Protein kinase D potentiates DNA synthesis and cell proliferation induced by bombesin, vasopressin, or phorbol esters in Swiss 3T3 cells.

We examined whether protein kinase D (PKD) overexpression in Swiss 3T3 cells potentiates the proliferative response to either the G protein-coupled receptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu). In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with retrovirus encoding murine PKD and green fluorescent protein (GFP) expressed as two separate proteins translated from the same mRNA. GFP was used as a marker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 cells was dramatically activated by cell treatment with bombesin or PDBu as judged by in vitro kinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, these stimuli induced PKD phosphorylation at Ser(744), Ser(748), and Ser(916). PKD activation and phosphorylation were prevented by exposure of the cells to protein kinase C-specific inhibitors. Addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in DNA synthesis and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulation of DNA synthesis in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results demonstrate that overexpression of PKD selectively potentiates mitogenesis induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells.

Neuropeptides stimulate DNA synthesis and cell proliferation in cultured cells and are implicated as growth factors in a variety of fundamental processes including development, inflammation, tissue regeneration, and tumorigenesis (1)(2)(3). In particular, the potent mitogens of the bombesin family (4,5) bind to a G protein-coupled receptor (GPCR) 1 (6,7) that pro-motes G␣ q -mediated activation of ␤-isoforms of phospholipase C (8,9) to produce 2 second messengers as follows: inositol 1,4,5-trisphosphate that mobilizes Ca 2ϩ from internal stores and diacylglycerol that activates PKC (1, 10 -12). There are multiple related PKC isoforms, which can be classified into three distinct subgroups on the basis of structural and regulatory differences as follows: the conventional PKCs (␣, ␤ I , ␤ II , and ␥), which are stimulated by calcium, diacylglycerol (DAG), and phospholipids; the novel PKCs (␦, ⑀, , and ), which are activated by DAG and phospholipids; and the atypical PKCs ( and ), whose regulation is less characterized but that have been proposed to be regulated by D-3 phosphoinositides (13)(14)(15). The DAG-regulated PKC isoforms all bind phorbol esters and are major cellular targets for this class of tumor promoter (16). PKCs, which have been implicated in the regulation of a wide range of biological responses including cell proliferation and carcinogenesis (17,18), play a pivotal role in neuropeptidemediated mitogenesis (2,19). Despite the recognized importance of PKCs in mitogenic signal transduction, the downstream targets that mediate PKC-induced cell proliferation remain largely undefined.
Protein kinase D (PKD)/protein kinase C is a serine/threonine protein kinase with structural, enzymological, and regulatory properties different from the PKC family members (20,21). The most distinct characteristics of PKD are the presence of a catalytic domain distantly related to Ca 2ϩ -regulated kinases, a pleckstrin homology region that regulates enzyme activity, and a highly hydrophobic stretch of amino acids in its N-terminal region (22)(23)(24). This N-terminal region also contains a tandem repeat of cysteine-rich, zinc finger-like motifs (CRD), which confer high affinity binding of phorbol esters, and plays a negative role in the regulation of catalytic kinase activity of PKD (25)(26)(27)(28). The recent identification of additional cDNA clones, similar in overall structure, primary amino acid sequence, and enzymological properties to PKD/PKC (29,30), supports the notion that PKD isoenzymes constitute a separate family of serine protein kinases.
PKD can be activated within intact cells by pharmacological agents like biologically active phorbol esters and cell-permeant DAGs as well as by physiological stimuli including GPCR agonists, growth factors, and antigen-receptor engagement (31)(32)(33)(34)(35)(36)(37)(38)(39). Treatment with PKC-selective inhibitors prevented PKD activation by all these factors (22,(31)(32)(33). Furthermore, cotransfection of PKD with constitutively active mutants of PKC ⑀ and dramatically increased the catalytic activity of PKD (24,31) and led to complex formation between PKD and PKC (24). In all cases, PKD activation appears to involve the phos-phorylation of Ser 744 and Ser 748 within the activation loop of the catalytic domain of PKD (40 -43). These findings revealed a link between PKCs and PKD and implied that PKD lies downstream of PKCs in a novel signal transduction pathway activated by multiple growth-promoting factors (22,41). PKD has been implicated in the regulation of a variety of cellular functions including EGF receptor and c-Jun signaling (44,45), Na ϩ /H ϩ antiport activity (46), Golgi organization and function (47,48), NFB-mediated gene expression (49), and cell migration (50). However, the precise role of PKD in neuropeptideinduced DNA synthesis and cell proliferation has not been elucidated.
