Cooperation of Gq, Gi, and G12/13 in Protein Kinase D Activation and Phosphorylation Induced by Lysophosphatidic Acid*

Jingzhen YuanDagger §, Lee W. SliceDagger , Jennifer Gu, and Enrique RozengurtDagger ||

From the Dagger  Department of Medicine, David Geffen School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095 and the  Division of Biology, California Institute of Technology, Pasadena, California 91125

Received for publication, October 31, 2002, and in revised form, December 10, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

To examine the contribution of different G-protein pathways to lysophosphatidic acid (LPA)-induced protein kinase D (PKD) activation, we tested the effect of LPA on PKD activity in murine embryonic cell lines deficient in Galpha q/11 (Galpha q/11 KO cells) or Galpha 12/13 (Galpha 12/13 KO cells) and used cells lacking rhodopsin kinase (RK cells) as a control. In RK and Galpha 12/13 KO cells, LPA induced PKD activation through a phospholipase C/protein kinase C pathway in a concentration-dependent fashion with maximal stimulation (6-fold for RK cells and 4-fold for Galpha 12/13 KO cells in autophosphorylation activity) achieved at 3 µM. In contrast, LPA did not induce any significant increase in PKD activity in Galpha q/11 KO cells. However, LPA induced a significantly increased PKD activity when Galpha q/11 KO cells were transfected with Galpha q. LPA-induced PKD activation was modestly attenuated by prior exposure of RK cells to pertussis toxin (PTx) but abolished by the combination treatments of PTx and Clostridium difficile toxin B. Surprisingly, PTx alone strikingly inhibited LPA-induced PKD activation in a concentration-dependent fashion in Galpha 12/13 KO cells. Similar results were obtained when activation loop phosphorylation at Ser-744 was determined using an antibody that detects the phosphorylated state of this residue. Our results indicate that Gq is necessary but not sufficient to mediate LPA-induced PKD activation. In addition to Gq, LPA requires additional G-protein pathways to elicit a maximal response with Gi playing a critical role in Galpha 12/13 KO cells. We conclude that LPA induces PKD activation through Gq, Gi, and G12 and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Protein kinase C (PKC),1 a major target for the tumor promoting phorbol esters, has been implicated in the signal transduction pathways regulating a wide range of biological responses, including changes in cell morphology, differentiation, and proliferation (1, 2). Molecular cloning has demonstrated the presence of multiple PKC isoforms (2-5), i.e. conventional PKCs (alpha , beta 1, beta 2, and gamma ), novel PKCs (delta , epsilon , eta , and theta ), and atypical PKCs (zeta  and lambda ) all of which possess a highly conserved catalytic domain.

PKD/PKCµ is a serine/threonine protein kinase (6, 7) with distinct structural, enzymological, and regulatory properties (8). In particular, PKD is rapidly activated in intact cells through a mechanism that involves phosphorylation (8). Specifically, exposure of intact cells to phorbol esters, cell-permeant diacylglycerols, or bryostatin induces rapid PKD phosphorylation and activation, which is maintained during cell lysis and immunoprecipitation (8-13). Several lines of evidence, including the use of PKC-specific inhibitors and co-transfection of PKD with constitutively active PKC mutants indicate that PKD is activated through a novel PKC-dependent signal transduction pathway in vivo (9-11). The residues Ser-744 and Ser-748 in the activation loop of PKD have been identified as critical phosphorylation sites in PKD activation (14, 15). Taken together, these results suggest an important connection between PKCs and PKD and indicate that PKD can function downstream of PKC in a novel signal transduction pathway.

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are composed of alpha , beta , and gamma  subunits and transduce external signals from heptahelical receptors to intracellular effectors (16). Mammalian G protein alpha  subunits are classified into four subfamilies: Gs, Gi, Gq, and G12. The alpha  subunit of Gq stimulates the beta  isoforms of phospholipase C (PLC), which catalyze the production of inositol 1,4,5-trisphosphate that triggers the release of Ca2+ from internal stores and diacylglycerol that activates the classical and novel isoforms of PKC (reviewed in Ref. 17). A variety of neuropeptide agonists that signal through heptahelical receptors and couple to heterotrimeric G proteins, including bombesin, bradykinin, endothelin, and vasopressin, induce rapid PKD activation in normal and neoplastic cells (11, 13, 18, 19). Although each of these receptors activates Gq and Galpha q signaling stimulates PKD activity (20), expression of a COOH-terminal fragment of Galpha q that acts in a dominant-negative fashion, attenuated (but did not eliminate) PKD activation in response to agonist stimulation of bombesin receptor (20). These results suggested that G protein-coupled receptors (GPCRs) stimulate PKD activation not only via Galpha q but also through other G protein-mediated signaling pathways.

Many GPCRs also interact with other heterotrimeric G proteins including members of the G12 family, which mediate activation of the low molecular weight G proteins of the Rho subfamily (21-26) via guanine nucleotide exchange factors that directly link the Galpha subunits to Rho (27-29). Rho plays a major role in promoting cytoskeletal responses including formation of actin stress fibers, assembly of focal adhesions, and tyrosine phosphorylation of focal adhesion proteins and has been implicated in gene expression, cell migration, proliferation, and transformation (30-32). Interestingly, a number of recent studies have suggested a convergence between Rho- and PKC-mediated signaling in yeast and mammalian cells (33-38). For example, Slater et al. (37) have demonstrated that Rho-GTP potently stimulates PKCalpha activity in vitro using recombinant proteins and Sagi et al. (38) reported that Galpha q and PLC signaling are synergistic with Rho. Recently, we demonstrated that in addition to Galpha q, Rho- and Galpha 13-mediated signaling can promote PKD activation in intact cells and that endogenous Rho and Galpha 13 contribute to PKD activation in response to bombesin GPCR stimulation (39). Thus, our results identified PKD as a novel downstream target in Galpha 13 and Rho signaling and indicated that GPCR stimulation promotes PKD activation via both Gq- and Galpha 13/Rho-dependent pathways.

Lysophosphatidic acid (LPA), a major bioactive lipid of serum (40), elicits a plethora of biological responses by activation of its specific GPCRs (41-43). Thus far, three genes (lpA1, lpA2, and lpA3) encoding LPA receptors, LPA1/EDG-2, LPA2/EDG-4, and LPA3/EDG-7, have been identified in mammals (43-45). All LPA receptors are coupled to Gi and Gq and, at least LPA1 and LPA2 are also coupled to G12 (42, 45, 46). LPA induces Ras activation leading to stimulation of Raf, MEK, and the ERKs via a pertussis toxin (PTx)-sensitive pathway that involves the beta gamma subunits of Gi (47-51) while the alpha  subunit of this trimeric G protein mediates inhibition of adenylate cyclase activity (42, 46). LPA stimulates PLC-mediated generation of inositol 1,4,5-trisphosphate and diacylglycerol, leading to Ca2+ mobilization from intracellular stores and PKC activation, respectively. These PLC-dependent responses are mediated by the alpha  subunit of Gq and/or the beta gamma subunits of the PTx-sensitive Gi (52). LPA also promotes PTx-insensitive stress fiber formation, assembly of focal adhesions, and tyrosine phosphorylation of focal adhesion proteins (53) via activation of Galpha 13 and Rho (24, 54). Because LPA receptors are expressed in most cell types, this bioactive lipid is a prototype agonist that promotes the activation of multiple endogenous G protein pathways that are responsible for transducing LPA signals into a broad spectrum of biological responses (43, 45, 55).

