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J. Biol. Chem., Vol. 276, Issue 42, 38619-38627, October 19, 2001

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Activation of Protein Kinase D by Signaling through Rho and the  Subunit of the Heterotrimeric G Protein G₁₃^*

Jingzhen Yuan, Lee W. Slice, and Enrique Rozengurt^§

From the Department of Medicine, School of Medicine and Molecular Biology Institute, UCLA, Los Angeles, California 90095

Received for publication, June 15, 2001, and in revised form, August 15, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Protein kinase D (PKD/PKCµ) immunoprecipitated from COS-7 cells transiently transfected with either a constitutively active mutant of Rho (RhoQ63L) or the Rho-specific guanine nucleotide exchange factor pOnco-Lbc (Lbc) exhibited a marked increase in basal activity. Addition of aluminum fluoride to cells co-transfected with PKD and wild type G₁₃ also induced PKD activation. Co-transfection of Clostridium botulinum C3 toxin blocked activation of PKD by RhoQ63L, Lbc, or aluminum fluoride-stimulated G₁₃. Treatment with the protein kinase C inhibitors GF I or Ro 31-8220 prevented the increase in PKD activity induced by RhoQ63L, Lbc, or aluminum fluoride-stimulated G₁₃. PKD activation in response to G₁₃ signaling was also completely prevented by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Co-expression of C. botulinum C3 toxin and a COOH-terminal fragment of G_q that acts in a dominant-negative fashion blocked PKD activation in response to agonist stimulation of bombesin receptor. Expression of the COOH-terminal region of G₁₃ also attenuated PKD activation in response to bombesin receptor stimulation. Our results show that G₁₃ contributes to PKD activation through a Rho- and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both G_q and G₁₃ in response to bombesin receptor stimulation.

	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 (, ₁, ₂, and ), novel PKCs (, , , and ), and atypical PKCs ( and ), 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 DAGs, 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 induced by phorbol esters (14). 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 , , and  subunits and transduce external signals from heptahelical receptors to intracellular effectors (15). Mammalian G protein  subunits are classified into four subfamilies: G_s, G_i, G_q, and G₁₂. The  subunit of G_q stimulates the  isoforms of phospholipase C (PLC) that catalyze the production of inositol 1,4,5-trisphosphate that triggers the release of Ca²⁺ from internal stores and diacylglycerol (DAG) that activates the classical and novel isoforms of PKC (reviewed in Ref. 16). We reported that 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, 17, 18). Although each of these receptors activates G_q and G_q signaling stimulates PKD activity (19), the kinetics and degree of PKD activation differ among the different receptors even in the same cell line (11). Furthermore, expression of a COOH-terminal fragment of G_q that acts in a dominant-negative fashion attenuated (but did not eliminate) PKD activation in response to agonist stimulation of bombesin receptor (19). Taken together, these results prompted us to consider the possibility that G protein-coupled receptors (GPCRs) stimulate PKD activation not only via G_q but also through other, as yet unidentified, G protein-mediated signaling pathways.

Many G_q-coupled receptors 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 (20-25) via guanine nucleotide exchange factors that directly link the G subunits to Rho (26-28). 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 (29-31). Interestingly, a number of recent studies have suggested a convergence between Rho- and PKC-mediated signaling in yeast and mammalian cells (32-37). In the budding yeast, the homologues of mammalian PKC and RhoA, Pkc1p and Rho 1p, respectively, act through a common mechanism that appears to involve a direct interaction between these proteins (32, 33). In epithelial and endothelial cells, treatment with Clostridium difficile toxin, which inactivates all members of the Rho subfamily, prevented PKC translocation and activation (34). Rho signaling has been shown to enhance AP-1 transcription in T lymphocytes, and a molecular association between Rho and PKC has been demonstrated in these cells (35). Recently, Slater et al. (36) have demonstrated that Rho-GTP potently stimulates PKC activity in vitro using recombinant proteins, and Sagi et al. (37) reported that G_q and PLC signaling are synergistic with Rho. In addition, Rho has been implicated in the stimulation of pathways leading to lipid-derived second messenger synthesis and PKC activation (38, 39) including phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) generation (40, 41), PLD activation (42, 43), and inhibition of DAG kinase isoforms (44). These considerations prompted us to examine whether, in addition to G_q, Rho- and G₁₃-mediated signaling can promote PKD activation in intact cells and whether endogenous Rho and G₁₃ could contribute to PKD activation in response to bombesin receptor stimulation.