In the present study, we examined whether increased PKD expression potentiates the proliferative response to the GPCR agonists bombesin and vasopressin and the biologically active phorbol ester PDBu. We used high efficiency retroviral mediated transfer of PKD into Swiss 3T3 cells, a cell line that undergoes reversible arrest in the G 0 phase of the cell cycle and has been used extensively as a model system to elucidate signal transduction pathways in the action of mitogenic GPCR agonists (1,2,19). Our results show that PKD overexpression strikingly and selectively potentiates the stimulation of DNA synthesis and cell division induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells. These findings support the hypothesis that PKD mediates neuropeptide-induced mitogenesis in these cells.

EXPERIMENTAL PROCEDURES
Cell Culture-Stock cultures of untransfected and transfected Swiss 3T3 cells were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO 2 and 90% air. For experimental purposes, cells were plated in 100-mm dishes at 6 ϫ 10 5 cells/ dish or 35-mm dishes at 1 ϫ 10 5 cells/dish and grown in DMEM containing 10% fetal bovine serum for 7-9 days until they became confluent and quiescent (51). Phoenix packaging cells (kindly provided by Dr. G. Nolan, Stanford University, Stanford, CA) were cultured in the same media in a humidified atmosphere containing 5% CO 2 .
Production of Retrovirus-pMSCVneo retroviral vector was engineered to include a single cassette that expresses the murine PKD and green fluorescent protein (GFP) from the same promoter. The MSCV-IRES-GFP plasmid was constructed by substituting the neo gene in the vector with the IRES-GFP fragment. PKD cDNA (spanning from position ϩ50 to ϩ2910, from the published GenBank TM sequence) was inserted into EcoRI site of the MSCV-IRES-GFP plasmid, upstream of IRES. This fragment of PKD cDNA, which contains all coding sequences but lacks the polyadenylation signal, was amplified by polymerase chain reaction. Polymerase chain reaction was initiated from bipartite primers 5Ј-CTGGAATTCCTCCCGGAAAGTTTGGTGGTT (sense) and 5Ј-ATCGAATTCGTGTTTTGACAGATTAGAGG (antisense) that introduced EcoRI restriction sites at 5Ј-and 3Ј-ends of the fragment. The nucleotide sequence of the amplified PKD coding region was confirmed by sequencing which revealed the presence of A and not T at position 2172, correcting previously published data. This correction leads to the appearance of Arg rather than Trp at position 606 in the amino acid sequence of the wild-type PKD.
For retrovirus production, logarithmically growing Phoenix ecotropic cells were transfected with either MSCV-PKD-IRES-GFP or MSCV-IRES-GFP using Fugene 6 Transfection Reagent as per protocol of the manufacturer. Virus-containing supernatants were collected 48 h after transfection and used immediately. Logarithmically growing Swiss 3T3 cells were incubated with the virus-containing supernatants in the presence of 5 g/ml Polybrene for 5 h. Cells were collected 48 -72 h later, and GFP-positive fractions were FACS-sorted using a Becton Dickinson FACStar PLUS machine. GFP-positive cells were propagated, and multiple aliquots were frozen. A fresh batch of cells was restarted every 2 months. Following sorting, GFP-positive Swiss 3T3 cells were maintained as described above.
Immunoblotting and Detection of PKD-Confluent, quiescent Swiss 3T3-GFP cells and Swiss 3T3-PKD.GFP cells were lysed in 2ϫ SDSpolyacrylamide gel electrophoresis sample buffer (20 mM Tris/HCl, pH 6.8, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol) and boiled for 10 min. After SDS-PAGE, proteins were transferred to Immobilon-P membranes as described previously (31) and blocked by overnight incubation with 5% non-fat dried milk in PBS, pH 7.2. Membranes were incubated at room temperature for 2 h with antisera specifically recognizing either the C terminus of PKD at a dilution of 1 g/ml or the phospho-specific pS916 antibody recognizing PKD phosphorylated at Ser 916 (1:500), or the phospho-specific pS748 antibody recognizing PKD phosphorylated at Ser 748 (1:500), in PBS containing 3% non-fat dried milk, or the phospho-specific pSpS744/748 antibody recognizing PKD phosphorylated at Ser 744 and Ser 748 (1:1000) in PBS, 0.1% Tween 20 containing 5% bovine serum albumin. Our recent results indicate that the phospho-specific pSpS744/748 antibody recognizes primarily PKD phosphorylated at Ser 744 (52). Immunoreactive bands were visualized using horseradish peroxidase-conjugated antirabbit IgG and enhanced chemiluminescence methods. Autoradiograms were scanned using a GS-710 scanner (Bio-Rad), and the labeled bands were quantified using the Quantity One software program (Bio-Rad).
