Protein kinase D potentiates DNA synthesis induced by Gq-coupled receptors by increasing the duration of ERK signaling in swiss 3T3 cells.

Protein kinase D (PKD) potentiates cellular DNA synthesis in response to G protein-coupled receptor (GPCR) agonists but the mechanism(s) involved has not been elucidated. Here, we examined whether PKD overexpression in Swiss 3T3 cells regulates the activation/inactivation kinetics of the extracellular-regulated protein kinase (ERK) in response to the mitogenic GPCR agonists bombesin and vasopressin. Addition of bombesin or vasopressin to Swiss 3T3 cells overexpressing PKD induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of either control Swiss 3T3 cells or Swiss 3T3 cells expressing a kinase-inactive PKD mutant. In contrast, the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not increased. Furthermore, bombesin or vasopressin promoted a striking increase in phosphorylation (at Ser-374) and accumulation of c-Fos (the c-fos proto-oncogene product) in Swiss 3T3 cells overexpressing wild-type (but not kinase-inactive) PKD. Inhibition of the sustained phase of ERK/RSK activation abrogated the increase in c-Fos accumulation and DNA synthesis induced by bombesin or vasopressin in PKD-overexpressing cells. Our results demonstrate that PKD selectively potentiates mitogenesis induced by bombesin or vasopressin in Swiss 3T3 cells by increasing the duration of MEK/ERK/RSK signaling.

PKD (also initially known as PKC) is a serine/threonine protein kinase with structural, enzymology, and regulatory properties different from the PKC family members (20,21). PKD most distinct characteristics are the presence of a catalytic domain distantly related to Ca 2ϩ -regulated kinases, a pleckstrin homology (PH) region that regulates enzyme activity and a highly hydrophobic stretch of amino acids in its Nterminal region (22)(23)(24). This N-terminal region also contains a tandem repeat of cysteine-rich, zinc finger-like motifs, which confers high affinity for phorbol esters and plays a negative role in the regulation of catalytic kinase activity (25)(26)(27)(28). The identification of PKD-2 and PKD-3, similar in overall structure, primary amino acid sequence, and enzymology properties to PKD/PKC (29 -32), supports the notion that PKD isoenzymes constitute a separate family of serine protein kinases.
PKD can be activated in intact cells through multiple G protein pathways, including G q , G i , and G 12 (33)(34)(35)(36)(37)(38)(39), as well as by biologically active phorbol esters, growth factors and antigen-receptor engagement (36, 37, 39 -45). In all these cases, rapid PKD activation is mediated by PKC-dependent phosphorylation of Ser-744 and Ser-748 within the activation loop of the catalytic domain of PKD (33, 46 -48). PKD activation is associated with its translocation to the plasma membrane and subsequent transient accumulation in the nucleus (25,28,50,51). These findings revealed that PKD is activated by multiple growth-promoting factors (22,52) suggesting that it functions in mitogenic signaling. Indeed, we reported that PKD overexpression markedly potentiates DNA synthesis induced by the GPCR agonists bombesin and vasopressin in Swiss 3T3 cells (53), a cell line that has been used extensively as a model system to elucidate signal transduction pathways in the mitogenic action of GPCR agonists (1,54,55). These results suggest that PKD plays an important role in mediating cellular DNA synthesis in response to GPCR agonists, but the mechanism(s) involved has not been elucidated.
One of the major signaling pathways involved in the mitogenic response induced by both receptor tyrosine kinases and GPCRs is the ERK cascade (56,57). The ERKs (ERK-1 and ERK-2) are directly activated by phosphorylation on specific tyrosine and threonine residues by the dual-specificity ERK kinase (or MEK). Studies on the mechanisms by which mitogenic GPCRs activate ERK revealed considerable heterogeneity, depending on receptor and cell context (58). It is increasingly recognized that the duration and intensity of ERK pathway activation is of critical importance for determining specific biological outcomes, including proliferation, differentiation, and transformation (59,60). For example, sustained activation of ERK for several hours is associated with re-initiation of DNA replication in fibroblasts whereas transient ERK activation (20 -30 min) is not sufficient to promote mitogenesis in these cells (60,61). The protein products of immediate early genes (e.g. c-Fos) have been proposed to function as molecular sensors of ERK1/2 signal duration (62). As a first step to elucidate the mechanism(s) by which PKD facilitates GPCR-induced mitogenesis, we examined whether PKD overexpression influences the duration or intensity of ERK signaling in response to mitogenic GPCR agonists.
The results presented here demonstrate that overexpression of wild-type (but not kinase-inactive) PKD dramatically increases the duration of MEK/ERK/RSK activation and the accumulation of c-Fos (the c-fos proto-oncogene product) induced by either bombesin or vasopressin in Swiss 3T3 cells. Inhibition of the sustained phase of ERK activation by treatment with either U0126 or PD98059 abrogated the accumulation of c-Fos protein and the potentiation of DNA synthesis induced by bombesin or vasopressin in Swiss 3T3 cells overexpressing PKD. Our results indicate that an increase in the duration of the ERK signal is one of the mechanisms by which PKD facilitates GPCR-induced mitogenesis.

