombesin-induced PKD activation were examined in Rat-1 cells stably transfected with the bombesin/GRP receptor (Results not shown). Thus, GF I and Ro 31-8220 did not inhibit PKD directly but interfered with bombesin-mediated PKD activation in intact cells by blocking PKC.
Next, we examined whether other signaling pathways contribute to bombesin-mediated PKD activation in Swiss 3T3 cells. To determine the role of Ca 2ϩ mobilization in PKD activation by bombesin, quiescent Swiss 3T3 cells were treated with the tumor promoter thapsigargin. This agent specifically inhibits the endoplasmic reticulum Ca 2ϩ -ATPase and thereby depletes Ca 2ϩ from intracellular stores (45). Treatment with 30 nM thapsigargin for 30 min abolished the increase in cytosolic Ca 2ϩ induced by subsequently added bombesin (results not shown) but did not prevent PKD activation induced by bombesin. Similarly, chelation of extracellular Ca 2ϩ with EGTA to prevent Ca 2ϩ influx did not affect PKD activation. Furthermore, a combination of thapsigargin and EGTA did not inhibit bombesin-induced PKD activation (Fig. 8A). Thus inhibition of shown. Approximately six prominent 32 P-labeled tryptic peptide spots were found to be reproducibly induced by either phorbol ester (PDB) or bombesin (Bom) treatments. Interestingly, at least two of these prominent spots also appeared on maps generated from unstimulated cells upon long term exposure of films to thin layer plates. Approximately 1000 cpm were spotted to control plates, and approximately 5000 cpm to plates were used for maps of PKD activated within cells by PDB or bombesin treatments. The control autoradiogram shown was exposed to the thin layer plate for 6 days, and the PDB-or bombesin-stimulated autoradiograms were exposed to the respective thin layer plates for 2 days. Results shown are typical of four similar experiments.

FIG. 4. Time-and dose-dependent activation of PKD by bombesin in Rat-1 cells transfected with bombesin/GRP-receptor.
Confluent and quiescent Rat-1 cells stably transfected with the bombesin/GRP preferring receptor were treated for different times as indicated with 10 nM bombesin and lysed. The lysates were subjected to immunoprecipitation with PA-1 antiserum, and PKD activity was determined by autophosphorylation followed by SDS-PAGE and autoradiography. The results shown are the mean of two independent experiments. The autoradiogram shown is from a single representative experiment. Inset, confluent and quiescent Rat-1 cells expressing the bombesin receptor were treated with various concentrations of bombesin as indicated for 10 min and lysed. PKD activity was determined by an in vitro kinase assay followed by SDS-PAGE and autoradiography. The results shown are the mean of two independent experiments. Autoradiogram, confluent, and quiescent Rat-1 cells were incubated for 20 min with either 100 nM bombesin antagonist [D-F 5 -Phe 6 ,D-Ala 11 ]bombesin-(6 -13)-OMe or an equivalent amount of solvent for 20 min. Cells were left unstimulated (Ϫ) or were stimulated for 10 min with 10 nM bombesin in the absence (B) or in the presence of the antagonist (AϩB). The cultures were lysed, and the lysates were immunoprecipitated with PA-1 antiserum and analyzed by an in vitro kinase assay followed by SDS-PAGE and autoradiography. The autoradiogram shown is from a representative experiment; two additional experiments gave similar results. Ca 2ϩ influx or mobilization from intracellular stores did not affect bombesin-mediated PKD activation.
Bombesin stimulates activation of p42 mapk /p44 mapk (46, 47) and p70 s6k (48) and tyrosine phosphorylation of p125 fak and other substrates (49 -51) in Swiss 3T3 cells. Inhibition of p70 s6k activation with rapamycin, of p42 mapk and p44 mapk with the selective MEK-1 inhibitor PD 098059, and of tyrosine phosphorylation of p125 fak with either cytochalasin D or genistein did not affect PKD activation in response to bombesin (Fig. 8B). In contrast, pretreatment with GF I or Ro 31-8220 markedly reduced subsequent PKD activation induced by bombesin in parallel cultures of Swiss 3T3 cells (Fig. 8B).