LPA has also been shown to induce a rapid PKC-dependent PKD activation in intact Swiss 3T3, Rat-1, and IEC-6 cells (56, 57). Interestingly, treatment of these cells with PTx markedly attenuated PKD activation in response to LPA. These results identified the involvement of an additional Gi-dependent pathway leading to PKD activation and indicated that the endogenous Gq pathway is not sufficient to promote PKD activation in response to LPA. Because our previous studies demonstrated that PKD can be activated through Gq- and G12-dependent pathways (see above) and because LPA receptors are also coupled to Gq and G12, we hypothesized that LPA induces PKD through multiple G protein signal transduction pathways. Here, we tested this hypothesis using mouse embryonic cell lines deficient in Galpha q/11 (Galpha q/11 KO cells) or Galpha 12/13 (Galpha 12/13 KO cells), and used a cell line lacking rhodopsin kinase (RK cells), as a control. Based on our results, we conclude that LPA induces PKD activation through the cooperation of Gq with either Gi or G12 and that PKD activation is a point of convergence in the action of multiple G protein pathways.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cell Culture-- Mouse embryonic fibroblasts were generated from genetically engineered mice (58, 59) that contained gene knockout for rhodopsin kinase, Galpha q/11, and Galpha 12/13. Stock cultures of these cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 250 µg/ml G418 in a humidified atmosphere containing 10% CO2 and 90% air at 37 °C. For experimental purposes, cells were plated in 60- or 100-mm dishes at about 30% confluency in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 250 µg/ml G418 and were allowed to grow to near confluency (6-7 days) and then changed to serum-free Dulbecco's modified Eagle's medium for 8-24 h prior to the experiment.

Cell Transfection and cDNA Constructs Used in Transfections-- To transfect the cells, RK cells and Galpha q/11 KO cells were subcultured to 80-90% confluency in 10-cm dishes. All transfections and cotransfections were carried out with equivalent amounts of DNA (20 µg/dish). Transfections were carried out in Opti-MEM using LipofectAMINE 2000 reagent according to the protocol from the manufacturer (Invitrogen). Cells were used for experiments 48-72 h after of transfection. Chimeric fusion proteins between GFP and PKD (GFP-PKD) have been described previously (69, 70).

Immunoprecipitation-- Cultures of RK, Galpha q/11, and Galpha 12/13 cells, treated as described in the individual experiments, were washed and lysed in lysis buffer (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). 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 antiserum (1:100) or with GFP antibody for GFP-PKD transfected cell lysates, as previously described (9, 10). The immune complexes were recovered using protein A coupled to agarose.

Kinase Assay of PKD-- PKD autophosphorylation 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) 100 µM [gamma -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 1 ml 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 (9, 60). The gels were dried and the 110- or 140-kDa radioactive band corresponding to autophosphorylated PKD or GFP-PKD was visualized by autoradiography. Autoradiographs were scanned in a GS-710 Calibrated Imaging Densitometer (Bio-Rad), and the labeled band was quantified using the Quantity OneTM software program.

Exogenous substrate phosphorylation by immunoprecipitated PKD was carried out by mixing 20 µl of the washed immunocomplexes with 20 µl of a phosphorylation mixture containing 2.5 mg/ml syntide-2 (PLARTLSVAGLPGKK), a peptide based on phosphorylation site two of glycogen synthase. After 10 min of incubation at 30 °C, the reaction was stopped by adding 100 µl of 75 mM H3PO4, and 75 µl of the supernatant was spotted on P-81 phosphocellulose paper. Free [gamma -32P]ATP was separated from the labeled substrate by washing the P-81 paper four times for 5 min, in 75 mM H3PO4. The papers were dried, and the radioactivity incorporated into syntide-2 was determined by Cerenkov counting.

Western Blot Analysis for pS744/748 and ERK-2/ERK-1 Activation-- Samples of cell lysates were directly solubilized by boiling in 2× SDS-PAGE sample buffer. Following SDS-PAGE on 8% gels, proteins were transferred to Immobilon-P membranes (Millipore) and blocked by 3-6 h incubation with 5% nonfat milk in phosphate-buffered saline, pH 7.2. Membranes were then incubated 3 h with the respective antibodies. PKD phosphorylation was determined by incubating the membrane with an antibody that specifically recognizes the phosphorylated state of serine 744 and serine 748 of PKD at a dilution of 1:1000 in phosphate-buffered saline, 0.1% Tween 20 containing 5% bovine serum albumin.

Activation of ERK-2 and ERK-1 occurs through phosphorylation of specific threonine and tyrosine residues, resulting in slower migrating forms in SDS-PAGE gels. These activated forms were monitored by using a specific antiphospho-ERK-1/ERK-2 monoclonal antibody that recognizes the phosphorylated state of Thr-202 and Tyr-204 of ERL-1/2 at a dilution of 1:1000 in phosphate-buffered saline, 0.1% Tween 20 containing 5% nonfat dried milk. The same membranes were stripped and probed in a similar fashion with goat anti-ERK-2 polyclonal antibody.

Horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5000, Amersham Biosciences) was then applied for 1 h at room temperature after washing 3 times with phosphate-buffered saline containing 0.05% Tween. Immunoreactive bands were detected by enhanced chemiluminescence Western blotting ECLTM reagents.

Materials-- [gamma -32P]ATP (370 MBq/ml), horseradish peroxidase-conjugated donkey anti-rabbit IgG, and enhanced chemiluminescence reagents were from Amersham Biosciences. Protein-A agarose and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride were from Roche Molecular Biochemicals. LPA, PDB, GF 109203X, GF V, U-0126, PD 098059, and Clostridium difficile toxin B were obtained from Sigma. Ro 31-8220, U73122, and PTx were purchased from Calbiochem (La Jolla, CA). Opti-MEM and LipofectAMINE 2000 reagent were from Invitrogen. PA-1 antiserum was raised against the synthetic peptide EEREMKALSERVSIL that corresponds to the carboxyl-terminal region of the predicted amino acid sequence of PKD, as previously described (9, 60). Phospho-PKD Ser-744/748 antibody was obtained from Cell Signaling Technology (Beverly, MA). Anti-phospho-ERK-1/2 monoclonal antibody was obtained from New England Biolabs. Anti-Galpha q/11, anti-Galpha 12/13, anti-GFP, and anti-ERK-2 polyclonal antibodies were from Santa Cruz Biotechnologies (Palo Alto, CA). Other items were from standard suppliers or as specifically indicated.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

LPA Induces PKD Activation in RK Cells through a PKC-dependent Pathway-- To examine the contribution of different G protein pathways to LPA-induced PKD activation, we decided to test the effect of this agonist on PKD activity isolated from murine embryonic cell lines lacking RK, Galpha q/11, and Galpha 12/13. Initially, we determined whether LPA induces PKD activation in RK cells, used as controls. Cultures of these cells were stimulated with increasing concentrations of LPA (0.01-10 µM) for 10 min and lysed. PKD was immunoprecipitated from the extracts of these cells and the immune complexes were incubated with [gamma -32P]ATP, subjected to SDS-PAGE, and analyzed by autoradiography to detect the prominent 110-kDa band corresponding to autophosphorylated PKD.

The results presented in Fig. 1A show that LPA induced a marked increase in PKD autophosphorylation activity in a concentration-dependent fashion in RK cells. Half-maximal and maximal stimulation (6-fold increase) were achieved at 0.07 and 3 µM, respectively. As illustrated in the inset to Fig. 1, similar results were obtained when PKD activity in immunocomplexes was determined by phosphorylation of syntide-2 (61, 62), a synthetic peptide previously demonstrated to be an excellent substrate for PKD (6).