EXPERIMENTAL PROCEDURES
Cell Culture-Stock cultures of Swiss 3T3-PKD.GFP cells, which overexpress PKD, Swiss 3T3-PKDK618N.GFP cells, which overexpress the kinase-inactive PKD mutant PKDK618N, and control Swiss 3T3-GFP cells generated as previously described (53), 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 cells became confluent and quiescent (63). Phoenix packaging cells (kindly provided by Dr. G. Nolan, Stanford University, Stanford, CA) were cultured in the same medium in a humidified atmosphere containing 5% CO 2 .
Production of Retrovirus-To generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were transduced with retrovirus encoding either wild-type or kinase-inactive murine PKD, in which PKD and GFP were translated from the same bicistronic mRNA and expressed as two separate proteins. After transduction, cells expressing higher levels of GFP were sorted by FACS, collected and propagated for further studies.
Specifically, construction of the retroviral plasmid expressing wildtype PKD (MSCV-PKDiresGFP) has been previously described (53). To generate retroviral plasmid containing cDNA of the kinase-inactive PKD (MSCV-PKDK618NiresGFP), the fragment of the wild-type PKD cDNA spanning from ϩ1660 to ϩ2910 of the published sequence and containing the K618N mutation was amplified by PCR from the previously described kinase-inactive construct (40). The 5Ј-PCR primer contained the endogenous HpaI restriction site. The 3Ј-PCR primer was designed to introduce an additional HpaI site at the 3Ј-end of the amplified fragment. The HpaI digest of the MSCV-PKDiresGFP construct released the ϩ1660/ϩ2910 fragment of the wild-type PKD. The PCR-amplified fragment of the kinase-inactive PKD (ϩ1660 to ϩ2910) was introduced into HpaI sites of the MSCV-PKDiresGFP by ligation, thus producing MSCV-PKDK618NiresGFP. The nucleotide sequence of the MSCV-PKDK618NiresGFP was validated by sequencing.
For retrovirus production, logarithmically growing Phoenix ecotropic cells were transfected with MSCV-PKDiresGFP, MSCV-PKDK618-NiresGFP or MSCV-iresGFP 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 transduced cells was generated every 2 month. Following sorting, GFPpositive cells were maintained as described earlier in this section.
Immunoblotting and Detection of MEK, ERK, Ribosomal S6 Kinases (RSK), FAK, PKD, and c-Fos-Confluent, quiescent Swiss 3T3-GFP, Swiss 3T3-PKD.GFP and Swiss 3T3-PKDK618N.GFP cells were lysed in 2ϫ SDS-polyacrylamide 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. 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, and 20% CH 3 OH. For detection of proteins, membranes were blocked using 5% nonfat dried milk in phosphate-buffered saline (pH 7.2) and then incubated for at least 2 h with the desired antibodies diluted in phosphate buffered saline (pH 7.2) containing 3% nonfat dried milk. Bound primary antibodies to immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection with horseradish peroxidase conjugated anti-mouse or anti-rabbit. The phosphospecific antibodies used were as follows; the phospho-ERK1/2 monoclonal antibody recognizes the ERK-1/2 only when they are phosphorylated on Thr-202 and Tyr-204 (pERK1/ERK2); the phospho MEK1/2 polyclonal antibody recognizes MEK1/2 only when they are phosphorylated on Ser-217 and Ser-221 (pMEK1/2); the phospho-p90RSK polyclonal antibody is specific to p90RSK only when it is phosphorylated on Thr-574; the phospho-MARCKS polyclonal antibody is specific to the phosphorylated state of MARCKS only when it is phosphorylated on Ser-152 and Ser-156 (pMARCKS); the phospho-FAK910 polyclonal antibody recognizes FAK only when it is phosphorylated on Ser-910; the phospho-FAK397 polyclonal antibody recognizes FAK only when it is phosphorylated on Tyr-397, the major autophosphorylation site of FAK; the phospho FAK577 polyclonal antibody recognizes FAK only when it is phosphorylated on Tyr-577; the phospho-PKD polyclonal antibody PKDS916 recognizes PKD only when it is autophosphorylated on Ser-916. The phospho c-Fos polyclonal antibody recognizes c-Fos only when it is phosphorylated on Ser-374. Autoluminograms were scanned using a GS-710 scanner (Bio-Rad), and the bands were quantified using the Quantity One software program (Bio-Rad).
Immunoprecipitation of FAK and c-Fos-Cultures of Swiss 3T3 cells, treated as described in the individual experiments, were washed and lysed in 50 mM Tris/HCl pH 7.6, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and 1% Triton X-100 (lysis buffer A). Cell lysates were clarified by centrifugation at 15,000 ϫ g for 10 min at 4°C. Immunoprecipitations were carried out at 4°C for 2-4 h using a FAK C-20 polyclonal antibody for FAK and a c-Fos polyclonal antibody for c-Fos. The immune complexes were recovered using protein-A coupled to agarose and solubilized with 2ϫ sample buffer.