The compound R 59 949, a selective inhibitor of DAG kinase, enhances DAG accumulation in a variety of cell types including Swiss 3T3 cells (52,53). As shown in Fig. 8C, R59 949 potentiated the ability of a submaximal concentration of bombesin (0.4 nM) to stimulate PKD activation in these cells. The results presented in Figs. 7 and 8 suggest that bombesin-induced DAG accumulation stimulates PKD activation via PKC.
Vasopressin, Endothelin, Bradykinin, and PDGF Also Induce PKD Activation in Swiss 3T3 Cells-To investigate whether bombesin was unique in its ability to activate PKD within Swiss 3T3 cells or whether agonist-mediated activation of other G q -coupled receptors would also lead to PKD activation, we also tested a variety of other biologically active peptides. Vasopressin, like bombesin, also induced striking and rapid increases in PKD activity (Fig. 9A). Maximal stimulation (13-fold over base-line values) of PKD activation by vasopressin occurred within 2 min of exposure, and this stimulation was again concentration-dependent, with half-maximal activation occurring at 0.6 nM (Fig. 9B). Similarly, endothelin also induced a time-and concentration-dependent activation of PKD in Swiss 3T3 cells (Fig. 9, C and D) with half-maximal activation achieved at 3 nM. Interestingly, whereas stimulation of PKD activity by bombesin and vasopressin peaked rapidly and then declined to somewhat lower constant values, endothelin treatment of Swiss 3T3 cells induced stimulation of PKD activity somewhat more gradually, such that maximal stimulation (8fold over base-line values) consistently occurred after approximately 5 min of exposure to this agonist (Fig. 9C). Endothelin also induced PKD activation in Rat-1 cells which also express receptors for this agonist (results not shown).
Bradykinin is known to induce rapid but transient stimulation of diacylglycerol production and PKC stimulation in Swiss 3T3 cells (30). As shown in Fig. 10C, bradykinin also induced PKD activation in Swiss 3T3 cells although it was consistently less effective in promoting PKD activation than bombesin, vasopressin, or endothelin.
To examine the role of PKC in PKD activation induced by vasopressin, endothelin, and bradykinin, we tested the effect of GF I and Ro 31-8220 on the increase in PKD activity induced by these agonists. As shown in Fig. 10, treatment of Swiss 3T3 cells with either 3.5 M GF I or 3.5 M Ro 31-8220 prevented PKD activation induced by subsequent exposure to vasopressin, endothelin, and bradykinin.
To determine whether other growth factors can also induce PKD activation, the effect of exposure of cells to either insulin or PDGF was examined. Saturating doses of insulin (1 g/ml), which synergistically stimulate DNA synthesis in Swiss 3T3 cells but are unable to activate PKC (54), did not induce PKD activation when measured 10 min after treatment (results not shown). In contrast, exposure of the cells to increasing concentrations of PDGF caused a dose-dependent increase in PKD activity (Fig. 11A). Half-maximal and maximal (13-fold over base-line values) effects were achieved at 3 and 10 ng/ml, respectively. PKD activation in response to PDGF could also be demonstrated in porcine aortic endothelial cells (37) stably transfected with the PDGF-␤ receptor.
PDGF-induced PKD activation in Swiss 3T3 cells was prevented by treatment with 50 M genistein, but it was not affected by inhibition of phosphatidylinositol 3-kinase activity with either 100 nM wortmannin or 20 M Ly294002 (Fig. 11B). To examine whether PKC also mediated PKD activation induced by PDGF, we tested the effect of GF I or Ro 31-8220 on the activation of PKD induced by PDGF. Treatment of Swiss 3T3 fibroblasts or endothelial cells with these PKC inhibitors markedly inhibited PKD activation in response to PDGF (Fig.  11C). DISCUSSION Recently, we reported that treatment of intact cells with biologically active phorbol esters and cell-permeant diacylglycerols induces PKD activation through a PKC-dependent signal transduction pathway (24). These findings revealed a novel connection between PKCs and PKD and have important implications for the understanding of signal transduction pathways mediating the action of the second messenger DAG. In the present study we determined whether physiological activation of PKC via occupancy of specific membrane receptors for mitogenic neuropeptides and growth factors can also increase the activity of PKD in intact cells.