The results presented here demonstrate that co-expression of PKD with constitutively activated Rho, the Rho-specific guanine nucleotide exchange factor pOnco-Lbc (45, 46), or G₁₃ stimulated by aluminum fluoride induced a marked increase in PKD activity. The Clostridium botulinum C3 toxin, which inactivates Rho, selectively prevented PKD activation in response to Rho, Lbc, and G₁₃ and attenuated PKD activation in response to bombesin GPCR activation. PKD activation induced by G₁₃/Rho signaling was prevented by PKC inhibitors and by mutation of Ser-744 and Ser-748 in the kinase activation loop of PKD to alanine, a non-phosphorylatable residue. Expression of a COOH-terminal fragment of G_q that acts in a dominant-negative fashion together with the C3 toxin virtually abolished PKD activation induced by agonist stimulation of the bombesin receptor. Thus, our results identify PKD as a novel downstream target in G₁₃ and Rho signaling and indicate that bombesin GPCR stimulation promotes PKD activation via both G_q- and G₁₃/Rho-dependent pathways.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cell Culture and Transfections-- COS-7 cells were maintained by subculture in 10-cm tissue culture plates every 3-4 days in Dulbecco's modified Eagle's medium supplemented with 10% FBS at 37 °C in a humidified atmosphere containing 10% CO₂. For experimental dishes, cells were subcultured at 6 × 10⁴ cells/ml in 6- (5 ml) or 10-cm (10 ml) dishes on the day prior to transfections. All transfections and co-transfections were carried out with equivalent amounts of DNA (6 µg/6-cm dish and 12 µg/10-cm dish). Transfections were carried out in Opti-MEM (Life Technologies, Inc.) using Lipofectin (Life Technologies, Inc.) at 10 µl/6-cm dish or 20 µl/10-cm dish, added to cells in a final volume of 2.5 ml/6-cm dish or 5 ml/10-cm dish, following formation of DNA-Lipofectin complexes according to the protocol provided by the manufacturer. Cells were allowed to take up complexes in the absence of FBS for 5-6 h or overnight and then FBS (10% final concentration) in Opti-MEM was added to the dishes to yield a final volume of 5 ml/6-cm dish or 10 ml/10-cm dish. Cells were used for experiments after a further 48-72 h of incubation.

cDNA Constructs Used in Transfections-- The wild type G_q subunit cDNAs in the eukaryotic expression vector pcDNA-1 (Invitrogen) (47) were obtained from the American Type Tissue Collection (Manassas, VA). The murine wild type G₁₃ subunit cDNAs in pcDNA-1 were gifts from Dr. H. R. Bourne (University of California, San Francisco) (48). The constructs pcDNA3-PKD encoding PKD (49) and pcDNA3-PKD mutants encoding PKD with site-specific mutations within the activation loop in the catalytic domain including the single mutants (PKD-S744A and PKD-S748A) and double mutants (PKD-S744A/S748A and PKD-S744E/S748E) have been described previously (14). BNR-pCD2 containing the cDNA encoding the bombesin/gastrin-releasing peptide receptor was kindly provided by Dr. J. F. Battey, Jr. (NIDCD, National Institutes of Health, Bethesda). Expression vectors for Rho, RhoQ63L, Ras, RasV12, Rac, and RacV12 in pcDNA3 were provided by Dr. D. Chang (UCLA). The production of the vector GFP-_qCT encoding a fusion protein of GFP containing G_q (residues 305-359) at its carboxyl terminus have been described previously (14). The plasmids pEF-LacZ and pEF-C3 were provided by Dr. R. Treisman (Imperial Cancer Research Fund, London, UK). The pOnco-Lbc was provided by Dr. D. Toksoz (Tufts University, Boston) and was described previously (50).

Polymerase chain reaction was used to generate DNA encoding for the carboxyl-terminal region of G₁₃ (residues 333-377) using the murine G₁₃ cDNA as a template with sense (5'-GCTCAAGCTTCGAAACGCCGGGACCAGCAGCAG-3') and antisense (5'-GGTGGATCCTCACTGCAGCATGAGCTGCTTCAG-3') primers. The resulting DNA fragment was subcloned into the BamHI and HindIII restriction sites of pcDNA-3. The fidelity of the polymerase chain reaction was confirmed by DNA sequencing. The BamHI/HindIII DNA fragment was cloned in pGFP-C1 (CLONTECH, Inc., La Jolla, CA) such that the resulting fusion protein produced by this plasmid would be a hybrid GFP containing G₁₃ (residues 333-377) at its carboxyl terminus.