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 1.   LPA activates PKD in a dose-dependent and PLC/PKC-dependent manner in RK cells. A, LPA activates PKD in a dose-dependent manner. Confluent and quiescent cultures of RK cells were incubated with various concentrations of LPA for 10 min at 37 °C. Cells were lysed with lysis buffer and immunoprecipitated with PA-1 antiserum. Top panel, in vitro kinase (IVK), PKD activity was determined by an in vitro kinase assay as described under "Experimental Procedures." followed by SDS-PAGE and autoradiography. The autoradiogram shown is representative of three independent experiments. Middle panel, pS744, cell lysates from the experiments described above were analyzed by SDS-PAGE and transferred to Immobilon membranes. Western blot analysis was carried out using pS744/748 PKD antibody. Bottom panel, scanning densitometry. The results shown are the values (mean ± S.E. n = 3) of the level of PKD activation by in vitro kinase obtained from scanning densitometry expressed as a percentage of the maximum increase in phosphorylation obtained with 3 µM LPA. Inset, PKD activity in the immunocomplexes was measured by syntide-2 phosphorylation, as described under "Experimental Procedures." The results expressed as an increased -fold over control in phosphorylation represent the mean ± S.E. obtained from three independent experiments, each performed in duplicate. B, LPA induces PKD activation through a PLC/PKC-dependent pathway. Confluent and quiescent RK cells were incubated for 1 h with the PKC inhibitor GF-109203X (GF1, 3.5 µM) or Ro 31-8220 (Ro, 2.5 µM), or with PLC inhibitor, U73122. Control cells (-) received equivalent amounts of solvent or GFV. The cultures were subsequently stimulated for 10 min with 3 µM LPA or 200 nM PDB at 37 °C. Cells were lysed with lysis buffer and immunoprecipitated with PA-1 antiserum. Top panels, in vitro kinase (IVK), PKD activity was determined by an in vitro kinase assay, as described under "Experimental Procedures." The autoradiogram shown is representative of three independent experiments. Middle panels, pS744, cell lysates from the experiments described as the above were analyzed by SDS-PAGE and transferred to Immobilon membranes. Western blot analysis was carried out using pS744/748 PKD antibody. Bottom panels, PKD activity in the immunocomplexes was measured by syntide-2 phosphorylation, as described under "Experimental Procedures." The results expressed as an increased -fold over control in phosphorylation represent the mean ± S.E. obtained from three independent experiments, each performed in duplicate.

We previously proposed that phosphorylation of Ser-744 and Ser-748 within the PKD activation loop plays a critical role in PKD activation (14, 15, 20). Recently, a novel antibody recognizing predominantly the phosphorylated state of Ser-744 (pS744/pS748) in PKD was used in our laboratory to monitor activation loop phosphorylation in response to phorbol ester and bombesin (15). To examine whether LPA induces activation loop phosphorylation in the RK cells, lysates from these cells treated with increasing concentrations of LPA were analyzed by SDS-PAGE, followed by Western blotting with the pS744/748 antibody. As shown in Fig. 1A, LPA induced PKD Ser-744 phosphorylation in a concentration-dependent fashion in RK cells with maximal stimulation achieved at 3 µM.

To determine whether LPA induces PKD activation and activation loop phosphorylation through a PKC-mediated pathway in RK cells, we used inhibitors that discriminate between PKCs and PKD. RK cells were treated for 1 h with the inhibitors of phorbol ester-sensitive isoforms of PKC, GF I (also known as GF 109203X or bisindolylmaleimide I) or Ro 31-8220 (64, 65), prior to stimulation with 3 µM LPA. As shown in Fig. 1B (left panels), exposure to either GF I or Ro 31-8220 prevented PKD activation (top) and Ser-744 phosphorylation (middle) induced by LPA. In contrast, the compound GFV, which is structurally related to GF I but biologically inactive, did not prevent PKD activation and phosphorylation in response to LPA in RK cells. Similar results were obtained when PKD activity in immunocomplexes was determined by assays of syntide-2 phosphorylation (bottom). Previously, we demonstrated that GF I and Ro 31-8220 do not inhibit PKD activity when added directly to the in vitro kinase assay at identical concentrations to those required to block PKD activation by LPA in RK cells (9, 11). Thus, the results shown in Fig. 1B imply that GF I and Ro 31-8220 do not inhibit PKD activity directly but interfere with LPA-induced PKD activation and activation loop phosphorylation in intact RK cells by blocking PKC.

To determine whether LPA induces PKD activation and phosphorylation through a PLC-dependent pathway in RK cells, cultures of these cells were treated with the aminosteroid U73122, an inhibitor of PLC (66, 67), prior to stimulation with LPA. As shown in Fig. 1B (right, top and middle panels), 2 µM U73122 markedly reduced PKD activation and Ser-744 phosphorylation in response to the subsequent addition of LPA. Similar results were obtained when PKD activity in immunocomplexes was determined by assays of syntide-2 phosphorylation (Fig. 1B, right bottom panel). The inhibitory effect of U73122 was selective because treatment with this compound did not interfere with PKD activation and Ser-744 phosphorylation induced by PDB (Fig. 1B).

Effect of Treatment with PTx on LPA-induced PKD Activation in RK Cells-- Previous studies demonstrated that LPA induces PKD activation through a PTx-sensitive pathway in 3T3 (18) and IEC-6 cells (57) but through a pathway that was only partially attenuated by PTx in IEC-18 cells (57). Because PTx catalyzes the ADP-ribosylation and inactivation of members of the Galpha i family (68), these results indicated that Gi contributes to PKD activation to a different degree in different cell contexts.

To assess the contribution of Gi to LPA-induced PKD activation in RK cells, cultures of these cells were treated with increasing concentrations of PTx (1-100 ng/ml) for 3 h and then challenged with 3 µM LPA for 10 min. As illustrated by Fig. 2A, treatment of RK cells with PTx at a concentration as high as 100 ng/ml attenuated only slightly PKD activation and Ser-744 phosphorylation in response to LPA (the maximal attenuation was only ~25%). Similar attenuation of PKD activation was obtained when PKD activity in immunocomplexes was determined by phosphorylation of the exogenous substrate syntide-2 rather than by autophosphorylation (Fig. 2B). These results suggest that LPA induces PKD activation predominantly through Gi-independent pathways in RK cells, presumably involving Galpha q/11 and/or Galpha 12/13.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of PTx on the activation of PKD induced by LPA in RK cells. A and B, confluent and quiescent RK cells were incubated with different concentrations of PTx as indicated for 3 h. Cells were then stimulated with (+) or without (-) 3 µM LPA (left panels) or 200 nM PDB (right panels) for 10 min at 37 °C. Cultures were lysed with lysis buffer and then immunoprecipitated with PA-1 antiserum. A, top panels, in vitro kinase (IVK), immunoprecipitates were subjected to in vitro kinase assay, SDS-PAGE, and autoradiography. The autoradiograph shown is representative of three independent experiments. Middle panels, pS744, cell lysates from the experiments described as above were analyzed by SDS-PAGE and transferred to Immobilon membranes. Western blot analysis was carried out using pS744/748 PKD antibody. Bottom panels, scanning densitometry, the results shown are the values (means ± S.E. n = 3) of the level of PKD activation obtained from scanning densitometry expressed as a percentage of the maximum increase in phosphorylation obtained with 3 µM LPA for 10 min at 37 °C. , values corresponding to PKD activity from cells incubated with PTx, and then stimulated with LPA; , values corresponding to PKD activity from cells incubated with PTx but without LPA. B, PKD activity in the immunocomplexes was measured by syntide-2 phosphorylation, as described under "Experimental Procedures." The results expressed as an increased -fold over control in phosphorylation represent the mean ± S.E. obtained from three independent experiments, each performed in duplicate.