Autophosphorylation reactions were initiated by combining 20 l of immune complexes with 5 l of a phosphorylation mixture containing 100 M [␥-32 P]ATP (specific activity, 400 -600 cpm/pmol) in kinase buffer. Following incubation at 30°C for 10 min, the reactions were terminated by addition of 1 ml of ice-cold kinase buffer and placed on ice. Immune complexes were recovered by centrifugation, and the proteins were extracted for SDS-PAGE analysis by addition of 2ϫ SDS-PAGE sample buffer (200 mM Tris/HCl, pH 6.8, 0.1 mM sodium orthovanadate, 1 mM EDTA, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol). Dried SDS-PAGE gels were subjected to autoradiography to visualize radiolabeled protein bands.
For assays of exogenous substrate phosphorylation, immune complexes were processed as for autophosphorylation reactions, and then substrate (syntide-2, final concentration 2.5 mg/ml) was added in the presence of 100 M [␥-32 P]ATP (400 -600 cpm/pmol) in kinase buffer (final reaction volume, 30 l). After incubation at 30°C for 10 min, the reactions were terminated by adding 100 l of 75 mM H 3 PO 4 , and 75 l of the mixed supernatant was spotted to Whatman P-81 phosphocellulose paper. Papers were washed thoroughly in 75 mM H 3 PO 4 , dried, and radioactivity incorporated into syntide-2 was determined by detection of Cerenkov radiation in a scintillation counter.
Assay of DNA Synthesis-Confluent and quiescent cultures of Swiss 3T3-PKD.GFP cells or Swiss 3T3-GFP cells were washed twice with DMEM and incubated with DMEM/Waymouth's medium (1:1, v/v) containing [ 3 H]thymidine (0.2 Ci/ml, 1 M) and various agonists as described in the figure legends. After 40 h of incubation at 37°C, cultures were washed twice with PBS and incubated in 5% trichloroacetic acid at 4°C for 20 min to remove acid-soluble radioactivity, washed with ethanol, and solubilized in 1 ml of 0.1 M NaOH, 0.1% SDS. The acidinsoluble radioactivity was determined by scintillation counting in 6 ml of Beckman Readysafe.
Measurement of Cell Number-Swiss 3T3-PKD.GFP cells, Swiss 3T3-GFP cells, and untransfected Swiss 3T3 cells were seeded in 35-mm Nunc Petri dishes at a density of 2 ϫ 10 4 with 2 ml of DMEM containing 10% FBS. At day 0 (24 h after plating), cultures were washed twice with DMEM and replaced with DMEM/Waymouth's medium (1:1, v/v) containing 1% FBS and 1 g/ml insulin (to preserve cell viability in low serum) and supplemented with bombesin, vasopressin, PDBu, or EGF, as described in the legend to Fig. 6. Cell number was determined by removing the cells from the dish with a trypsin/EDTA solution (0.5% trypsin in a Ca 2ϩ -and Mg 2ϩ -free PBS with EDTA) and counting a portion of the resulting cell suspension in a Coulter counter. Cell counts were obtained at day 0 (24 h after plating) and at days 3, 5, and 7 after plating.

Establishment of Swiss 3T3 Cells Stably Expressing
PKD-In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with MSCV retrovirus encoding murine PKD linked via an internal ribosome entry site (IRES) to GFP. This bicistronic retroviral vector expresses PKD and GFP as two separate proteins. Since transcription of both genes is driven by the same promoter, cells expressing higher levels of GFP also express higher levels of PKD. Consequently, GFP was used as a marker for selection of PKD-positive cells (termed Swiss 3T3-PKD.GFP cells). After infection, cells expressing higher levels of GFP were sorted by FACS, collected, and propagated for further studies (Fig. 1A, left). A major advantage of this expression system is that it eliminates phenotypic variation(s) resulting from clonal selection. As a control, parallel cultures of Swiss 3T3 cells were infected with MSCV retrovirus encoding only GFP and then FACS-sorted, collected, and propagated to generate Swiss 3T3-GFP cells (Fig. 1B, right). As expected, both Swiss 3T3-PK-D.GFP cells and Swiss 3T3-GFP cells express GFP, as revealed by fluorescence microscopy (Fig. 1B).
