INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

LYSOPHOSPHATIDIC ACID (LPA), a major bioactive lipid of serum, elicits a broad spectrum of biological responses, including platelet activation, smooth muscle contraction, changes in neuronal cell shape, and induction of cell proliferation and differentiation (14). LPA binds to a seven-transmembrane domain receptor(s) and activates several heterotrimeric G proteins, which are responsible for transducing LPA signals into multiple biological responses (5, 15). LPA stimulates Ras activation, leading to stimulation of Raf, MEK, and the ERKs via a pertussis toxin (PTx)-sensitive pathway that involves the subunits of G_i (2, 3, 11, 12, 26), whereas the  subunit of this trimeric G protein mediates inhibition of adenylate cyclase activity (5, 15). LPA induces PTx-insensitive stress fiber formation, assembly of focal adhesions, and tyrosine phosphorylation of focal adhesion proteins (20) via activation of G₁₃ (6, 16). LPA also stimulates phospholipase C (PLC)-mediated polyphosphoinositide breakdown that leads to generation of inositol 1,4,5-trisphosphate and diacylglycerol, the second messengers responsible for Ca²⁺ mobilization from intracellular stores and activation of protein kinase C (PKC), respectively. These PLC-dependent responses are thought to be mediated by PTx-insensitive G proteins of the G_q family (14, 15). It is also recognized that some of the downstream responses induced by LPA, including the activation of transcription factors NF-B (21) and serum response factor (1) are elicited by interaction of complementary pathways activated by G_q, G_i and G₁₂.

The PKC family consists of multiple related isoforms, i.e., conventional PKCs (, ₁, ₂, and ), novel PKCs (, , , and ) and atypical PKCs ( and ), all of which possess a highly conserved catalytic domain (17). Protein kinase D (PKD) (25), also named PKCµ (9), is a serine-threonine protein kinase with distinct structural, enzymological, and regulatory properties. In particular, PKD can be rapidly activated in intact cells through a phosphorylation-dependent mechanism (29). Treatment of intact cells with biologically active phorbol esters (29), bryostatin (13), or neuropeptide agonists, including bombesin, endothelin, and vasopressin (30), induces PKD activation that persists during cell disruption and immunoprecipitation. Several lines of evidence, including the use of selective PKC inhibitors and cotransfection of PKD with constitutively active mutants of PKC  and , indicate that PKD is activated by phosphorylation in living cells through a PKC-dependent signal transduction pathway (13, 29, 30). More recently, Ser⁷⁴⁴ and Ser⁷⁴⁸ have been identified as critical phosphorylation sites in the activation loop of the kinase catalytic domain of PKD (8). These findings reveal an unsuspected connection between PKCs and PKD and imply that PKD can function downstream of PKCs in signal transduction.

In the present study, we examined the effect of the multifunctional agonist LPA on the regulation of PKD activity in intact Swiss 3T3 and Rat-1 cells, which have been used extensively as model systems to elucidate the biological effects of this bioactive lipid. We report for the first time that LPA induces a rapid PKC-dependent PKD activation in these cells. Surprisingly, treatment of the cells with PTx markedly and selectively attenuated PKD activation in response to LPA. Our results identify a novel PTx-sensitive event in the action of LPA and provide the first example of a G_i-dependent pathway leading to PKD activation in any cell type.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell culture. Stock cultures of Swiss 3T3 cells and Rat-1 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 10% CO₂ and 90% air at 37°C. For experimental purposes, cells were plated in 100-mm dishes at 5 × 10⁵ cells/dish in DMEM containing 10% FBS and used after 6-8 days when the cells were confluent and quiescent.

Immunoprecipitation. Quiescent cultures of 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 µg/ml aprotinin, 10 µg/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 g for 10 min at 4°C. PKD was immunoprecipitated at 4°C for 2-4 h with the PA-1 antipeptide antiserum (1:100), as previously described (29). The immune complexes were recovered using protein A coupled to agarose.