Immunoprecipitations-- Transfected COS-7 cells were washed twice with Dulbecco's modified Eagle's medium and equilibrated in 5 ml of the same medium at 37 °C for 1-2 h. Some dishes were treated with various pharmacological agents during this equilibration period or with agonists for 10 min at the end of this period, as indicated in the corresponding figure legends. Cells were lysed in buffer A (50 mM Tris-HCl, pH 7.6, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 100 µg/ml leupeptin, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, hydrochloride (Pefabloc), and 1% Triton X-100). PKD was immunoprecipitated at 4 °C for 3 h with the PA-1 antiserum (1:50 dilution) raised against the synthetic peptide EEREMKALSERVSIL that corresponds to the COOH-terminal region of PKD as described previously (6, 49). The immune complexes were recovered using protein-A coupled to agarose.

In Vitro Kinase Assays-- Immune complexes were washed twice with lysis buffer and then twice with kinase buffer consisting of 30 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 1 mM dithiothreitol. Autophosphorylation reactions were initiated by combining 20 µl of immune complexes with 5 µl of a phosphorylation mixture containing 100 µM [-³²P]ATP (specific activity, 400-600 cpm/pmol) in kinase buffer. Following incubation at 30 °C for 10 min, the reactions were terminated by addition of 1 ml of ice-cold kinase buffer and placed on ice. Immune complexes were recovered by centrifugation, and the proteins were extracted for SDS-PAGE analysis by addition of 2× SDS-PAGE sample buffer (200 mM Tris-HCl, pH 6.8, 0.1 mM sodium orthovanadate, 1 mM EDTA, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol). Dried SDS-PAGE gels were subjected to autoradiography to visualize radiolabeled protein bands.

For assays of exogenous substrate phosphorylation, immune complexes were processed as for autophosphorylation reactions, and then substrate (syntide-2, final concentration 2.5 mg/ml) was added in the presence of 100 µM [-³²P]ATP (400-600 cpm/pmol) in kinase buffer (final reaction volume, 30 µl). After incubation at 30 °C for 10 min, the reactions were terminated by adding 100 µl of 75 mM H₃PO₄, and 75 µl of the mixed supernatant was spotted to Whatman P-81 phosphocellulose paper. Papers were washed thoroughly in 75 mM H₃PO₄ and dried, and radioactivity incorporated into syntide-2 was determined by detection of Cerenkov radiation in a scintillation counter.

Western Blot Analysis-- Samples of cell lysates were directly solubilized by boiling in SDS-PAGE sample buffer. Following SDS-PAGE on 8% gels (for PKD) or 10% gel (for G proteins), proteins were transferred to Immobilon-P membranes (Millipore), as described previously (9, 23) and blocked by overnight incubation with 5% non-fat dried milk in PBS, pH 7.2. Membranes were incubated at room temperature for 3 h with antisera specifically recognizing either PKD, the different G proteins (G_q or G₁₃), or GFP at 1:250-1:500 dilution in phosphate-buffered saline containing 3% non-fat dried milk. Immunoreactive bands were visualized using either horseradish peroxidase-conjugated anti-rabbit IgG and subsequent enhanced chemiluminescence detection or ¹²⁵I-labeled protein A followed by autoradiography. The G_q antiserum was raised against a synthetic peptide corresponding to the COOH-terminal decapeptide of this G protein that was cross-linked to keyhole limpet hemocyanin with glutaraldehyde. The G₁₃ antiserum was raised against the synthetic peptide CLHDNLKQLMLQ (which corresponds to the carboxyl-terminal peptide 367-377 of murine G₁₃ with an amino-terminal cysteine added for coupling) cross-linked to keyhole limpet hemocyanin with the hetero-bifunctional reagent sulfosuccinimidyl 4-(p-maleimidophenyl) butyrate, as described (52). The antibody for GFP was raised in rabbits to a glutathione S-transferase-GFP fusion protein, as described recently (53).