Our results demonstrate, for the first time, that stimulation of Swiss 3T3 cells with bombesin induces a striking activation of PKD. The concentration dependence and sensitivity of this response to a specific receptor antagonist indicates that the effects of bombesin are mediated through the same receptors that elicit other molecular responses and stimulate DNA synthesis in these cells. PKD recovered by immunoprecipitation from bombesin-stimulated cells is fully active in the absence of lipid effectors (i.e. phosphatidylserine and PDB) as shown by autophosphorylation assays as well as by phosphorylation of the exogenous substrate syntide-2. The conversion of PKD into this activated state that persists during cell disruption and protein isolation occurs within seconds of bombesin stimulation of Swiss 3T3 cells and thus is one of the early events induced by this neuropeptide agonist in these cells.
The results of transfection experiments reveal that bombesin-mediated activation of PKD is not restricted to the Swiss 3T3 cell line. Thus, stable expression of bombesin/GRP receptor in Rat-1 cells or transient co-expression of PKD and bombesin/ GRP receptor in COS-7 cells allowed rapid activation of PKD via bombesin stimulation of cells. The low endogenous levels of PKD expression in COS-7 cells was also advantageous in that this made it possible to verify that the inducible kinase activity in PKD immunoprecipitates was indeed due to PKD activation rather than to the stimulation of a co-precipitating kinase. Hence, bombesin did not stimulate kinase activity in COS-7 cells co-transfected with bombesin/GRP receptor and a PKD kinase-deficient mutant. A prominent early event induced by bombesin is the rapid generation of DAG and consequent activation of PKC (31)(32)(33)55). Since direct PKC stimulation by phorbol esters has been shown to activate PKD in intact cells, bombesin could induce PKD activation through a PKC-dependent pathway. Consistent with this possibility, a DAG kinase inhibitor potentiates PKD activation induced by submaximal concentration of bombesin, and PKD peptide maps give rise to identical patterns whether PKC activity was stimulated through bombesin-mediated generation of DAG or directly through PDB. Furthermore, pretreatment of either Swiss 3T3 cells or Rat-1 cells transfected with the bombesin/GRP receptor with the inhibitors of PKC, GF I, and Ro 31-8220 before stimulation with bombesin strikingly prevents PKD activation. Crucially, neither GF I nor Ro 31-8220 inhibits PKD activity when added directly in vitro, even at the concentrations used in intact cells to prevent bombesin-induced PKD activation. In addition, inhibition of many other signaling events induced by bombesin, including Ca 2ϩ influx and/or mobilization, MEK-1-mediated mitogen-activated protein kinase activation, p70 s6k activation, and p125 fak tyrosine phosphorylation, does not interfere with bombesin-induced PKD activation. Taken together these results strongly suggest that persistent PKD activation induced by bombesin is mediated by PKC.
Recent studies demonstrated that co-transfection of PKD with constitutively activated mutants of PKC⑀ and -strongly induced PKD activation in COS-7 cells (24). The dissociation of PKD activation from Ca 2ϩ fluxes in bombesin-treated Swiss 3T3 cells shown in the present study is consistent with a role for Ca 2ϩ -insensitive isoforms of PKC in mediating PKD activation in these cells. Further experimental work will be required to elucidate whether PKCs directly phosphorylate and activate PKD or stimulate an intermediary kinase(s) that leads to activation of PKD.
To substantiate the results obtained with bombesin, we also examined the effect of agonist activation of other seven transmembrane domain receptors that stimulate phospholipase C ␤ through G␣ q . Vasopressin, endothelin, and bradykinin bind to receptors that rapidly promote hydrolysis of inositol phospholipids, Ca 2ϩ mobilization, and PKC activation in Swiss 3T3 cells (29 -31, 56 -58). In the present study, we demonstrate that these agonists also induce PKD activation in these cells via a PKC-dependent pathway.