Kinase assay of PKD. The kinase activity of PKD was determined in an in vitro kinase assay by mixing 20 µl of PKD immunocomplexes with 10 µl of a phosphorylation mixture containing (final concentration) 10 µM [-³²P]ATP (specific activity 400-600 cpm/pmol), 30 mM Tris · HCl, pH 7.4, 10 mM MgCl₂, and 1 mM dithiothreitol. After 10 min of incubation at 30°C, the reaction was stopped by washing with 200 µl of kinase buffer and then adding an equal volume of 2× SDS-PAGE sample buffer (200 mM Tris · HCl, pH 6.8, 2 mM EDTA, 0.1 M Na₃VO₄, 6% SDS, 10% glycerol, and 4% 2-mercaptoethanol), followed by SDS-PAGE analysis (27, 29). The gels were dried, and the 110-kDa radioactive band corresponding to autophosphorylated PKD was visualized by autoradiography. Autoradiographs were scanned in a ScanJet 6100C/T (Hewlett Packard), and the labeled band was quantified using the National Institutes of Health image software program.

Phospholipase B treatment of LPA and FBS. LPA and lysophosphatidates bound to albumin in serum are inactivated by treatment with phospholipase B (PLB) (10). Here, 100 µl of a 500-µM LPA stock solution were dissolved in PBS-0.01% bovine serum albumin (wt/vol) or 100 µl of FBS were incubated with or without 100 IU of PLB for 1 h at 37°C. The PLB-treated LPA or FBS were used immediately, as indicated in Fig. 1. In control experiments the activity of PLB was destroyed by heating to 75°C for 1 h before incubation with LPA or FBS.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Lysophosphatidic acid (LPA) induces protein kinase D (PKD) activation. A: PKD activation induced by LPA and fetal bovine serum (FBS) in intact Swiss 3T3 cell line is blocked by phospholipase B (PLB). Confluent and quiescent Swiss 3T3 cells were washed with DMEM and subsequently stimulated with DMEM supplemented with either 5% FBS or 5 µM LPA for 10 min and then lysed. Parallel cultures were also challenged with either LPA or FBS, which had been treated with PLB (as described in MATERIALS AND METHODS) for 10 min and then lysed. All lysates were immunoprecipitated with PA-1 antiserum. To determine PKD activity, immunoprecipitates were incubated with [-32P]ATP in phosphorylation mixture and products of the reaction were further analyzed by SDS-PAGE and autoradiography. All other experimental details were as described in MATERIALS AND METHODS. Autoradiograms were scanned to quantify phosphoprotein in terms of peak area. Values correspond to autophosphorylation of PKD expressed over the unstimulated value. Results shown are means ± SE of 2 independent experiments. B: LPA induces PKD activation in Swiss 3T3 cells in a time- and dose-dependent manner. Confluent and quiescent cells were washed and incubated with 5 µM LPA for various times as indicated. Cells were then lysed and extracts were incubated with PA-1 antiserum and subjected to in vitro kinase assay, SDS-PAGE, and autoradiography. Inset shows cells treated with various concentrations of LPA for 10 min, whereas autoradiogram depicts representative dose-response experiment. Quantification of level of PKD autophosphorylation was performed in both cases by scanning densitometry, and results shown are expressed as increase over unstimulated values. In all cases autoradiograms are representative of 2 independent experiments. C: LPA induces PKD activation in Rat-1 cells in dose-dependent manner. Confluent and quiescent cells were washed and treated with various concentrations of LPA for 10 min as indicated. Cells were then lysed and extracts were incubated with PA-1 antiserum and subjected to in vitro kinase assay, SDS-PAGE, and autoradiography. Autoradiogram depicts representative dose-response experiment.