Exogenous substrate phosphorylation by immunoprecipitated ERK1/2 was determined as previously described (20). Briefly, the immunocomplexes were washed twice with lysis buffer A and twice with kinase buffer (30 mM Tris-HCL, pH 7.6, 10 mM MgCl 2 , 1 mM dithiothreitol). Then, 20 l of the washed ERK immunocomplexes were mixed with 20 l of kinase buffer containing 100 M final concentration of [␥-32 P]ATP (specific activity 400 -600 cpm/pmol) and 20 g of MBP. After 10 min of incubation at 30°C, the reaction was stopped by adding 100 l of 75 mM H 3 PO 4 and spotting 75 l of the supernatant on 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, in 75 mM H 3 PO 4 . The papers were dried, and the radioactivity incorporated into the MBP substrate was determined by Cerenkov counting.
Assay of DNA Synthesis-Confluent and quiescent cultures of trans- The following antibodies were purchased from Signaling Technology (Beverly, MA), phospho-p44 mapk /p42 mapk (pERK1/2), phospho-MEK1/2, phospho-p90 RSK , and phospho-MARCKS. The phosphospecific polyclonal antibodies to Ser-910, Tyr-397, and Tyr-577 of FAK were obtained from BioSource International (Camarillo, CA). Anti-c-Fos polyclonal and the ERK1/2 kinase assay were from Upstate Biotechnology. Anti-ERK-2 and anti-FAK C-20 polyclonal antibodies were obtained from Santa Cruz Biotechnology (San Diego, CA). The phosphospecific monoclonal antibody to Ser-374 of c-Fos was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Phospho-PKD916 (53) and the PA-1 (64) antisera were generated by the Antibody Core of the CURE: Digestive Diseases Research Center. All other materials were of the highest grade available.

Protein Kinase D Overexpression Selectively Increases the Duration of ERK Signaling Induced by Bombesin in Swiss 3T3
Cells-In an effort to elucidate the mechanism(s) by which PKD overexpression potentiates the mitogenic activity of GPCR agonists, we examined the intensity and duration of ERK activation in bombesin-stimulated Swiss 3T3-PKD.GFP cells, which overexpress PKD, and control Swiss 3T3-GFP cells, generated as previously described (53). We previously demonstrated that PKD overexpressed in Swiss 3T3 cells retains tight, signal-dependent, regulation of multisite phosphorylation and kinase catalytic activity (53).
Quiescent monolayers of Swiss 3T3-PKD.GFP cells and control Swiss 3T3-GFP cells were challenged with 10 nM bombesin at 37°C for various times and then lysed. The active forms of ERK-1 and ERK-2 in the extracts were detected by Western blotting using an antibody that detects the dually phosphorylated forms of these enzymes at the regulatory threonine and tyrosine residues. As shown in Fig. 1, ERK activation loop phosphorylation in both Swiss 3T3-PK-D.GFP cells and Swiss 3T3-GFP cells increased dramatically to similar levels within 5 min of bombesin stimulation. In Swiss 3T3-GFP cells, the ERK signal was transient, i.e. ERK phosphorylation declined rapidly after 5 min, and returned to near baseline levels by 60 min. Similar kinetics were obtained in untransfected Swiss 3T3 cells (results not shown). In striking contrast, bombesin-induced ERK activation in Swiss 3T3 cells overexpressing PKD is dramatically prolonged as compared with control Swiss 3T3 cells. Indeed, ERK phosphorylation in Swiss 3T3-PKD.GFP cells was robust even after 240 min of bombesin stimulation (Fig. 1A, upper panel). These results were corroborated when ERK phosphorylation was examined in response to increasing concentrations of bombesin added for either 5 or 240 min. Bombesin stimulation for 5 min increased ERK phosphorylation in both Swiss 3T3-PKD.GFP cells and Swiss 3T3-GFP cells, in a concentration-dependent manner (Fig. 1A). Bombesin stimulation for 240 min induced ERK phosphorylation in Swiss 3T3-PKD.GFP cells in a concentration-dependent manner with maximal effect at 3 nM whereas no effect of bombesin at this concentration was detected in control Swiss 3T3-GFP cells and only a slight increase in ERK phosphorylation was seen in these cells at 10 nM (Fig. 1A, lower panel). These results indicate that overexpression of PKD dramatically increases the duration of ERK signaling in response to bombesin, especially in cells stimulated by low concentrations of this neuropeptide (1-3 nM).
To substantiate that overexpression of PKD promotes sustained ERK catalytic activity in response to bombesin, the ERKs were immunoprecipitated from lysates of either Swiss 3T3-PKD.GFP cells or Swiss 3T3-GFP cells treated with or without 10 nM bombesin for 5 min or 240 min and the resulting immunocomplexes were subjected to in vitro kinase assays using MBP, as a substrate. As shown in Fig. 1B, bombesin stimulation for 5 min induced a striking increase (ϳ8-fold) in ERK kinase activity in both Swiss 3T3-PKD.GFP cells and Swiss 3T3-GFP cells. After 240 min of incubation with bombesin, ERK catalytic activity from Swiss 3T3-PKD.GFP cells was markedly increased as compared with that immunoprecipitated from Swiss 3T3-GFP cells. Thus, the duration of ERK phosphorylation and catalytic activation in response to bombesin is markedly increased in cells overexpressing PKD.