Materials-- [-³²P]ATP (370 MBq/ml), ¹²⁵I-labeled protein A (15 mCi/ml), horseradish peroxidase-conjugated donkey anti-rabbit IgG, and enhanced chemiluminescence reagents were from Amersham Pharmacia Biotech. Protein-A agarose and Pefabloc were from Roche Molecular Biochemicals. Opti-MEM and Lipofectin were from Life Technologies, Inc. GF I and cytochalasin D were obtained from Sigma. Ro 31-8220 and 1-(5-isoquinolinesulfonyl)-homopiperazine (HA1077) were from Calbiochem. All other reagents were from standard suppliers or as described in the text and were the highest grade commercially available.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Expression of Constitutively Activated Rho Induces PKD Activation-- To test whether PKD activation can be induced by signaling pathway(s) initiated by the low molecular weight G proteins of the Rho subfamily, COS-7 cells were co-transfected with PKD and expression vectors encoding wild type Rho, Ras, Rac or constitutively active forms of the three proteins, RhoQ63L, RasVal12, or RacVal12. PKD was immunoprecipitated from the lysates of transfected cells with PA-1 antiserum, and the immune complexes were incubated with [-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 for the first time that cells co-transfected with RhoQ63L and PKD exhibit a marked increase in PKD activity compared with cells transfected with either PKD and wild type Rho or with PKD alone. Similar results were obtained when PKD activity in immunocomplexes was determined by phosphorylation of syntide-2 (54, 55), a synthetic peptide demonstrated previously (6) to be an excellent substrate for PKD. We verified that the level of PKD expression in cells co-transfected with Rho (either wild type or QL) and PKD was similar to those transfected with PKD (Fig. 1C). In contrast, PKD activity was increased only slightly by overexpression of wild type or constitutively activated mutants of Rac or Ras (Fig. 1, A and B).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   The constitutively activated mutant RhoQ63L (RhoQL) induces PKD activation in COS-7 cells, and the Rho inhibitor C3 toxin blocks this induction. Exponentially growing COS-7 cells were co-transfected with pcDNA3-PKD (PKD) and pcDNA3 or pcDNA3 encoding wild type Rho (Rhowt), Ras (Raswt), and Rac (Racwt) or encoding the constitutively activated mutants of Rho (RhoQL), Ras (RasV12), and Rac (RacV12). Two of these cell dishes were also co-transfected with pEF-LacZ (, pEF) or pEF-C3 (+, C3), as indicated in this figure. Three days after transfection, the cultures were lysed. A, the lysates were immunoprecipitated with PA-1 antiserum, 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 is shown. The position of autophosphorylated PKD at apparent Mr 110,000 is indicated by the arrow to the left. Similar results were obtained in three independent experiments. The bar graph shows the quantification of the level of PKD autophosphorylation in these experiments performed by scanning densitometry. The results expressed as an increased fold over control in phosphorylation are means ± S.E. (n = 3). B, syntide-2 phosphorylation in immune complexes. 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. C, levels of expression of PKD in each transfection were analyzed by Western blotting (W. Blot) aliquots of total cell lysates with antiserum against PKD.

Expression of the C. botulinum C3 toxin, which specifically ADP-ribosylates Rho at residue 41 and impairs its function (46), markedly attenuated PKD activation induced by Rho QL (Fig. 1, A and B, right). Expression of the C3 toxin did not interfere with the expression of PKD (Fig. 1, C, right). The results presented in Fig. 1 indicate that Rho activation is a potential pathway leading to PKD activation.