In order to examine the expression of PKD in the FACSsorted cells, lysates of these cell populations were analyzed by SDS-PAGE and Western blotting using an antibody directed against the C-terminal region of this enzyme. As shown in Fig.  1C, lysates of Swiss 3T3-PKD.GFP cells exhibited a marked increase (8.7 Ϯ 1.8-fold increase; n ϭ 12) in the expression of an immunoreactive band of 115 kDa, which corresponds to the molecular mass of PKD (20), as compared with Swiss 3T3-GFP cells. The detection of this band was completely blocked when the immunoblots were incubated with the antibody in the presence of the immunizing peptide EEREMKALSERVSIL that corresponds to the C-terminal region of the predicted amino acid sequence of PKD. A prominent PKD band was also obtained when lysates from Swiss 3T3-PKD.GFP cells were immunoprecipitated with the PA-1 antiserum (see "Experimental Procedures"), and the immunoprecipitates were analyzed by Western blotting using a different antibody directed against PKD (results not shown). Detection of this 115-kDa band was blocked by the inclusion of the immunizing peptide during the immunoprecipitation. Thus, using high efficiency retrovirally mediated transfection we generated Swiss 3T3 cells overexpressing PKD.
PKD Activation in Response to Bombesin, Vasopressin, or PDBu in Swiss 3T3-PKD.GFP Cells-We reported that cell stimulation with neuropeptide agonists that signal through heptahelical receptors coupled to G␣ q , including bombesin and vasopressin, induces rapid PKC-dependent conversion of PKD from an inactive to an active state (32,35,39,43,54). Here, we examined whether the activity of PKD overexpressed in Swiss 3T3 cells is regulated in a similar manner.
To determine whether bombesin induces PKD activation in intact Swiss 3T3-PKD.GFP cells, cultures of these cells were treated with increasing concentrations of this agonist for 10 min and lysed, and PKD was immunoprecipitated with PA-1 antiserum. The resulting immunocomplexes were incubated with [␥-32 P]ATP, and the incorporation of 32 P into PKD was analyzed by SDS-PAGE and autoradiography. As shown in Fig.  2A, PKD isolated from unstimulated Swiss 3T3-PKD.GFP cells had very low catalytic activity, indicating that overexpression did not lead to constitutive activation of this enzyme. Stimulation of these cells with bombesin induced a striking dose-dependent increase in PKD kinase activity that was maintained during cell lysis and immunoprecipitation. Half-maximal and maximal increases in catalytic PKD activity were achieved at 0.3 and 3 nM. PKD activation was also induced when Swiss 3T3-PKD.GFP cells were stimulated with PDBu instead of bombesin.
Next, we examined whether PKD activation induced by bombesin or PDBu in Swiss 3T3-PKD.GFP cells occurs through a PKC-dependent pathway. Cultures of these retrovirally transfected cells were treated with either Ro 31-8220 or bisindolylmaleimide GF I, selective inhibitors of phorbol-ester sensitive isoforms of PKC (55-57), but not PKD (31)(32)(33), before bombesin or PDBu stimulation. As illustrated in Fig. 2B, the increase in PKD activity induced by these stimuli was markedly attenuated by treatment with either Ro 31-8220 or GF I.
As an independent measure of PKD activation induced by GPCR agonists, PDBu, or growth factors in Swiss 3T3-PK-D.GFP cells, we also examined phosphorylation of an exogenous substrate using syntide-2 (58, 59), a peptide identified as an excellent model substrate for PKD (20,24,53,60). Consistent with the results of in vitro autophosphorylation assays, syntide-2 phosphorylation assays also showed that PKD isolated from unstimulated Swiss 3T3-PKD.GFP cells had low catalytic activity (Fig. 2C). PKD activity was markedly activated by stimulation of these cells with bombesin or PDBu (solid bars), as compared with the activity immunoprecipitated from lysates of either unstimulated cells or of Swiss 3T3-GFP cells (open bars).