The binding of bombesin to its heptahelical GPCR in Swiss 3T3 cells induces a complex array of signaling events, including PKC-dependent phosphorylation of MARCKS (65,66) and rapid tyrosine phosphorylation of the non-receptor tyrosine kinase FAK, via a Rho-dependent but PKC-independent pathway (3,55). As shown in Fig. 1C, PKD overexpression did not increase the intensity or duration of either MARCKS phosphorylation at Ser-152/Ser-156 or FAK tyrosine phosphorylation in response to bombesin. These results indicate that PKD overexpression does not have a generalized effect on the kinetics of bombesin receptor signaling but selectively increases the duration of ERK activation.

PKD Overexpression Selectively Increases the Duration of ERK Signaling Induced by Vasopressin in Swiss 3T3 Cells-
Vasopressin, which acts in Swiss 3T3 cells via an endogenously expressed G q -coupled V1 receptor subtype (67,68), also induces a marked increase in PKD activity (36,53). In contrast, EGF does not induce any significant increase in the phosphorylation or catalytic activity of PKD (53). To examine the notion that PKD overexpression selectively increases the duration of ERK signaling by G q -coupled agonists that act via PKD, we determined the kinetics of ERK phosphorylation and catalytic activation in response to either vasopressin or EGF in Swiss 3T3-PKD.GFP cells and Swiss 3T3-GFP cells.
As shown in Fig. 2A, ERK phosphorylation in both Swiss 3T3-PKD.GFP cells and Swiss 3T3-GFP cells increased within 5 min of vasopressin stimulation. In Swiss 3T3-GFP cells, ERK phosphorylation declined to near baseline levels by 30 min. In striking contrast, vasopressin-induced ERK phosphorylation in Swiss 3T3 cells overexpressing PKD was strikingly prolonged and it was still evident even after 240 min of vasopressin stimulation ( Fig. 2A, upper panel). Similarly, vasopressin stimulation for 5 min induced a striking increase in ERK kinase activity in both Swiss 3T3-PKD.GFP cells and Swiss 3T3-GFP cells (Fig. 2B). After 240 min of incubation with vasopressin, ERK activity from Swiss 3T3-PKD.GFP cells was markedly increased as compared with that from Swiss 3T3-GFP cells, which was virtually identical to the activity of unstimulated cells. These results indicate that the duration of ERK phosphorylation and catalytic activation in response to vasopressin are markedly increased in cells overexpressing PKD. In sharp contrast, the kinetics of ERK phosphorylation ( Fig. 2A, lower panel) and activation (Fig. 2B) induced by EGF in Swiss 3T3-PKD.GFP cells was identical to that obtained in Swiss 3T3-GFP cells.
We verified that PKD overexpression did not increase the duration of either MARCKS phosphorylation or FAK tyrosine phosphorylation in response to either vasopressin or EGF (Fig.  2C). The results presented in Fig. 2 are consistent with the hypothesis that PKD overexpression selectively increases the duration ERK signaling in Swiss 3T3 cells stimulated by G qcoupled receptor agonists.
The Increase in the Duration of ERK Signaling Induced by Bombesin and Vasopressin in PKD-overexpressing Cells Requires the Catalytic Activity of PKD-Next, we examined whether the PKD-induced increase in the duration of ERK signaling in response to GPCR stimulation requires the catalytic activity of PKD. Previously, we reported that mutation of Lys 618 in the catalytic domain of PKD renders this enzyme non-functional (40). To generate Swiss 3T3 cells stably overexpressing kinase-inactive PKD, cultures of these cells were infected with retrovirus encoding PKD-K618N and GFP expressed as two separate proteins translated from the same bicistronic mRNA. After infection, cells expressing higher levels of GFP (termed Swiss 3T3-PKDK618N.GFP cells) were sorted by FACS, collected and propagated for further studies.
An antiserum specifically recognizing the phosphorylated form of a PKD C-terminal residue, Ser-916, has been used to detect in vivo autophosphorylation at this site by active PKD (69). Thus, this antibody detects the conversion of PKD from an inactive state to an active form within intact cells. Here, lysates from Swiss 3T3-PKD.GFP, Swiss 3T3-PKDK618N.GFP and Swiss 3T3-PKD.GFP cells stimulated with bombesin, vasopressin or EGF for 5 min or 240 min were analyzed by SDS-PAGE followed by Western blot analysis using the pS916 antiserum. Bombesin and vasopressin stimulation for either 5 min or 4 h induced a dramatic increase in the immunoreactivity of the wild-type PKD band indicative of phosphorylation at Ser 916 (Fig. 3A). In contrast, EGF did not produce any increase in PKD immunoreactivity, either at early or late times of exposure, consistent with the notion that this tyrosine kinase receptor agonist does not induce any significant activation of PKD. As expected, we only detected faint bands in lysates from Swiss 3T3-PKDK618N.GFP cells, confirming that the mutation K618N renders PKD inactive in intact cells. We verified that wild-type and kinase-deficient PKD were expressed at similar levels in Swiss 3T3 cells (Fig. 3B). Our next objective was to determine whether the catalytic activity of PKD is necessary for prolonging the duration of ERK signaling in response to GPCR agonists. We analyzed ERK1/2 phosphorylation and catalytic activity in lysates from Swiss 3T3-PKD.GFP, Swiss 3T3-PKDK618N.GFP and Swiss 3T3-PKD.GFP cells stimulated with bombesin, vasopressin or EGF for 5 or 240 min. As shown in Fig. 3 (C and D), the increase in the duration of ERK phosphorylation and catalytic activity in response to GPCR agonists requires the catalytic activity of PKD because it was seen in Swiss 3T3-PKD.GFP but not in Swiss 3T3 cells overexpressing a kinase-inactive PKD.