Measurement of intracellular calcium concentration. Intracellular calcium concentration ([Ca²⁺]_i) was measured with the fluorescent indicator fura 2. Confluent and quiescent cultures of Swiss 3T3 cells, grown on 9 × 22 mm coverslips, were washed twice with DMEM and then incubated for 10 min in DMEM containing 1 µM fura 2-AM at 37°C. The cultures were then washed twice with Hanks' buffered salt solution, pH 7.2, supplemented with NaHCO₃ (35 mM), CaCl₂ (1.3 mM), MgCl₂ (0.5 mM), MgSO₄ (0.4 mM), and 0.1% bovine serum albumin (calcium buffer). The coverslips were then incubated for a further 15 min in calcium buffer and then transferred to a quartz cuvette containing 2 ml of the same buffer, and fluorescence was monitored using a Hitachi F-2000 fluorospectrophotometer with dual excitation wavelengths of 340 nm (1) and 380 nm (2) and an emission wavelength of 510 nm while the cells were continually stirred at 37°C. [Ca²⁺]_i was determined using the equation
where R, R_min, and R_max are the ratios of the emission at 510 nm following excitation at 340 nm and 380 nm, F_max is the fluorescence after the addition of 40 µM digitonin, F_min is the fluorescence after the Ca²⁺ in the solution has been chelated with 25 mM EGTA. The value of dissociation constant (K_d) used was 224.

Phospho p42^mapk (ERK-2) and p44^mapk (ERK-1) immunoblot. Quiescent cultures of Swiss 3T3 cells grown on 33-mm dishes were washed twice with DMEM and incubated with or without PTx (30 ng/ml) for 3 h in DMEM at 37°C. LPA (5 µM) was then added to the cultures and incubated at 37°C for a further 5 min. The cells were lysed in 2× SDS-PAGE sample buffer, and the lysates were analyzed by SDS-PAGE. After SDS-PAGE, the proteins were transferred to immobilon membranes as per the manufacturers instructions. Membranes were blocked with 5% nonfat milk and incubated overnight with monoclonal anti-phospho ERK-1 and ERK-2 antibody (0.2 µg/ml, E10, Bio-Labs). Immunoreactive p42^mapk (ERK-2) and p44^mapk (ERK-1) bands were detected by enhanced chemiluminescence Western blotting ECL reagents (Amersham).

Materials. [-³²P]ATP (370 MBq/ml) was from Amersham International. GF-I (also known as GF-109203X or bisindolylmaleimide I), Gö-7874, Ro-31-8220, U-73122, U-73343, and PTx were from Calbiochem. LPA, phorbol 12,13-dibutyrate (PDB), wortmannin, rapamycin, PD-098059, genistein, and PLB were from Sigma. The monoclonal anti-phospho-ERK-1 and ERK-2 antibody E10 was purchased from Bio-Labs. Protein A-agarose was from Boehringer Mannheim. PA-1 antiserum was raised against the synthetic peptide EEREMKALSERVSIL that corresponds to the carboxy terminal region of the predicted amino acid sequence of PKD, as previously described (27, 29). Other items were from standard suppliers or as indicated in the text.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

LPA induces PKD activation in Swiss 3T3 and Rat-1 cells. Quiescent Swiss 3T3 fibroblasts have proved to be a useful model system for elucidating signal transduction pathways in the action of multiple agonists, including LPA (19). To examine whether LPA induces PKD activation, confluent and quiescent cultures of these cells were stimulated with 5 µM LPA for 10 min and lysed. The extracts were immunoprecipitated with the PA-1 antibody raised against a peptide composed of the 15 carboxy terminal amino acids of PKD. The immunocomplexes were incubated with [-³²P]ATP and then analyzed by SDS-PAGE and autoradiography to examine the level of autophosphorylation. As illustrated in Fig. 1, stimulation of Swiss 3T3 cells with LPA induced a marked PKD activation that was maintained during cell disruption and immunoprecipitation. In 25 independent experiments, addition of 5 µM LPA to cultures of Swiss 3T3 cells induced a 7.6 ± 0.47 (means ± SE)-fold increase in PKD activity.