In addition to bombesin and PDBu, vasopressin, which acts in Swiss 3T3 cells via an endogenously expressed V1 receptor subtype, also induced a marked increase in PKD activity as shown by syntide-2 phosphorylation assays. In contrast, addition of either EGF or insulin, neither of which stimulate PKC activity in Swiss 3T3 cells, did not induce any significant increase in the activity of PKD (Fig. 2C).
The binding of bombesin to its heptahelical GPCR also induces rapid tyrosine phosphorylation of multiple substrates in Swiss 3T3 cells including FAK and paxillin (61)(62)(63)(64). As shown in Fig. 2D, PKD overexpression did not enhance bombesininduced FAK or paxillin tyrosine phosphorylation, which are known to be mediated via a Rho-dependent but PKC-independent pathway (62,65). These results indicate that PKD overexpression did not appear to have a generalized effect on bombesin signaling.
Multisite PKD Phosphorylation in Response to Bombesin or PDBu in Swiss 3T3-PKD.GFP Cells-Recently, an antiserum specifically recognizing the phosphorylated form of a PKD Cterminal residue, serine 916, was developed and used to detect in vivo autophosphorylation at this site by active PKD (66). Thus, the pS916 antiserum provides a novel approach for detecting conversion of PKD to an active form within intact cells. Here, lysates from Swiss 3T3-PKD.GFP cells stimulated with increasing concentrations of bombesin were analyzed by SDS-PAGE followed by Western blot analysis using the pS916 antiserum. Bombesin stimulation induced a dramatic increase in the immunoreactivity of the PKD band indicative of phosphorylation at Ser 916 (Fig. 3A). The dose response of bombesininduced Ser 916 phosphorylation is in excellent agreement with the dose response generated by measuring the in vitro kinase activity after immunoprecipitation.
Stimulation with either vasopressin or PDBu also induced a marked increase in PKD activity within cells as shown by Ser 916 phosphorylation (Fig. 3B). In contrast, neither EGF nor insulin induced any detectable increase in PKD autophosphorylation at Ser 916 .
The increase in PKD Ser 916 phosphorylation induced by either bombesin or PDBu was markedly attenuated by treatment with the PKC inhibitors Ro 31-8220 and GF I (Fig. 3C). In contrast, inhibition of other kinases including MEK with PD098059 (67), p38MAPK with SB202190, phosphoinositide 3-kinase with wortmannin (68, 69), p70 ribosomal S6 kinase with rapamycin (70 -72), Src family with PP-2 (73), Rho-associated kinases with HA 1077 (74), or disruption of the actin cytoskeleton with cytochalasin D did not affect PKD Ser 916 phosphorylation in response to bombesin in Swiss 3T3-PK-D.GFP cells (Fig. 3D). These results corroborate the notion that PKD activation in Swiss 3T3-PKD.GFP cells proceeds through a PKC-dependent pathway.
We proposed previously (40,41,43,75) that phosphorylation of Ser 744 and Ser 748 within the PKD activation loop plays a critical role in PKD activation. Specifically, a PKD mutant with both Ser 744 and Ser 748 altered to non-phosphorylatable alanines could not be activated in vivo by PKC stimulation. Recently, we developed novel phosphorylation state-specific antibodies directed against Ser 744 and Ser 748 in the PKD activation loop (52). By using these antibodies, we demonstrate here that PKD Ser 744 /Ser 748 phosphorylation is rapidly triggered in Swiss 3T3-PKD.GFP cells by stimulation of cells with either bombesin or PDBu (Fig. 3E). The increase in the phosphoryla- tion of these residues was also prevented by prior exposure to the PKC inhibitor GF I. The results presented in Fig. 3 show that PKD becomes phosphorylated at Ser 744 , Ser 748 , and Ser 916 in response to bombesin or PDBu stimulation in Swiss 3T3-PKD.GFP cells via a PKC-mediated pathway.
Taken together, the results illustrated in Figs. 2 and 3 demonstrate that PKD overexpressed in Swiss 3T3 cells retains tight, signal-dependent, regulation of multisite phosphorylation and kinase catalytic activity.
Overexpression of PKD Potentiates DNA Synthesis Induced by Bombesin, Vasopressin, or PDBu-Bombesin, vasopressin, PDBu, and EGF are potent mitogens for Swiss 3T3 cells through PKC-dependent and -independent signaling pathways (1,19). For instance, chronic treatment with PDBu, which downregulates conventional and novel PKCs, prevents the growthpromoting effects elicited by bombesin, vasopressin, or PDBu but does not interfere with the proliferative response induced by EGF in the presence of insulin. Since PKD has been identified as a downstream target of PKCs (31-39), we hypothesized that PKD could play a role in mediating PKC-dependent mitogenesis. To test this hypothesis, we examined whether PKD overexpression in Swiss 3T3 cells selectively potentiates the proliferative response to bombesin, vasopressin, and PDBu in these cells.