PKD Overexpression Increases the Duration of MEK Activation Induced by Bombesin in Swiss 3T3 Cells-Subsequently, we examined whether PKD overexpression also prolongs the activation of MEK induced by bombesin. Activation of MEK1 and MEK2 occurs through phosphorylation of Ser-217 and Ser-221, located in the kinase activation loop (70). Consequently, MEK activation in response to bombesin (10 nM) added for various times (from 2.5 to 120 min) was evaluated by Western blotting using an antibody that detects the active forms of MEK.
As shown in Fig. 4A, bombesin stimulation for 2.5 min induced a striking increase in MEK phosphorylation in Swiss 3T3-PKD.GFP, Swiss 3T3-PKDK618N.GFP, and Swiss 3T3-PKD.GFP cells. After 60 and 120 min of incubation with bombesin, MEK from Swiss 3T3-PKD.GFP cells was markedly phosphorylated at the activation loop as compared with that from Swiss 3T3-GFP cells or Swiss 3T3-PKDK618N.GFP cells, indicating that the duration of MEK activation in response to bombesin is also increased in cells overexpressing PKD.
To test whether the increase in the duration of ERK activation requires functional MEK, cultures of Swiss 3T3-PKD.GFP and Swiss 3T3-PKD.GFP cells were treated with the selective inhibitors of MEK activation U0126 (71) and PD98059 (72). In agreement with the previous results, bombesin and vasopressin induced sustained ERK activation in cells overexpressing PKD. Treatment with the MEK inhibitors prevented ERK-1 and ERK-2 activation in response to bombesin or vasopressin treatment for either 5 min or 240 min (Fig. 4, B and C). We verified that treatment with U0126 did not exert any detectable effect on PKD activation, as shown by immunoblotting with antibodies that detect the phosphorylated state of PKD at The cells were washed twice with cold PBS and lysed in ice-cold buffer A as described under "Experimental Procedures." ERK1/2 were immunoprecipitated using an anti-ERK1/2 antibody bound to protein A-agarose, and subsequently a kinase assay was performed using MBP as a substrate with the protein A-ERK1/2 immunocomplex as described under "Experimental Procedures." The results shown here are the mean cpm of 32 P incorporated into MBP from three separate dishes Ϯ S.E. Shown here is a representative plot from one experiment. Similar results were obtained in three independent experiments. C, PKD overexpression does not affect MARCKS or FAK phosphorylation induced by vasopressin or EGF in Swiss 3T3 cells. Confluent and quiescent cultures of Swiss 3T3-PKD.GFP cells (ϩ) and Swiss 3T3-GFP cells (Ϫ) were washed and incubated at 37°C in 2 ml of DMEM containing 50 nM VP or 5 ng/ml EGF for 5 or 240 min as indicated and then lysed with 2ϫ SDS-PAGE sample buffer. The cultures were then lysed with 2ϫ SDS-PAGE sample buffer for the detection of phospho-MARCKS. For the detection of tyrosine phosphorylation of the cultures were lysed with ice-cold buffer A and FAK was immunoprecipitated as described under "Experimental Procedures." The samples were analyzed by SDS-PAGE and immunoblotted with either an antibody that detects the phosphorylated state of MARCKS at Ser-152/156 (pMARCKS) or with 4G10 antiphosphotyrosine monoclonal (pY-FAK). Shown here are representative autoluminograms, similar results were obtained in three independent experiments.
Ser-916, a major autophosphorylation site (Fig. 4D). Collectively, the results depicted in Fig. 4 indicate that PKD promotes sustained ERK activation by prolonging the activation of MEK.