LPA-induced PKD Activation Requires Functional Gq-- Most cell types express multiple members of the Gq and G12 families with overlapping functions, thus rendering it difficult to analyze the contribution of these G proteins to heptahelical receptor signaling. To circumvent this problem and in view of the results presented above, we next examined whether LPA induces PKD activation in murine embryonic fibroblasts generated from double knockout mice for Galpha q/11 and Galpha 12/13. As illustrated by the Western blot analysis of cell lysates shown in Fig. 3A, Galpha q/11 KO cells did not express either Galpha q or Galpha 11 whereas the Galpha 12/13 KO cells did not express either Galpha 12 or Galpha 13 while, as expected, the RK cells express the alpha  subunits of these G proteins.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3.   Comparison of three cell lines (RK, Gq/11 KO, or G12/13 KO) in Galpha subunit expression and PKD activation induced by LPA or PDB. A, Western blot analysis (W. Blot) of RK, Gq/11, and G12/13 knockout cell lysates. Confluent and quiescent cultures of the three type cells were solubilized with 2× sample buffer. The lysates were analyzed by SDS-PAGE and transferred to Immobilon membranes. Western blot analysis was carried out using the antibody against alpha q/11, alpha 12, or alpha 13. The positions of immunoreactive Galpha subunits at apparent Mr 43,000 are indicated by the arrows to the left. B, confluent and quiescent cultures of RK cells were incubated with (+) or without (-) 3 µM LPA or 200 nM PDB for 10 min at 37 °C. Cells were lysed with lysis buffer and immunoprecipitated with PA-1 antiserum. Upper panel, IVK, PKD activity was determined by an in vitro kinase assay as described under "Experimental Procedures," followed by SDS-PAGE and autoradiography. The autoradiogram shown is representative of three independent experiments. Middle panel, scanning densitometry. The results shown are the values (mean ± S.E. n = 3) of the level of PKD activation by in vitro kinase obtained from scanning densitometry expressed as a percentage of the maximum increase in phosphorylation obtained with 200 nM PDB. Lower panel, PKD activity in the immunocomplexes was then measured by syntide-2 phosphorylation, as described under "Experimental Procedures." The results expressed as an increased -fold over control in phosphorylation represent the mean ± S.E. obtained from three independent experiments, each performed in duplicate.

In agreement with the results shown in Figs. 1 and 2, treatment of RK cells with either LPA or PDB induced a marked increase in PKD catalytic activity measured either by autophosphorylation or syntide-2 phosphorylation assays (Fig. 3B). In striking contrast, addition of LPA to cultures of Galpha q/11 KO cells did not induce any significant increase in PKD activity, as measured in immunocomplexes by either autophosphorylation or syntide-2 phosphorylation assays. PDB, which acts directly on PKC and thus, bypasses the receptor-G protein interaction, induced robust stimulation of PKD activity in Galpha q/11 KO cells, indicating that the block in LPA action in these cells is upstream of the PKC/PKD cascade.

To further confirm the requirement of functional Gq in LPA-induced PKD activation, we examined whether transfection of Gq/11 KO cells with wild type Galpha q restores the ability of LPA to induce PKD activation. Because PKD is abundantly expressed in Gq/11 KO cells and RK cells, we used GFP-tagged PKD (GFP-PKD) for these co-transfection experiments and GFP antibody to immunoprecipitate the ectopically expressed PKD. Previously, we demonstrated that the GFP tag does not interfere with the regulatory properties of PKD (69, 70). Gq/11 KO cells were co-transfected with GFP-PKD, either alone or together with pcDNA1-Galpha q, and the RK cells transfected with GFP-PKD were used as the control. Three days after transfection, the transfected cells were stimulated without or with 3 µM LPA or 200 nM PDB. PKD was immunoprecipitated from the lysates of transfected cells with GFP antibody, and the immune complexes were incubated with [gamma -32P]ATP, subjected to SDS-PAGE, and analyzed by autoradiography to detect the prominent 140-kDa autophosphorylated GFP-PKD. The results presented in Fig. 4 show that Gq/11 KO cells cotransfected with wild type Galpha q and PKD exhibit a marked increase in LPA-induced PKD activity (Fig. 4A) and Ser-744 phosphorylation (Fig. 4B) compared with Gq/11 KO cells transfected with GFP-PKD alone. The increased PKD activity and phosphorylation in Gq/11 KO cells cotransfected with wild type Galpha q is comparable with that in the control RK cells transfected with GFP-PKD, which express endogenous Galpha q. These results demonstrate that a functional Galpha q/11 pathway is necessary for mediating LPA-induced PKD activation.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 4.   LPA induces PKD activation in Gq/11 KO cells transfected with Galpha q. A and B, Gq/11 KO cells co-transfected with GFP-PKD and pcDNA1 or pcDNA1 encoding wild type Galpha q. The control cells (RK cells) were transfected with GFP-PKD and pcDNA1, as indicated in this figure. Three days after transfection, the cultures were left unstimulated (-) or stimulated (+) either with 3 µM LPA or with 200 nM PDB for 10 min and lysed. A, upper panel, the lysates were immunoprecipitated with GFP antibody and PKD activity in the immunocomplexes was determined by an in vitro kinase assay (IVK) as described under "Experimental Procedures," followed by SDS-PAGE and autoradiography. A representative autoradiogram of three independent experiments is shown. Lower panel, PKD activity in immunocomplexes was measured by syntide-2 phosphorylation. The results expressed as an increased -fold over control in phosphorylation represent the mean ± S.E. obtained from three independent experiments. B, cell lysates from the experiments described as above were analyzed by SDS-PAGE and transferred to Immobilon membranes. Western blot (W. Blot) analysis was carried out using pS744/748 PKD antibody (upper panel) or GFP antiserum (lower panel).

LPA-induced Maximal PKD Activation Requires Functional Galpha 12/13-- Recently, we demonstrated that Galpha 13 contributes to PKD activation through a Rho- and PKC-dependent signaling pathway and hypothesized that PKD activation is mediated by both Galpha q and Galpha 13 in response to acute bombesin receptor stimulation (39). To assess the role of Galpha 12/13 in PKD activation induced by LPA, we examined the effect of this agonist on PKD activity in Galpha 12/13 KO cells. As shown in Fig. 3, addition of 5 µM LPA to cultures of Galpha 12/13 KO cells induced a lower level of PKD activation than that induced by either PDB in these cells or by LPA in RK cells. These results imply that whereas Galpha q/11 are necessary for LPA-induced PKD activation, Galpha 12/13 are required for eliciting maximal LPA-induced PKD activation.