PKC mediates LPA-stimulated PKD activation. Next, we determined the role of PKCs in PKD activation induced by LPA. Quiescent cultures of Swiss 3T3 cells were treated with various concentrations of GF-I (also known as GF-109203X or bisindolylmaleimide I), a potent inhibitor of phorbol ester-sensitive isoforms of PKC (24) but not PKD (29, 30), before PDB stimulation. As shown in Fig. 2, treatment of the cells with GF-I potently blocked PKD activation induced by subsequent addition of LPA, in a concentration-dependent fashion. In contrast, GF-I added directly to the in vitro kinase assay, even at the concentrations (0.5-2.5 µM) required to abrogate LPA-mediated PKD activation in intact 3T3 cells, did not inhibit PKD activity.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Protein kinase C (PKC) inhibitors GF-I, Ro-31-8220, and Gö-7874 prevent PKD activation by LPA. Confluent and quiescent Swiss 3T3 cells were washed with DMEM and incubated for 1 h with different concentrations (µM) of selective PKC inhibitors GF-109203X (GF-I), Ro-31-8220 (Ro), or Gö-7874 (Go) as indicated (top, Pretreat). Control cells received an equivalent amount of solvent (). Cultures were subsequently stimulated with 5 µM LPA for 10 min, lysed, and extracts were immunoprecipitated with PA-1 antiserum. PKD activity was determined by in vitro kinase assay carried out in absence () or in presence (+) of indicated concentrations of GF-I, Ro-318220, Gö-7874 added directly to incubation mixture (bottom, in vitro). Control PKD immunoprecipitates received equivalent amount of solvent (). Reactions were analyzed by SDS-PAGE and autoradiography. Results shown are representative of 3 independent experiments.

LPA induces PKD activation via PLC. To determine whether LPA induces PKC-dependent PKD activation through a PLC-dependent pathway, Swiss 3T3 cells were treated with the aminosteroid U-73122, an inhibitor of PLC, prior to stimulation with LPA. As shown in Fig. 3A, U-73122 markedly reduced PKD activation in response to the subsequent addition of LPA in a concentration-dependent fashion. Maximal inhibition of LPA-stimulated PKD activation was achieved at 2.5 µM. The inhibitory effect of U-73122 was selective because this agent, at similar concentrations, did not interfere with PKD activation induced by PDB. Furthermore, U-73343, an inactive analog of U-73122, did not affect LPA stimulation of PKD activation when added at an identical concentration (Fig. 3B).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Phospholipase C (PLC) inhibitor U-73122 abrogates PKD activation in Swiss 3T3 cells. A: confluent and quiescent Swiss cells were washed with serum-free medium and then incubated with DMEM containing increasing concentrations of selective PLC inhibitor U-73122 (0.5, 1, and 2.5 µM) for 1 h. Control cultures received equivalent amount of solvent (). Cells were subsequently stimulated with 200 nM phorbol 12,13-dibutyrate (PDB) or 5 µM LPA for 10 min and lysed. Lysates were immunoprecipitated with PA-1 antiserum, and PKD activity was determined by in vitro kinase assay as described in MATERIALS AND METHODS, followed by SDS-PAGE and autoradiography. B: confluent and quiescent cells were washed and treated with 2.5 µM Ro-31-8220 (Ro), 2.5 mM U-73122, 2.5 mM U-73343, 100 nM wortmannin (Wor), 20 nM rapamycin (Rap), 25 µM PD-098059 (PD), 50 µM genistein (Gen), or an equivalent volume of solvent for 1 h. Cells were then incubated with 5 µM LPA for 10 min. Cultures were lysed, extracts were immunoprecipitated with PA-1 antiserum, and immunocomplexes of PKD were subjected to in vitro kinase assay, SDS-PAGE, and autoradiography. Results shown are means ± SE of 3 independent experiments.