The binding of PDGF to individual receptor chains stimulates their dimerization and subsequent transphosphorylation (59). Phospholipase C␥ and phosphatidylinositol 3-kinase, as other cytoplasmic effector proteins, associate with specific phosphorylated tyrosine residues on the receptor and are phosphorylated on tyrosine by the intrinsic tyrosine kinase activity  (End, B), and for 1 min with 50 nM bradykinin (BK, C). Cells were lysed, and the lysates were immunoprecipitated with PA-1 antiserum, and PKD activity was determined by an in vitro kinase assay as described under "Experimental Procedures," followed by SDS-PAGE and autoradiography. The results shown are the mean Ϯ S.E. of three independent experiments. of the receptor (35,60). Activated PLC␥ stimulates polyphosphoinositide hydrolysis leading to PKC activation, whereas the putative second messengers generated by phosphatidylinositol 3-kinase, phosphatidylinositol (3,4)-bisphosphate, and phosphatidylinositol (3,4,5)-trisphosphate have been proposed to activate novel isoforms of PKC (61,62) and the protein kinase encoded by the akt proto-oncogene (63). Here we demonstrate that PDGF stimulates PKD activation in either Swiss 3T3 cells or in porcine aortic endothelial cells stably transfected with the PDGF-␤ receptor. Given that the potent phosphatidylinositol 3-kinase inhibitors wortmannin and Ly 294002 did not inter-fere with PKD activation induced by PDGF, it is unlikely that either phosphatidylinositol 3-kinase or Akt initiates a major pathway leading to PKD activation. In contrast, treatment of these cells with either GF I or Ro 31-8220 before stimulation with PDGF prevented PKD activation suggesting that PKC activation is once again a critical step.
In conclusion, our findings demonstrate that PKD can be rapidly activated in response to multiple signaling peptides in a variety of cell types. The results indicate that bombesin, vasopressin, endothelin, bradykinin, and PDGF induce PKD activation via PKC. We conclude that PKD activation is a novel early event in the action of multiple regulatory peptides and growth factors. FIG. 11. A, PDGF activates PKD in a dose-dependent manner. Confluent and quiescent Swiss 3T3 cells were treated for 10 min with various concentrations of PDGF as indicated and lysed. PKD activity was determined by an in vitro kinase assay followed by SDS-PAGE and autoradiography. The level of autophosphorylation was quantified by scanning densitometry as described under "Experimental Procedures." The results shown are the mean Ϯ S.E. of three independent experiments. Inset shows a representative autoradiogram; two additional experiments gave similar results. B, effect of genistein, wortmannin, and Ly 294002 on PKD activation induced by PDGF. Confluent and quiescent Swiss 3T3 cells were incubated for 1 h with either 50 M genistein (Ge), 100 nM wortmannin (Wo), or 20 M Ly 294002 (Ly). Control cells received an equivalent amount of solvent (Ϫ). Cells were subsequently stimulated for 10 min with 6 ng/ml PDGF and lysed, and the lysates were immunoprecipitated with PA-1 antiserum. PKD activity was determined by an in vitro kinase assay as described under "Experimental Procedures," followed by SDS-PAGE and autoradiography. The labeled 110-kDa band corresponding to PKD was quantified by scanning densitometry. Values shown are the mean Ϯ S.E. of three independent experiments. C, PDGF induces PKD activation through a PKC-dependent pathway. Confluent and quiescent Swiss 3T3 cells and porcine aortic endothelial cells (PAE␤) stably transfected with PDGF-␤ receptor were incubated for 1 h with either 3.5 M GF I or 3.5 M Ro 31-8220 (Ro). Control cells received an equivalent amount of solvent (Ϫ). The cultures were subsequently stimulated for 10 min with either 6 ng/ml PDGF (Swiss 3T3 cells) or 30 ng/ml PDGF (PAE ␤). Cells were lysed, and the lysates were immunoprecipitated with PA-1 antiserum, and PKD activity was determined by an in vitro kinase assay as described under "Experimental Procedures," followed by SDS-PAGE and autoradiography. The autoradiograms are from a representative experiment; two additional experiments gave similar results.