The results presented in Fig. 5 were designed to examine in more detail the stimulation of PKD activity in response to LPA in Galpha 12/13 KO cells. LPA promoted PKD activation in Galpha 12/13 KO cells in a concentration-dependent fashion with half-maximal and maximal stimulation achieved at 0.07 and 3 µM. The maximal increase in PKD autophosphorylation activity (4-fold) was lower than that achieved by LPA in RK cells (6-fold). These results confirmed that LPA stimulated PKD activation in Galpha 12/13 KO cells with reduced effectiveness, even at the concentrations that induced a maximal response, and implied that the expression of Galpha 12/13 is necessary for maximal stimulation of PKD in response to LPA. The results shown in Fig. 5A also demonstrate that LPA induced PKD Ser-744 phosphorylation and syntide-2 phosphorylation activity (inset) in a concentration-dependent manner in Galpha 12/13 KO cells.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 5.   LPA activates PKD in a dose-dependent and PLC/PKC-dependent manner in G12/13 KO cells. A, LPA activates PKD in a dose-dependent manner. Confluent and quiescent cultures of G12/13 knockout cells were incubated with various concentrations of LPA for 10 min at 37 °C. Cells were lysed with lysis buffer and immunoprecipitated with PA-1 antiserum. Top panel, PKD activity was determined by an in vitro kinase (IVK) assay as described under "Experimental Procedures," followed by SDS-PAGE and autoradiography. The autoradiogram shown is representative of three independent experiments. Middle panel, pS744, cell lysates from the experiments described as above were analyzed by SDS-PAGE and transferred to Immobilon membranes. Western blot analysis was carried out using pS744/748 PKD antibody. Bottom panel, scanning densitometry. The results shown are the values (mean ± S.E. n = 3) of the level of PKD activation by in vitro kinase obtained from scanning densitometry expressed as a percentage of the maximum increase in phosphorylation obtained with 3 µM LPA. Inset, PKD activity in the immunocomplexes was measured by syntide-2 phosphorylation, as described under "Experimental Procedures." The results expressed as an increased -fold over control in phosphorylation represent the mean ± S.E. obtained from three independent experiments, each performed in duplicate. B, LPA induces PKD activation through a PLC/PKC-dependent pathway. Confluent and quiescent G12/13 knockout cells were incubated for 1 h with the PKC inhibitor GF-109203X (GF1, 3.5 µM) or Ro 31-8220 (Ro, 2.5 µM), or with PLC inhibitor, U73122. Control cells (-) received equivalent amounts of solvent or GFV. The cultures were subsequently stimulated for 10 min with 3 µM LPA or 200 nM PDB at 37 °C. Cells were lysed with lysis buffer and immunoprecipitated with PA-1 antiserum. Top panels, IVK, PKD activity was determined by an in vitro kinase assay, as described under "Experimental Procedures." The autoradiogram shown is representative of three independent experiments. Middle panels, pS744, cell lysates from the experiments described as the above were analyzed by SDS-PAGE and transferred to Immobilon membranes. Western blot analysis was carried out using pS744/748 PKD antibody. Bottom panels, PKD activity in the immunocomplexes was measured by syntide-2 phosphorylation, as described under "Experimental Procedures." The results expressed as an increased -fold over control in phosphorylation represent the mean ± S.E. obtained from three independent experiments, each performed in duplicate.

We verified that LPA induces PKD activation in these cells through a PKC- and PLC-dependent pathway. As shown in Fig. 5B, left, exposure to either GF I or Ro 31-8220 prevented PKD activation and Ser-744 phosphorylation induced by LPA whereas the biologically inactive analog GFV did not prevent PKD activation in response to LPA. Similarly, treatment with the PLC inhibitor U73122 (66, 67) prevented PKD activation induced by LPA but not by PDB, as shown by assays of autophosphorylation, Western blot analysis, or syntide-2 phosphorylation (Fig. 5B, right).

LPA-induced PKD Activation and Phosphorylation Requires Functional Gi in Galpha 12/13 KO Cells-- We hypothesized that PKD activation and activation loop phosphorylation are elicited by LPA and other GPCR agonists through interaction of complementary G protein pathways, namely, Gq, Galpha 12/13, and Gi (see Introduction for references). Although the effect of Gi is not prominent in LPA-induced PKD activation in RK cells, our hypothesis predicts that in Galpha 12/13 KO cells, members of the Gq family interact with members of the Gi family in mediating LPA-induced PKD activation. To test the contribution of Gi to LPA-induced PKD activation in Galpha 12/13 KO cells, cultures of these cells were treated with increasing concentrations of PTx (1-100 ng/ml) and PKD activity in immunocomplexes was measured by either autophosphorylation or syntide-2 phosphorylation assays.

As shown in Fig. 6A, treatment with PTx dramatically inhibited both PKD activation and Ser-744 phosphorylation elicited by LPA in a concentration-dependent fashion. Indeed, treatment with 30-100 ng/ml PTx completely abolished PKD activation and Ser-744 phosphorylation in response to LPA. In contrast, treatment with 50 ng/ml PTx did not interfere with PKD activation and Ser-744 phosphorylation induced by PDB in these cells. As illustrated in Fig. 6B, similar results were obtained when PKD activity in immunocomplexes was determined by assays of syntide-2 phosphorylation. These findings contrast with those shown in Fig. 2 with RK cells (PTx inhibited LPA-induced PKD activation by only ~25%) and indicate that Gi plays a critical role in promoting PKD activation in Galpha 12/13 KO cells. We conclude that in Galpha 12/13 KO cells, Gi cooperates with Gq in mediating the PKC/PKD phosphorylation cascade in response to LPA.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of PTx on the activation of PKD induced by LPA in G12/13 KO cells. A and B, confluent and quiescent G12/13 KO cells were incubated with different concentration of PTx as indicated for 3 h. Cells were then stimulated with (+) or without (-) 3 µM LPA (left panels) or 200 nM PDB (right panels) for 10 min at 37 °C. Cultures were lysed with lysis buffer and then immunoprecipitated with PA-1 antiserum. A, top panels, in vitro kinase (IVK), immunoprecipitates were subjected to in vitro kinase assay, SDS-PAGE, and autoradiography. The autoradiograph shown is representative of three independent experiments. Middle panels, cell lysates from the experiments described above were analyzed by SDS-PAGE and transferred to Immobilon membranes. Western blot analysis was carried out using pS744/748 PKD antibody. Bottom panels, scanning densitometry, the results shown are the values (mean ± S.E., n = 3) of the level of PKD activation obtained from scanning densitometry expressed as a percentage of the maximum increase in phosphorylation obtained with 3 µM LPA for 10 min at 37 °C. , values corresponding to PKD activity from cells incubated with PTx, and then stimulated with LPA; , values corresponding to PKD activity from cells incubated with PTx but without LPA. B, PKD activity in the immunocomplexes was measured by syntide-2 phosphorylation, as described under "Experimental Procedures." The results expressed as an increased -fold over control in phosphorylation represent the mean ± S.E. obtained from three independent experiments, each performed in duplicate.

Recently, we demonstrated that in addition to Galpha q, Galpha 13-mediated signaling contributes to PKD activation in bombesin-stimulated cells through endogenous Rho (39). C. difficile toxin monoglucosylates the threonine residue at position 35 in Rac and Cdc-42 and threonine 37 in Rho and thereby inactivates these small G proteins (71, 72). In view of the results shown in Fig. 6 with Galpha 12/13 KO cells, we hypothesized that inactivation of Rho GTPases with C. difficile toxin B in RK cells should enhance the sensitivity of LPA-induced PKD activation to treatment with PTx. To test this hypothesis, RK cells were treated with PTx either with or without C. difficile toxin B and then stimulated with either LPA or PDB, as indicated in Fig. 7. Treatment with C. difficile toxin B markedly enhanced the ability of PTx to inhibit LPA-induced PKD activation (Fig. 7A) and Ser-744 phosphorylation (Fig. 7B) in RK cells. These results provide an independent line of evidence indicating that PKD activation induced by LPA is mediated by cooperation of multiple G protein pathways.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 7.   PKD activation induced by LPA in RK cells is partially blocked by C. difficile toxin B (toxin B) and almost abolished by the combination treatments of the cells with toxin B and Ptx. A and B, confluent and quiescent RK cells were preincubated with (+) or without (-) PTx (50 ng/ml) for 3 h and then treated with different concentrations of toxin B as indicated for 1.5 h. Cells were then stimulated with (+) or without (-) 3 µM LPA or 200 nM PDB for 10 min at 37 °C. Cultures were lysed with lysis buffer and then immunoprecipitated with PA-1 antiserum. A, upper panel, in vitro kinase (IVK), immunoprecipitates were subjected to in vitro kinase assay, SDS-PAGE, and autoradiography. The autoradiograph shown is representative of three independent experiments. Lower panel, PKD activity in the immunocomplexes was measured by syntide-2 phosphorylation, as described under "Experimental Procedures." The results expressed as an increased -fold over control in phosphorylation represent the mean ± S.E. obtained from three independent experiments, each performed in duplicate. B, pS744, cell lysates from the experiments described above were analyzed by SDS-PAGE and transferred to Immobilon membranes. Western blot analysis was carried out using pS744/748 PKD antibody.