LPA stimulates PKD activation via a PTx-sensitive pathway. LPA activates several heterotrimeric G proteins including G_q and G_i, which are responsible for transducing LPA signals into multiple biological responses (6, 15). PLC-mediated polyphosphoinositide breakdown, leading to rapid Ca²⁺ mobilization and PKC activation, is thought to be mediated by PTx-insensitive G proteins of the G_q family, at least in rodent cell lines (14). We verified that treatment of Swiss 3T3 cells with 30 ng/ml PTx for 3 h, a condition known to promote ADP ribosylation and inactivation of G_i in these cells (22), did not interfere with the rapid and transient increase in [Ca²⁺]_i induced by LPA (Fig. 4A). In contrast, a similar treatment with PTx markedly inhibited ERK activation in response to LPA, as shown by Western blot analysis using an antibody that recognizes the dually phosphorylated and active p42^mapk (ERK-2) and p44^mapk (ERK-1) (Fig. 4A).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effect of pertussis toxin (PTx) on p42mapk (ERK-2) and p44mapk (ERK-1) phosphorylation, intracellular Ca2+ concentration ([Ca2+]i), and PKD activation induced by LPA in intact Swiss 3T3 cells. A, top: p42mapk (ERK-2) and p44mapk (ERK-1) phosphorylation induced by LPA is blocked by PTx. Confluent and quiescent cultures of Swiss 3T3 cells were washed twice with DMEM and then incubated without or with PTx (30 ng/ml) for 3 h in DMEM at 37°C. LPA (5 µM) was then added to cultures and incubated at 37°C for a further 5 min. Cells were then lysed in 2× SDS-PAGE sample buffer and analyzed by SDS-PAGE and immunoblotting with phospho-ERK antibody as described in MATERIALS AND METHODS. Cont., control. A, bottom: increase in [Ca2+]i induced by LPA is not inhibited by PTx. Confluent and quiescent cultures of Swiss 3T3 cells grown on 9 × 22 mm coverslips were washed twice with DMEM and then incubated without or with PTx (30 ng/ml) for 3 h in DMEM at 37°C. [Ca2+]i following addition of 5 µM LPA (arrows) was determined as described in MATERIALS AND METHODS. Calcium tracings show representative experiment. Bars are means ± SE of 8 individual experiments. B: PKD activation induced by LPA in intact Swiss cell line is blocked by PTx in dose-dependent manner. Confluent and quiescent Swiss 3T3 cells were washed with DMEM and incubated with different concentrations of PTx as indicated or equivalent amount of solvent for 3 h. Cells were then stimulated with 5 µM LPA for 10 min. Cultures were lysed and lysates were immunoprecipitated with PA-1 antiserum. Immunoprecipitates were subjected to in vitro kinase assay, SDS-PAGE, and autoradiography. Autoradiograms were scanned to quantify phosphoprotein in terms of peak area. Values correspond to autophosphorylation of PKD expressed as percentage of maximum value. Results shown are means ± SE (n = 6). Autoradiogram shown is representative of 6 independent experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   PTx selectively blocks PKD activation induced by LPA. A: effect of PTx on PKD activation induced by LPA, bombesin, or PDB in Swiss 3T3 cells. Cultures were washed with DMEM and incubated with different concentrations (15, 30, 60 ng/ml) of PTx or equivalent amount of solvent for 3 h. Cells were then stimulated with 5 µM LPA, 10 nM bombesin (Bom), or 200 nM PDB for 10 min. Cultures were lysed and lysates were immunoprecipitated with PA-1 antiserum. Immunoprecipitates were subjected to in vitro kinase assay, SDS-PAGE, and autoradiography. Results shown are representative of 7 similar experiments. B: PTx attenuates PKD activation induced by LPA in Rat-1 cells. Cultures were washed with DMEM and incubated with 100 ng/ml of PTx or equivalent amount of solvent for 3 h. Cells were then stimulated with 5 µM LPA, 200 nM PDB, or 10% FBS for 10 min. Cultures were lysed and lysates were immunoprecipitated with PA-1 antiserum. Immunoprecipitates were subjected to in vitro kinase assay, SDS-PAGE, and autoradiography. Results shown are representative of 2 similar experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

LPA promotes a broad range of biological responses and multiple molecular events in target cells (15). Consistent with the stimulation of multiple signaling pathways, LPA has been shown to bind to several heptahelical receptors (5) and activate several heterotrimeric G proteins, including G_q, G_i, and G₁₂ in Swiss 3T3 cells (6) and in Rat-1 cells (14). The results presented here demonstrate that LPA rapidly induces PKD activation in intact Swiss 3T3 and Rat-1 cells and thus identify a novel molecular response in LPA action. Our results also suggest that LPA is a major factor in serum that mediates PKD activation in these cell types.