PKD Overexpression Selectively Increases the Duration of FAK Phosphorylation at Ser-910 Induced by Bombesin or Vasopressin in Swiss 3T3 Cells-If PKD overexpression increases the duration of ERK pathway activation within cells in re-
sponse to GPCR agonists, we would expect a selective prolongation of the activation/phosphorylation of downstream targets of ERK. An analysis of the individual phosphorylation sites of FAK provides an attractive approach to test this possibility because GPCR agonists, including bombesin, stimulate FAK phosphorylation not only at multiple tyrosines but also at Ser-910, a direct target of ERK (73). Specifically, we determined here whether PKD overexpression differentially prolongs the kinetics of FAK phosphorylation at Ser-910 in bombesintreated Swiss 3T3 cells. Cultures of Swiss 3T3-PKD.GFP cells, Swiss 3T3-GFP cells or Swiss 3T3-PKDK618N.GFP cells were stimulated with bombesin for 60 or 240 min and lysed. The extracts were analyzed by SDS-PAGE and immunoblotted with an antibody that detect the phosphorylated state of FAK at Ser-910 (pS-910). As shown in Fig. 5A, bombesin-induced FAK phosphorylation at Ser-910 was strikingly enhanced in PKDoverexpressing cells as compared with control cells or cells overexpressing the PKDK618N kinase-deficient mutant.
To examine the specificity of the enhancement of FAK phosphorylation at Ser-910 in response to bombesin in Swiss 3T3-PKD.GFP cells, we also determined autophosphorylation of FAK at Tyr-397 (pY-397) and Src-mediated phosphorylation of FAK at Tyr-577 (pY-577), located in the activation loop of the kinase domain (74,75). We demonstrated previously that these residues are phosphorylated in response to bombesin through an ERK-independent pathway (76). As seen in Fig. 5A, bombesin-induced phosphorylation of FAK at either Tyr-397 or Tyr-577 was not altered by PKD overexpression, confirming that the enhancement of FAK phosphorylation is restricted to Ser-910, a residue directly targeted by ERK. We again verified that PKD overexpression increases the duration of ERK signaling in response to bombesin by immunoblotting the same membranes with the antibodies that detect the dually phosphorylated state of ERK (Fig. 5A).
In accord with our previous results, PKD overexpression also enhanced FAK phosphorylation at Ser-910 in response to cell stimulation with vasopressin but not with EGF (Fig. 5B). Furthermore, the increase in FAK phosphorylation at Ser-910 induced by bombesin in PKD-overexpressing cells was abrogated by treatment with the MEK inhibitor U0126 (Fig. 5C). Collectively, the results shown in Fig. 5 substantiate the notion that overexpression of catalytically competent PKD increases the duration of ERK signaling in Swiss 3T3 cells. were washed and incubated at 37°C in 2 ml of DMEM containing either 10 nM Bom, 50 nM VP or 5 ng/ml EGF for 5 or 240 min as indicated. The cells were washed twice with cold PBS and lysed in ice-cold buffer A as described under "Experimental Procedures." ERK1/2 were immunoprecipitated using an anti-ERK1/2 antibody bound to protein A-agarose, and subsequently a kinase assay was performed using MBP as a substrate with the protein A-ERK1/2 immunocomplex as described under "Experimental Procedures." The results shown here are the mean cpm of 32 P incorporated into MBP from three separate dishes Ϯ S.E. Shown here is a representative plot from one experiment. Similar results were obtained in three independent experiments. pressin in Swiss 3T3 Cells-The 90 kDa RSKs are serine/ threonine protein kinases that are activated via ERK-mediated phosphorylation (77,78). If PKD overexpression increases the duration of ERK catalytic activity within cells in response to GPCR agonists, we would also expect a prolongation of the activation/phosphorylation of RSK, a well defined downstream target of ERK. To test this possibility, we determined the effect of bombesin, vasopressin, and EGF on RSK activation in Swiss 3T3-PKD.GFP cells and Swiss 3T3-GFP cells, as revealed by immunoblotting with an antibody that detects phosphorylated Thr-574, a site directly phosphorylated by ERK and important for RSK activation (79).

Protein Kinase D Overexpression Selectively Increases the Duration of RSK Signaling Induced by Bombesin and Vaso-
As shown in Fig. 6A, stimulation for 5 min with bombesin, vasopressin or EGF induced a striking increase in RSK phosphorylation in both Swiss 3T3-PKD.GFP cells and Swiss 3T3-GFP cells, in agreement with the ability of these agonists to induce rapid ERK activation in these cells. After 240 min of treatment with bombesin or vasopressin (but not with EGF), the phosphorylation of RSK isolated from Swiss 3T3-PKD.GFP cells was markedly increased as compared with that from Swiss 3T3-GFP cells, which is entirely consistent with the increased duration of ERK signaling induced by G q -coupled receptor agonists in PKD-overexpressing cells. Furthermore, the increased phosphorylation of RSK induced by bombesin and vasopressin was abrogated by treatment with the MEK inhibitor U0126 (Fig. 6A).
PKD Overexpression Induces Striking Accumulation of c-Fos Protein in Response to Bombesin or Vasopressin-Immediate early gene products, including members of the c-fos protooncogene family, function as cellular sensors of ERK1/2 signal duration (62). When ERK activation is transient, its activity declines before the c-Fos protein accumulates, and c-Fos is degraded rapidly. However, when ERK signaling is sustained, c-Fos is phosphorylated by ERK and RSK at serines 374 and 362, respectively (80,81). As shown in Fig. 6, (panel B), PKD overexpression markedly increased the phosphorylation of c-Fos, as judged by immunoblotting of c-Fos immunoprecipitates with an antibody that detects the phosphorylated state of c-Fos at Ser-374. These C-terminal phosphorylations stabilize c-Fos (80,82) and could promote its cellular accumulation. We hypothesized that sustained bombesin-induced ERK1/2 and RSK signaling in PKD-overexpressing Swiss 3T3 cells leading to c-Fos phosphorylation should increase c-Fos protein accumulation in these cells.