LPA Induces Activation of Mitogen-activated Protein Kinases via Gi-dependent but PKC-independent Pathways in RK, Galpha q/11 KO, and Galpha 12/13 KO Cells-- To substantiate that the different profiles of PKD activation in response to LPA in the three cell lines used in our study is specific and reflects the participation of multiple G proteins, we examined other biological effects induced by LPA in these cells.

In most cell types, LPA stimulates Ras activation leading to stimulation of Raf, MEK, and the ERKs via a PTx-sensitive pathway that involves the beta gamma subunits of Gi (47-51). Here, we determined the effect of PTx on LPA-induced ERK activation in these three types of cells. As shown by Western blot analysis using an antibody that detects the dually phosphorylated and active ERK-1 and ERK-2, LPA induced robust activation of the ERKs in RK, Galpha 12/13 KO, and Galpha q/11 KO cells (Fig. 8). Treatment with PDB also induced strong ERK activation in the three cell lines, indicating the existence of a PKC-dependent pathway.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 8.   LPA induces activation of ERK-2 and ERK-1 in RK cells and Gq/11 KO or G12/13 KO cells. Confluent and quiescent RK cells (A) and Gq/11 KO cells (B) or G12/13 KO cells (C) were incubated with PTx for 3 h or with the other inhibitors, GF-109203X (GF1, 3.5 µM), or Ro 31-8220 (Ro, 2.5 µM), or U0126 (U, 2.5 µM), as indicated, for 1 h. Control cells (-) received an equivalent amount of solvent. Cells were then stimulated with (+) or without (-) 3 µM LPA or 200 nM PDB for 10 min at 37 °C. Cultures were lysed with lysis buffer. Cell lysates from the experiments were analyzed by SDS-PAGE and transferred to Immobilon membranes. A-C, upper panels, Western blot analysis with specific antiphospho-ERK-1/2 monoclonal antibody; lower panels, the Western blot was also probed for total ERK by ERK-2 polyclonal antibody.

Treatment of RK cells with PTx markedly inhibited ERK activation in response to LPA, whereas exposure to GF I or Ro 31-8220 did not interfere with ERK activation, indicating that LPA induces ERK through a PTx-sensitive, PKC-independent in RK cells. Virtually identical results were obtained with Galpha 12/13 KO cells and Galpha q/11 KO cells. Specifically, LPA promoted ERK activation in both Galpha 12/13 KO cells and Galpha q/11 KO cells through a Gi-dependent but PKC-independent pathway (Fig. 8). These results demonstrate that the lack of expression of these G proteins does not interfere with other biological effects of LPA mediated by Gi and provide further support to the conclusion that LPA utilizes different G protein pathways to activate parallel phosphorylation cascades leading to PKD and ERK activation in RK cells.

Concluding Remarks-- LPA promotes a broad range of biological responses and multiple molecular events in target cells (42). Consistent with the stimulation of multiple signaling pathways, LPA has been shown to activate several heterotrimeric G proteins including Gq, Gi, and G13 in Swiss 3T3 cells (54). PKC-dependent PKD activation has been identified as an early event in the action of LPA in intact Swiss 3T3, Rat-1, and IEC-6 cells (56, 57). Because our previous studies indicated that Galpha q- and Galpha 13-mediated signaling can promote PKD activation in intact cells (20, 39), it was surprising that treatment of these cells with PTx markedly inhibited PKD activation in response to LPA (56, 57). Because PTx catalyzes the ADP-ribosylation and inactivation of members of the Galpha i family (68), these results identified the involvement of an additional Gi-dependent pathway leading to PKD activation in response to LPA. However, other results also demonstrated that LPA induces PKD activation through a pathway that was only partially attenuated by PTx in IEC-18 cells (57) indicating that Gi contributes to PKD activation to different degrees in different cell contexts. In the present study, we tested the hypothesis that LPA induces PKD activation through multiple G protein signal transduction pathways, including Gq, Gi, and G13, using mouse embryonic cell lines deficient in Galpha q/11 and Galpha 12/13 and using a cell line deficient in rhodopsin kinase (RK cells), as a control.

Based on the results presented in this study, we propose a model that envisages that LPA induces PKD activation through Gq acting synergistically with G12 and Gi. Thus, inhibition of Gi signaling in RK cells by treatment with PTx only induces a modest attenuation of PKD activation in response to LPA because the remaining G protein pathways (Gq and G12) cooperate to mediate a substantial response to LPA. Similarly, treatment of RK cells with C. difficile toxin B that inactivates Rho GTPases, the effectors of the G12 pathway, had little effect on PKD activation by LPA because the remaining G protein pathways (in this case Gq and Gi) are sufficient to mediate PKD activation in response to LPA. An important result substantiating our hypothesis is that treatment of RK cells with both C. difficile toxin B and PTx completely prevented LPA-induced PKD activation, indicating that endogenous Gq appears necessary but not sufficient to mediate this response.

The salient features of this model are corroborated by the results obtained in the cell lines deficient in either Galpha q/11 or Galpha 12/13. A crucial role of Galpha q/11 in mediating LPA-induced PKD activation is clearly demonstrated by the fact that LPA-induced PKD activation was completely abrogated in Galpha q/11 KO cells and that transfection of Galpha q into the Gq/11 KO cells restored LPA-induced PKD activation to the similar level as the control cells. In Galpha 12/13 KO cells, LPA induced substantial PKD activation, a response predicted by the model to be mediated by cooperation of Gq and Gi. In line with this interpretation and in sharp contrast to the results obtained with RK cells, treatment of Galpha 12/13 KO cells with PTx dramatically inhibited PKD activation in response to LPA. These results are also consistent with the notion that endogenous Galpha q/11 is not sufficient to mediate PKD activation in response to LPA. Thus, our model integrates the PTx-sensitive and -insensitive effects obtained in Galpha 12/13 KO cells and RK cells with the striking suppression of LPA-induced PKD activation in Galpha q/11 KO cells and leads to the notion that LPA induces PKD activation through the synergistic interaction of pathways initiated by Gq, G12, and Gi. A corollary of these results is that the level of expression of the different alpha  subunits of heterotrimeric G proteins can be identified as one of the important molecular elements that determine the influence of cell context on agonist-induced PKD activation.

Recent work from other laboratories has shown that transcriptional responses induced by LPA also result from synergistic effects between parallel G protein signaling pathways. For example, the stimulation of the transcription factor NF-kappa B by LPA is mediated by Gq and Gi pathways (73) and the activation of the serum response element is mediated by cooperative effects between Gi and Galpha 13/Rho pathways (74). Furthermore, recent genetic studies indicate that the Gq-mediated signaling pathway functionally interacts with the G12-mediated signaling pathway to promote embryonic survival (63).