Quiescent cultures of either Swiss 3T3-PKD.GFP (solid bars) or Swiss 3T3-GFP (open bars) cells were washed and transferred to media containing bombesin, PDBu, vasopressin, EGF, or insulin. Incorporation of [ 3 H]thymidine into DNA was measured after 40 h of incubation. As illustrated by Fig. 4, addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in the level of DNA synthesis as compared with that produced by these stimuli in cultures of Swiss 3T3-GFP cells. In six independent experiments, the fold increases in DNA synthesis were 7.5 Ϯ 1.1, 7.3 Ϯ 1.2, and 7.8 Ϯ 1.2 in response to bombesin, vasopressin, or PDBu, respectively (Fig. 4, inset). In contrast, stimulation of DNA synthesis induced by EGF or insulin, which act via PKC/PKD-independent pathways, was not enhanced in Swiss 3T3-PKD.GFP cells. These results suggested that overexpression of PKD selectively facilitates PKC-dependent DNA synthesis in Swiss 3T3 cells.
In order to substantiate the notion that PKD overexpression selectively potentiates mitogenesis mediated through PKC, we examined in more detail the stimulation of DNA synthesis induced by bombesin, PDBu, and EGF in Swiss 3T3-PKD.GFP and Swiss 3T3-GFP cells. Stimulation of DNA synthesis in response to bombesin started after a lag period (G 1 ) of 12-14 h and reached a maximal level after 48 h of incubation (Fig. 5A). Bombesin-induced DNA synthesis was markedly enhanced in Swiss 3T3-PKD.GFP as compared with Swiss 3T3-GFP cells at all times examined, up to 48 h of incubation. We also determined [ 3 H]thymidine incorporation into Swiss 3T3-PKD.GFP and Swiss 3T3-GFP cells stimulated with increasing concentrations of bombesin (0.1-10 nM). As shown in Fig. 5B, bombesin-induced re-initiation of DNA synthesis in Swiss 3T3-PK-D.GFP was markedly higher than in Swiss 3T3-GFP even at the highest concentration of bombesin tested (10 nM).
As shown in Fig. 5C, PDBu-induced DNA synthesis was also strikingly potentiated in Swiss 3T3-PKD.GFP as compared with Swiss 3T3-GFP cells. The concentration of PDBu required to induce half-maximal concentration of [ 3 H]thymidine incorporation in Swiss 3T3-PKD.GFP cells and Swiss 3T3-GFP cells was shifted by ϳ10-fold (from 2 to 20 nM). In sharp contrast, the dose response of DNA synthesis induced by EGF in the presence of insulin in Swiss 3T3-PKD.GFP cells was identical to that generated in Swiss 3T3-GFP cells (Fig. 5D). The results presented in Fig. 5 substantiate the hypothesis that PKD selectively facilitates PKC-dependent mitogenesis in Swiss 3T3 cells.
PKD Overexpression Potentiates Cell Proliferation Induced by Bombesin, Vasopressin, or PDBu-We also determined if overexpression of PKD selectively enhances cell proliferation in response to bombesin, vasopressin, or PDBu. Sparse cultures of Swiss 3T3-PKD.GFP (Fig. 6, solid bars), Swiss 3T3-GFP (open bars), and untransfected Swiss 3T3 cells (hatched bars) were transferred to media containing bombesin, vasopressin, PDBu, or EGF. For comparison, parallel cultures were incubated with DMEM containing 10% fetal bovine serum. Cell number was determined by counting trypsinized cells using a Coulter counter. As illustrated in Fig. 6, addition of bombesin induced a dramatic increase in cell number of Swiss 3T3-PKD.GFP as compared with either Swiss 3T3-GFP or untransfected Swiss 3T3 cells. Addition of PDBu was as effective as bombesin in promoting cell proliferation of Swiss 3T3-PKD.GFP as compared with either Swiss 3T3-GFP or untransfected Swiss 3T3 cells. Vasopressin also induced proliferation of PKD-overexpressing cells, although it was less effective than bombesin or PDBu. In contrast, EGF induced cell proliferation to approximately the same degree in Swiss 3T3-PKD.GFP, Swiss 3T3-GFP, and untransfected Swiss 3T3 cells.