Treatment of the cells with the PKC inhibitors GF-I, Ro-31-8220, and Gö-7874 before stimulation with LPA strikingly prevented PKD activation. Importantly, these PKC inhibitors did not reduce PKD activity when added directly to the in vitro kinase assays, even at concentrations higher than those used in intact cells to block LPA-induced PKD activation. Furthermore, the PLC inhibitor U-73122 selectively prevented PKD activation by LPA. We conclude that LPA-induced PKD activation is downstream to PLC and PKC in Swiss 3T3 cells.

LPA-induced PLC and PKC activation is thought to be mediated by LPA receptor coupling to PTx-insensitive G_q (14). In line with this hypothesis, inositol phosphate production and Ca²⁺ mobilization in response to LPA is not prevented by treatment with PTx in rodent cell lines, including Swiss 3T3 cells. In addition, LPA has been shown to stimulate phosphorylation of the Rac exchange factor Tiam-1 via a PTx-insensitive PKC-dependent pathway in these cells (4).

Although LPA induces PLC and PKC activation through G_q and PKD activation in response to LPA is downstream to PKC, it could not be excluded that other signaling inputs also contribute to PKD activation induced by LPA. A surprising feature of our results is that PKD activation in response to LPA is attenuated markedly and selectively by prior treatment of either Swiss 3T3 cells or Rat-1 cells with low concentrations of PTx. These results indicate that the G_q pathway is not sufficient to promote PKD activation in response to LPA in these cells and identify for the first time the involvement of an additional G_i-dependent pathway leading to PKD activation in any cell type.

Interestingly, the concentration of LPA required for stimulation of PKD activation in Swiss 3T3 cells (EC₅₀ 1 µM) is similar to that needed for activation of the transcription factors NF-B (21) and serum response factor (1). Recent evidence indicates that LPA leads to the activation of these transcription factors through parallel signal transduction pathways. For example, the stimulation of NF-B by LPA is mediated by G_q and G_i pathways (21), and the activation of the serum response factor is mediated by cooperative effects between G_i and G₁₃-Rho pathways (1). We propose that LPA-induced PKD activation, which precedes the activation of these transcription factors, is also mediated by complementary pathways initiated by G_i and G_q.

It is well established that treatment with PTx almost completely blocks LPA-induced mitogenesis in a variety of cell types. LPA signaling through PTx-sensitive G_i activates the Ras-Raf-ERK kinase cascade (2, 3, 11, 12, 26) and phosphoinositide 3-kinase activity in cultured fibroblasts (18). Recently, Takeda et al. (23) demonstrated that LPA induces phosphoinositide 3-kinase-dependent activation of PKC via G_i. The results presented here identify PKD activation as a novel PTx-sensitive early molecular response in the action of LPA, which can be dissociated from either Ras-Raf-ERK or phosphoinositide 3-kinase signaling pathways. In addition, our previous results showed that cotransfection of PKD with a constitutively active form of PKC does not lead either to PKD activation (29) or to the formation of stable molecular complexes between PKC and PKD (28). The role of PKD in the PTx-sensitive biological responses induced by LPA warrants further experimental work.

In conclusion, our results demonstrate that LPA induces PKC-dependent PKD activation in Swiss 3T3 cells. In contrast to the model of PKD regulation by neuropeptide agonists through G_q (30), we propose that LPA stimulates PKD activation through both G_i and G_q in these cells. Our results identify a novel PTx-sensitive molecular response in the action of LPA and demonstrate for the first time the involvement of a G_i-dependent pathway leading to PKD activation in any cell type.