Here, we propose that PKD activation, which precedes the transcriptional responses, is also mediated by cooperative effects between events initiated by Gq, Gi, and G12. In this manner, PKD may constitute an early point of convergence of Gq, G12, and Gi signaling in LPA-treated cells. The scheme shown in Fig. 9 summarizes the multiple G protein signal transduction pathways involved in LPA-induced PKD activation proposed in this study and also illustrates the molecular, cellular, and pharmacological approaches used in our experiments.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 9.   Signal transduction pathways involved in LPA-induced PKD activation. The scheme proposes a model that envisages that PKD activation in response to LPA receptor signaling is mediated through interaction of complementary G protein pathways, namely, Gq/11, Gi, and G12/13, and also illustrates the molecular, cellular, and pharmacological approaches used in this study. As shown in this scheme, Galpha q/11 plays a crucial role (expressed by thick arrows) in mediating LPA-induced PKD activation through a PLC- and PKC-dependent pathway, which is clearly demonstrated by the fact that LPA-induced PKD activation and phosphorylation were completely blocked (dashv ) in Gq/11 knockout (Gq/11KO) cells and that transfection of Galpha q into the Gq/11 KO cells restores (right-arrow) LPA-induced PKD activation to the similar level as the control cells. However, LPA induced maximal PKD activation requires the synergistic interaction of pathways initiated by Gq, G12, and Gi. G12/13 mediates PKD activation through Rho and PKC, and Gi mediates PKD activation through PLC and PKC. In G12/13 knockout (G12/13 KO) cells, or in the RK cells treated with C. difficile toxin B (which inactivates Rho GTPases), or with Gi inhibitor, PTx, i.e. when either G12/13/Rho pathway alone or Gi pathway alone is blocked, LPA induces a reduced but substantial PKD activation. But, treatment of G12/13 KO cells with PTx, or a combination treatment of RK cells with Ptx and C. difficile toxin B, which resulted in the block of both G12/13 and Gi pathways, completely prevented LPA-induced PKD activation. In addition, this scheme also shows that LPA-induced signal transduction pathways initiated by Gq, G12, and Gi leading to PKD activation is PLC and PKC-dependent, which has been verified by the fact that in either RK cells or G12/13 KO cells, LPA induced-PKD activation and phosphorylation could be blocked (dashv ) by U73122 (PLC inhibitor) or GF1 or Ro 31-8220 (Ro) (selective inhibitors of PKC). Therefore, PKC/PKD activation is a point of convergence in the action of multiple G protein pathways. (Also see the text for details and abbreviations). Ins(1,4,5)P3, inositol 1,4,5-trisphosphate.


    ACKNOWLEDGEMENTS

We are very grateful to Dr. Melvin I. Simon, Division of Biology, California Institute of Technology, for the generous gift of fibroblast cell lines. We also thank Steven H. Young for assistance in cell transfection and J. Sinnett-Smith, Cliff Hurd, and Osvaldo Rey for helpful discussions and careful reading of the manuscript.

    FOOTNOTES

* This work was supported in part by National Health Institute Grants DK 55003, DK56930, DK 17294, and NCI Grant P50 CA 90388-01.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by National Institutes of Health National Research Service Award F32 CA84658-01A1.

|| To whom all correspondence should be addressed: Dept. of Medicine, UCLA School of Medicine, 900 Veterans Ave., Warren Hall Rm. 11-124, Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399; E-mail: erozengurt@mednet.ucla.edu.

Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M211175200

    ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; LPA, lysophosphatidic acid; G proteins, guanine nucleotide-binding regulatory proteins; GPCRs, G protein-coupled receptors; GFP, green fluorescent protein; PDB, phorbol 12,13-dibutyrate; PKD, protein kinase D; RK cells, murine embryonic cell line with rhodopsin kinase gene knockout; Galpha q/11 KO cells, murine embryonic cell line with Galpha q/11 gene knockout; Galpha 12/13 KO cells, murine embryonic cell line with Galpha 12/13 gene knockout; PTx, pertussis toxin; PLC, phospholipase C; ERK, extracellular signal-regulated kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Nishizuka, Y. (1992) Science 258, 607-614[Medline] [Order article via Infotrieve]
2. Newton, A. C. (1995) J. Biol. Chem. 270, 28495-28498[Free Full Text]
3. Dekker, L. V., and Parker, P. J. (1994) Trends Biochem. Sci. 19, 73-77[CrossRef][Medline] [Order article via Infotrieve]
4. Hug, H., and Sarre, T. F. (1993) Biochem. J. 291, 329-343[Medline] [Order article via Infotrieve]
5. Nishizuka, Y. (1995) FASEB J. 9, 484-496[Abstract/Free Full Text]
6. Valverde, A. M., Sinnett-Smith, J., Van Lint, J., and Rozengurt, E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8572-8576[Abstract]
7. Johannes, F. J., Prestle, J., Eis, S., Oberhagemann, P., and Pfizenmaier, K. (1994) J. Biol. Chem. 269, 6140-6148[Abstract/Free Full Text]
8. Rozengurt, E., Sinnett-Smith, J., and Zugaza, J. L. (1997) Biochem. Soc. Trans. 25, 565-571[Medline] [Order article via Infotrieve]
9. Zugaza, J. L., Sinnett-Smith, J., Van Lint, J., and Rozengurt, E. (1996) EMBO J. 15, 6220-6230[Abstract]
10. Yuan, J., Bae, D., Cantrell, D., Nel, A. E., and Rozengurt, E. (2002) Biochem. Biophys. Res. Commun. 291, 444-452[CrossRef][Medline] [Order article via Infotrieve]
11. Zugaza, J. L., Waldron, R. T., Sinnett-Smith, J., and Rozengurt, E. (1997) J. Biol. Chem. 272, 23952-23960[Abstract/Free Full Text]
12. Iglesias, T., and Rozengurt, E. (1998) J. Biol. Chem. 273, 410-416[Abstract/Free Full Text]
13. Abedi, H., Rozengurt, E., and Zachary, I. (1998) FEBS Lett. 247, 209-212[CrossRef]
14. Iglesias, T., Waldron, R. T., and Rozengurt, E. (1998) J. Biol. Chem. 273, 27662-27667[Abstract/Free Full Text]
15. Waldron, R. T., Rey, O., Iglesias, T., Tugal, T., Cantrell, D., and Rozengurt, E. (2001) J. Biol. Chem. 276, 32606-32615[Abstract/Free Full Text]
16. Offermanns, S., and Simon, M. I. (1996) Cancer Surv. 27, 177-198[Medline] [Order article via Infotrieve]
17. Exton, J. H. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 481-509[CrossRef][Medline] [Order article via Infotrieve]
18. Paolucci, L., and Rozengurt, E. (1999) Cancer Res. 59, 572-577[Abstract/Free Full Text]
19. Chiu, T., and Rozengurt, E. (2001) Am. J. Physiol. 280, C929-C942
20. Yuan, J., Slice, L., Walsh, J. H., and Rozengurt, E. (2000) J. Biol. Chem. 275, 2157-2164[Abstract/Free Full Text]
21. Hooley, R., Yu, C. Y., Symons, M., and Barber, D. L. (1996) J. Biol. Chem. 271, 6152-6158[Abstract/Free Full Text]
22. Klages, B., Brandt, U., Simon, M. I., Schultz, G., and Offermanns, S. (1999) J. Cell Biol. 144, 745-754[Abstract/Free Full Text]
23. Plonk, S. G., Park, S. K., and Exton, J. H. (1998) J. Biol. Chem. 273, 4823-4826[Abstract/Free Full Text]
24. Needham, L. K., and Rozengurt, E. (1998) J. Biol. Chem. 273, 14626-14632[Abstract/Free Full Text]
25. Seasholtz, T. M., Majumdar, M., and Brown, J. H. (1999) Mol. Pharmacol. 55, 949-956[Free Full Text]
26. Slice, L., Walsh, J. H., and Rozengurt, E. (1999) J. Biol. Chem. 274, 27562-27566[Abstract/Free Full Text]
27. Hart, M. J., Jiang, X., Kozasa, T., Roscoe, W., Singer, W. D., Gilman, A. G., Sternweis, P. C., and Bollag, G. (1998) Science 280, 2112-2114[Abstract/Free Full Text]
28. Kozasa, T., Jiang, X., Hart, M. J., Sternweis, P. M., Singer, W. D., Gilman, A. G., Bollag, G., and Sternweis, P. C. (1998) Science 280, 2109-2111[Abstract/Free Full Text]
29. Majumdar, M., Seasholtz, T. M., Buckmaster, C., Toksoz, D., and Brown, J. H. (1999) J. Biol. Chem. 274, 26815-26821[Abstract/Free Full Text]
30. Rozengurt, E. (1998) J. Cell. Physiol. 177, 507-517[CrossRef][Medline] [Order article via Infotrieve]
31. Hall, A. (1998) Science 279, 509-514[Abstract/Free Full Text]
32. Sah, V. P., Seasholtz, T. M., Sagi, S. A., and Brown, J. H. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 459-489[CrossRef][Medline] [Order article via Infotrieve]
33. Kamada, Y., Qadota, H., Python, C. P., Anraku, Y., Ohya, Y., and Levin, D. E. (1996) J. Biol. Chem. 271, 9193-9196[Abstract/Free Full Text]
34. Nonaka, H., Tanaka, K., Hirano, H., Fujiwara, T., Kohno, H., Umikawa, M., Mino, A., and Takai, Y. (1995) EMBO J. 14, 5931-5938[Abstract]
35. Hippenstiel, S., Kratz, T., Kruell, M., Seybold, J., Eichel-Streiber, C. V., and Suttorp, N. (1998) Biochem. Biophys. Res. Commun. 245, 830-834[CrossRef][Medline] [Order article via Infotrieve]
36. Chang, J.-H., Pratt, J. C., Sawasdikosol, S., Kapeller, R., and Burakoff, S. J. (1998) Mol. Cell. Biol. 18, 4986-4993[Abstract/Free Full Text]
37. Slater, S. J., Seiz, J. L., Stagliano, B. A., and Stubbs, C. D. (2001) Biochemistry 40, 4437-4445[CrossRef][Medline] [Order article via Infotrieve]
38. Sagi, S. A., Seasholtz, T. M., Kobiashvili, M., Wilson, B. A., Toksoz, D., and Brown, J. H. (2001) J. Biol. Chem. 276, 15445-15452[Abstract/Free Full Text]
39. Yuan, J., Slice, L. W., and Rozengurt, E. (2001) J. Biol. Chem. 276, 38619-38627[Abstract/Free Full Text]
40. Eichholtz, T., Jalink, K., Fahrenfort, I., and Moolenaar, W. H. (1993) Biochem. J. 291, 677-680[Medline] [Order article via Infotrieve]
41. Moolenaar, W. H. (1995) J. Biol. Chem. 270, 12949-12952[Free Full Text]
42. Moolenaar, W. H., Kranenburg, O., Postma, F. R., and Zondag, G. C. (1997) Curr. Opin. Cell Biol. 9, 168-173[CrossRef][Medline] [Order article via Infotrieve]
43. Contos, J. J. A., Ishii, I., and Chun, J. (2000) Mol. Pharmacol. 58, 1188-1196[Abstract/Free Full Text]
44. Hla, T., Lee, M.-J., Ancellin, N., Paik, J. H., and Kluk, M. J. (2001) Science 294, 1875-1878[Abstract/Free Full Text]
45. Yang, A. H., Ishii, I., and Chun, J. (2002) Biochim. Biophys. Acta 1582, 197-203[Medline] [Order article via Infotrieve]
46. Goetzl, E. J., and An, S. (1998) FASEB J. 12, 1589-1598[Abstract/Free Full Text]
47. Van Corven, E. J., Hordijk, P. L., Medema, R. H., Bos, J. L., and Moolenaar, W. H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1257-1261[Abstract]
48. Luttrell, L. M., van Biesen, T., Hawes, B. E., Koch, W. J., Touhara, K., and Lefkowitz, R. J. (1995) J. Biol. Chem. 270, 16495-16498[Abstract/Free Full Text]
49. Daub, H., Wallasch, C., Lankenau, A., Herrlich, A., and Ullrich, A. (1997) EMBO J. 16, 7032-7044[Abstract/Free Full Text]
50. Kranenburg, O., Verlaan, I., Hordijk, P. L., and Moolenaar, W. H. (1997) EMBO J. 16, 3097-3105[Abstract/Free Full Text]
51. Cunnick, J. M., Dorsey, J. F., Standley, T., Turkson, J., Kraker, A. J., Fry, D. W., Jove, R., and Wu, J. (1998) J. Biol. Chem. 273, 14468-14475[Abstract/Free Full Text]
52. Exton, J. H. (1997) Eur. J. Biochem. 243, 10-20[Abstract]
53. Seufferlein, T., and Rozengurt, E. (1994) J. Biol. Chem. 269, 9345-9351[Abstract/Free Full Text]
54. Gohla, A., Harhammer, R., and Schultz, G. (1998) J. Biol. Chem. 273, 4653-4659[Abstract/Free Full Text]
55. Kranenburg, O., and Moolenaar, W. H. (2001) Oncogene 20, 1540-1546[CrossRef][Medline] [Order article via Infotrieve]
56. Paolucci, L., Sinnett-Smith, J., and Rozengurt, E. (2000) Am. J. Physiol. 278, C33-C39[Abstract/Free Full Text]
57. Chiu, T., and Rozengurt, E. (2001) Am. J. Physiol. 280, C929-C942
58. Xu, J., Dodd, R. L., Makino, C. L., Simon, M. I., Baylor, D. A., and Chen, J. (1997) Nature 389, 505-509[CrossRef][Medline] [Order article via Infotrieve]
59. Offermanns, S., Zhao, L. P., Gohla, A., Sarosi, I., Simon, M. I., and Wilkie, T. M. (1998) EMBO J. 17, 4304-4312[Abstract/Free Full Text]
60. Van Lint, J. V., Sinnett-Smith, J., and Rozengurt, E. (1995) J. Biol. Chem. 270, 1455-1461[Abstract/Free Full Text]
61. Mochizuki, H., Ito, T., and Hidaka, H. (1993) J. Biol. Chem. 268, 9143-9147[Abstract/Free Full Text]
62. Lorca, T., Cruzalegui, F. H., Fesquet, D., Cavadore, J. C., Méry, J., Means, A., and Dorée, M. (1993) Nature 366, 270-273[CrossRef][Medline] [Order article via Infotrieve]
63. Gu, J. L., Muller, S., Mancino, V., Offermanns, S., and Simon, M. I. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 9352-9357[Abstract/Free Full Text]
64. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grandperret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., and Kirilovsky, J. (1991) J. Biol. Chem. 266, 15771-15781[Abstract/Free Full Text]
65. Yeo, E. J., and Exton, J. H. (1995) J. Biol. Chem. 270, 3980-3988[Abstract/Free Full Text]
66. Stauffer, T. P., Ahn, S., and Meyer, T. (1998) Curr. Biol. 8, 343-346[Medline] [Order article via Infotrieve]
67. Strassheim, D., Law, P. Y., and Loh, H. H. (1998) Mol. Pharmacol. 53, 1047-1053[Abstract/Free Full Text]
68. Staddon, J. M., Bouzyk, M. M., and Rozengurt, E. (1991) J. Cell Biol. 115, 949-958[Abstract]
69. Matthews, S., Iglesias, T., Cantrell, D., and Rozengurt, E. (1999) FEBS Lett. 457, 515-521[CrossRef][Medline] [Order article via Infotrieve]
70. Matthews, S. A., Iglesias, T., Rozengurt, E., and Cantrell, D. (2000) EMBO J. 19, 2935-2945[Abstract/Free Full Text]
71. Just, I., Wilm, M., Selzer, J., Rex, G., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) J. Biol. Chem. 270, 13932-13936[Abstract/Free Full Text]
72. Aktories, K. (1997) J. Clin. Invest. 99, 827-829[Free Full Text]
73. Shahrestanifar, M., Fan, X., and Manning, D. R. (1999) J. Biol. Chem. 274, 3828-3833[Abstract/Free Full Text]
74. An, S., Bleu, T., Hallmark, O. G., and Goetzl, E. J. (1998) J. Biol. Chem. 273, 7906-7910[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.