To test this hypothesis, cultures of Swiss 3T3.PKD-GFP and Swiss 3T3.GFP cells were treated with bombesin for various times, lysed, and the levels of c-Fos protein were determined in cell extracts by Western blot analysis. As shown in Fig. 6C PKD (Fig. 6C). These results are consistent with the hypothesis that sustained bombesin-induced ERK1/2 and RSK signaling in PKD-overexpressing Swiss 3T3 cells leads to c-Fos protein accumulation in these cells.
If the dramatic increase in c-Fos accumulation induced by bombesin in Swiss 3T3.PKD-GFP cells is caused by sustained ERK signaling, inhibition of this pathway should abrogate c-Fos protein accumulation. Exposure to either U0126 (Fig. 6D) or PD98059 (results not shown) prior to bombesin or vasopressin stimulation completely blocked the persistent increase in the level of c-Fos protein in Swiss 3T3.PKD-GFP cells.
ERK1/2 are known to mediate rapid activation of c-fos gene transcription (83,84), which is completed within 30 -45 min of stimulation (85). Therefore, the inhibition of c-Fos protein accumulation by U0126 could be attributed to inhibition of c-fos gene transcription in response to bombesin rather than to stabilization of the c-Fos protein. To determine whether sustained ERK signaling is responsible for c-Fos accumulation in response to bombesin in Swiss 3T3.PKD-GFP cells, we also added U0126 2 h after bombesin stimulation; i.e. well after bombesin-induced transcriptional induction of c-fos is completed. As shown in Fig. 6D, the addition of U0126 to cells 2h after bombesin stimulation also suppressed the accumulation of c-Fos protein measured 4 h after bombesin stimulation. Similar results were obtained with vasopressin (Fig. 6D). These results indicate that the dramatic accumulation of c-Fos protein observed in Swiss 3T3.PKD-GFP cells in response to bombesin or vasopressin requires sustained ERK signaling.
Increased Duration of ERK Signaling Mediates the Potentiation of Mitogenic Activity of Bombesin and Vasopressin in PKD-overexpressing Cells-Recently, we reported that PKD overexpression markedly potentiates the induction of DNA synthesis induced by the G q -coupled receptor agonists bombesin and vasopressin in Swiss 3T3 cells, but the mechanism(s) involved was not defined. To determine whether the increase in the mitogenic activity of these agonists in PKD-overexpressing cells is mediated by an increase in the duration of ERK signaling, cultures of Swiss 3T3-PKD.GFP and Swiss 3T3-GFP cells were treated with U0126. As illustrated by Fig. 7 and in agreement with our previous results, stimulation with either bomb-esin or vasopressin of Swiss 3T3 cells overexpressing PKD induced a striking increase in the level of [ 3 H]thymidine incorporation into DNA as compared with that produced by these stimuli in cultures of Swiss 3T3-GFP cells. Addition of U0126 together with bombesin or vasopressin completely blocked the enhancement of DNA synthesis seen in Swiss 3T3-PKD.GFP cells stimulated with these agonists.
To demonstrate that the sustained phase of ERK activation was required to mediate the enhancement of agonist-induced DNA synthesis in PKD-overexpressing cells, ERK activity was inhibited by adding U0126 to cultures that have been stimulated with either bombesin or vasopressin for 3 h. As shown in Fig. 7, addition of U0126 after bombesin or vasopressin stimulation also prevented the increase in DNA synthesis produced in cells overexpressing PKD. Similar results were obtained when the MEK inhibitor PD98059 was used instead of U0126 (Fig. 7, inset). These results strongly suggest that the sustained phase of ERK signaling is critical for the enhancement of agonist-induced DNA synthesis in PKD-overexpressing cells.
Concluding Remarks-A substantial body of evidence indicates that G q -coupled receptor activation leads to the stimulation of the ERKs which are implicated in the regulation of such fundamental cellular processes as proliferation, differentiation and apoptosis (56,57). It is increasingly recognized that the activation/inactivation kinetics of the ERK pathway is critical for determining specific biological outcomes (62). As a first step to elucidate the mechanism(s) by which PKD potentiates GPCR-induced mitogenesis and to identify signaling pathways that regulate the kinetics of ERK activation, we examined whether PKD overexpression influences the intensity or duration of GPCR-promoted ERK activation.