In order to substantiate further the results presented in Fig.  6A, we also examined cell proliferation of Swiss 3T3-PKD.GFP and Swiss 3T3-GFP in response to bombesin or EGF at various times. As shown in Fig. 6B, the increase in cell proliferation induced by bombesin was clearly evident after 5 and 7 days of incubation, whereas EGF-induced cell proliferation was similar in Swiss 3T3-PKD.GFP and Swiss 3T3-GFP at all times examined. The results presented in Fig. 6 corroborate the hypothesis that PKD facilitates cell proliferation induced through the PKC pathway in Swiss 3T3 cells.
Concluding Remarks-Neuropeptides including bombesin/ gastrin-releasing peptide are multifunctional agonists that act as potent cellular growth factors for a variety of cell types (1, 4, 19, 76 -79). Several lines of evidence including gene knock-out studies indicate that the mitogenic effects of neuropeptides are relevant for a variety of normal biological processes including development, inflammation, and cell proliferation under physiological conditions (2). It is also increasingly recognized that multiple neuropeptides, including those of the bombesin family, play an important role as autocrine/paracrine growth factors for human cancer cells (3,79). Consequently, it is important to identify the intracellular signal transduction pathways that mediate the mitogenic effects induced by these multifunctional agonists.
Although the PKC family occupies a pivotal role in the signal transduction pathways that mediate numerous cellular responses including neuropeptide-induced cell proliferation, the events occurring downstream of specific isoforms of PKC remain elusive. Recently, PKD has emerged as a putative downstream target of novel isoforms of PKC. Accordingly, PKC-dependent PKD activation within cells has been demonstrated in a wide variety of systems. These include engagement of GPCRs (32,35,36,38,39,43), tyrosine kinase receptors (32,34), or antigen receptors (25,28,37) with cognate ligands, induction of cellular oxidative stress (75), transfection with G␣ q which mediates signal transduction leading to phospholipase C activation (43), transfection of constitutively activated novel PKC isoforms (24,31), or by direct PKC stimulation by phorbol esters, membrane-permeant diacylglycerols, or bryostatin-1 (24, 31-33, 35-40, 43, 75). These findings established PKD as a novel downstream target of PKC-mediated signal transduction and raised the possibility that PKD lies in a signal transduction pathway that mediates PKC-dependent mitogenesis.
In an effort to clarify the contribution of PKD to PKC-dependent mitogenic signal transduction, we examined whether PKD overexpression in Swiss 3T3 cells potentiates the proliferative response to the GPCR agonists, bombesin and vasopressin, or the biologically active phorbol ester PDBu. We generated Swiss 3T3 cells stably overexpressing PKD using MSCV retrovirus encoding PKD and GFP as two separate proteins translated from the same mRNA, and we used GFP as a marker for selection of PKD-positive cells. Initially, we verified that PKD overexpressed in Swiss 3T3 cells was dramatically activated by cell treatment with either bombesin or PDBu, as judged by in vitro kinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, bombesin or PDBu stimulation induced PKD phosphorylation at multiple sites including Ser 744 and Ser 748 in the kinase activation loop and at Ser 916 , an autophosphorylation site located in the C terminus of the molecule. PKD activation and phosphorylation were prevented by exposure of the cells to PKC-specific inhibitors. These results established that PKD overexpressed in Swiss 3T3 cells retains tight, PKC-dependent, regulation of multisite phosphorylation and kinase catalytic activity. Our next step was to use this model system to determine whether increased expression of PKD enhances neuropeptide-induced DNA synthesis and cell proliferation.
The salient feature of the results presented here is that PKD overexpression in Swiss 3T3 cells selectively potentiated the stimulation of DNA synthesis induced by bombesin, vasopressin, or PDBu in these cells. These stimuli also induced a striking increase in cell number in Swiss 3T3 overexpressing PKD as compared with either Swiss 3T3 transfected with GFP or untransfected Swiss 3T3 cells. In contrast, stimulation of DNA synthesis and cell proliferation in response to EGF, which acts via PKC/PKD-independent pathways, was not enhanced. Collectively, our results demonstrate that overexpression of PKD selectively facilitates PKC-dependent mitogenesis in Swiss 3T3 cells and thus indicate that PKD plays a role in mediating GPCR agonist-induced cell proliferation.