In agreement with many other previous studies, our results show that the duration of ERK activation induced by the GPCR agonists bombesin or vasopressin is short-lived in Swiss 3T3 cells. In striking contrast, we demonstrate, for the first time, that MEK/ERK/RSK activation induced by these agonists is dramatically prolonged in Swiss 3T3 cells overexpressing wildtype PKD. PKD overexpression did not increase the duration of either MARCKS phosphorylation (Ser-152 and Ser-156) or FAK tyrosine phosphorylation (Tyr-397 and Tyr-577) in re- sponse to bombesin or vasopressin, indicating that PKD overexpression did not have a generalized effect on the kinetics of bombesin or vasopressin receptor signaling. In contrast, PKD overexpression prolonged bombesin-induced phosphorylation of FAK at Ser-910, a direct target of ERK. These results indicate that PKD overexpression selectively increased the duration of ERK activation.
EGF does not induce any significant increase in the phosphorylation or catalytic activity of PKD in Swiss 3T3 cells (53), but stimulates ERK1/2 activation through a Ras-dependent, PKC-independent pathway in these cells (86). Here, we demonstrate that the duration of ERK/RSK activation induced by EGF in Swiss 3T3-PKD.GFP cells was identical to that generated by this growth factor in Swiss 3T3-GFP cells. These results indicate that PKD selectively prolongs ERK signaling in response to G q -coupled receptor agonists in Swiss 3T3 cells.
As indicated above, it is increasingly recognized that the duration of ERK pathway activation is of critical importance for determining specific biological outcomes, including proliferation and differentiation in fibroblasts, thymocytes, PC12 cells and neurons (59,60,62,(87)(88)(89). For example, sustained activation of ERK for several hours is associated with re-initiation of DNA replication in fibroblasts whereas transient ERK activation for minutes is not sufficient to promote mitogenesis in these cells (62). The protein products of immediate early genes (e.g. c-fos) have been proposed to function as cellular sensors of ERK1/2 signal duration (62). Thus, when ERK activation is transient, its activity declines before the c-Fos protein accumulates, and unphosphorylated c-Fos is degraded rapidly. However, when ERK signaling is sustained, ERK and RSK phosphorylate the c-Fos protein at Ser-374 and Ser-362 (80,81), leading to its stabilization (62,80,82). Thus, we hypothesized that sustained bombesin-induced ERK1/2 and RSK signaling in PKD overexpressing Swiss 3T3 cells increases c-Fos protein phosphorylation thereby promoting its stability and accumulation in these cells. In line with this hypothesis, we found a dramatic accumulation of c-Fos protein in response to bombesin in Swiss 3T3 cells overexpressing wild-type PKD but not in either control cells or in Swiss 3T3 cells overexpressing a kinase-inactive PKD. Furthermore, the accumulation of c-Fos was completely prevented by treatment with either U0126 or PD98059, added either simultaneously or after agonist stimulation. These results indicate that sustained bombesin-induced ERK and RSK signaling in PKD-overexpressing Swiss 3T3 cells leads to c-Fos protein phosphorylation and accumulation in these cells.
Our previous results demonstrated that PKD overexpression facilitates DNA synthesis induced by the GPCR agonists bombesin or vasopressin but not by EGF. Consequently, we hypothesized that the increase in the duration of MEK/ERK/RSK signaling leading to the accumulation of protein products of immediate early genes plays a critical role in the mechanism by which PKD enhances the mitogenic effects of these agonists. Several lines of evidence support this possibility. 1) Inhibition of ERK activation with either U0126 or PD98059 abrogated the potentiation of bombesin-induced DNA synthesis seen in Swiss 3T3 cells overexpressing PKD. In particular, these MEK inhibitors completely blocked the enhancement of DNA synthesis induced by bombesin when added to the cells 3 h after bombesin, demonstrating the importance of the sustained phase of ERK signaling for DNA synthesis. 2) EGF, which does not activate PKD, neither increased the duration of ERK/RSK signaling nor enhanced the level of DNA synthesis in PKD-overexpressing cells. 3) Prolongation of ERK pathway activation, c-Fos accumulation and enhancement of DNA synthesis in response to bombesin or vasopressin require catalytically ac-tive PKD, i.e. these effects were not observed in cells overexpressing a kinase-inactive mutant of PKD. These results support the hypothesis that the increased duration of ERK pathway signaling is one of the mechanisms by which PKD facilitates GPCR-induced mitogenesis.
In conclusion, our results identify PKD as an important element in the control of the duration of the MEK/ERK/RSK pathway activation in response to GPCR agonists, a parameter of crucial importance in defining the biological outcomes of ERK activation. Our study supports the hypothesis that an increase in the duration of the ERK signaling leading to accumulation of immediate gene products is, at least, one of the mechanisms by which PKD overexpression selectively enhances re-initiation of DNA synthesis by G q -coupled receptor activation. Interestingly, three independent laboratories, including our own, have demonstrated attenuation of pro-apoptotic JNK signaling by PKD (49, 90 -92). For example, induced expression of an activated mutant of PKD was sufficient to suppress EGF-stimulated c-Jun phosphorylation at Ser-63, a natural substrate of JNK (91). In view of the opposite effects exerted by PKD on the activity and duration of the ERK and JNK pathways, PKD emerges as a critical element in regulating such fundamental cellular functions as cell fate and proliferation.