Low molecular weight G proteins of the Rho subfamily cycle between an inactive form bound to GDP and an active GTP-bound state. Guanine nucleotide exchange factors positively modulate the small GTPases by catalyzing the dissociation of the bound GDP to allow the association of GTP. The lbc oncogene encodes a specific guanine nucleotide exchange factor for Rho and causes cellular transformation through activation of the Rho signaling pathway (45, 46). In order to substantiate the conclusions drawn from data in Fig. 1, we transfected COS-7 cells with PKD either with or without constitutively activated Lbc (pOnco-Lbc), and we examined the effects of Lbc-mediated activation of endogenous Rho on PKD activity in immunoprecipitates. As shown in Fig. 2, expression of pOnco-Lbc (Lbc) induced a significant increase in PKD activity, as shown by assays of either autophosphorylation (Fig. 2A) or syntide-2 phosphorylation (Fig. 2B). PKD activation induced by Lbc was virtually abrogated by co-expression of C. botulinum C3 toxin, confirming that Lbc leads to PKD activation via Rho. Endogenous Rho was sufficient to mediate the effects of Lbc since the expression of wild type Rho with Lbc did not produce a significant further increase in PKD activity. The expression of Lbc with or without C3 toxin did not affect the level of PKD expression (Fig. 2C).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   pOnco-Lbc induces PKD activation in COS-7 cells, and this induction is blocked by the Rho inhibitor C3 toxin. Exponentially growing COS-7 cells were co-transfected with pcDNA3-PKD (PKD) and pSR or pSR encoding pOnco-Lbc (Lbc) or wild type Rho (Rho). Two of these cell dishes were also co-transfected with pEF-LacZ () or pEF-C3 (+), as indicated in this figure. Three days after transfection, the cultures were lysed. A, the lysates were immunoprecipitated with PA-1 antiserum, 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 is shown. The position of autophosphorylated PKD at apparent Mr 110,000 is indicated by the arrow to the left. Similar results were obtained in four independent experiments. The bar graph shows the quantification of the level of PKD autophosphorylation in these experiments performed by scanning densitometry. The results expressed as an increased fold over control in phosphorylation are means ± S.E. (n = 4). B, syntide-2 phosphorylation in immune complexes. 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. C, levels of expression of PKD in each transfection were analyzed by Western blotting (W. Blot) aliquots of total cell lysates with antiserum against PKD.

Aluminum Fluoride Stimulates PKD Activation in COS-7 Cells Transfected with G₁₃-- Recently, the G₁₂ subfamily has been implicated in pathways leading to activation of the low molecular weight G proteins of the Rho subfamily (20-25). Specifically, G₁₃ stimulates the nucleotide exchange activity of p115 GEF for Rho thereby leading to Rho activation (26-28). In order to examine the effect of G₁₃ signaling on PKD activity, we transiently transfected COS-7 cells with vector or wild type G₁₃, as well as G_q, and then stimulated the cells with either 10 µM aluminum fluoride or PDB as a positive control. Aluminum fluoride activates heterotrimeric G proteins due to its ability to mimic the -phosphoryl group of GTP when complexed with the GDP-bound  subunit (56). PKD activity in immunocomplexes was determined by autophosphorylation or by phosphorylation of syntide-2.

As shown in Fig. 3, PKD isolated from unstimulated COS-7 cells had low catalytic activity that was markedly activated by PDB stimulation of intact cells (~10-fold increase). Overexpression of G_q or G₁₃ in COS-7 cells did not induce any detectable PKD activation. Addition of aluminum fluoride to cells transfected with PKD alone failed to induce any significant increase in PKD activity, whereas addition of aluminum fluoride to cells co-transfected with PKD and wild type G_q induced a marked increase in PKD activity, in agreement with our recent results (19). A salient feature of the results shown in Fig. 3 is that aluminum fluoride stimulation of cells transfected with G₁₃ also induced a significant increase in PKD activity in immunocomplexes as measured by autophosphorylation (Fig. 3A) or syntide-2 phosphorylation assays (Fig. 3B). Western blot analysis confirmed that the cells transfected with the G_q or G₁₃ expression plasmids overexpressed these G subunits (Fig. 3C).

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Aluminum fluoride induces PKD activation in COS-7 cells transfected with wild type G13 proteins: selective inhibition by C3 toxin. Exponentially growing COS-7 cells were co-transfected with pcDNA3-PKD (PKD), pcDNA1 encoding wild type G13 (13), or Gq (q), pEF-LacZ, or pEF-C3 (C3). Three days after transfection, the cultures were left unstimulated () or stimulated (+) either with 200 nM PDB for 10 min or with 10 µM aluminum fluoride (10 mM NaF, 10 µM AlCl3) (AlF) for 30 min and lysed. The lysates were immunoprecipitated with PA-1 antiserum, and PKD activity in the immunocomplexes was determined either by autophosphorylation (A, in vitro kinase assay (IVK)) or by phosphorylation of the synthetic peptide syntide-2 (B), as described under "Experimental Procedures." A, the autoradiogram shown is representative of at least three independent experiments. The position of autophosphorylated PKD at apparent Mr 110,000 is indicated by the arrow to the left; the bar graph shows the quantification of the level of PKD autophosphorylation in these experiments performed by scanning densitometry. The results expressed as a percentage of the maximum increase in phosphorylation are means ± S.E. of three independent experiments. B, syntide-2 phosphorylation in immune complexes. The results expressed as an increased fold in phosphorylation represent the mean ± S.E. obtained from three independent experiments, each performed in duplicate. C, levels of expression of G proteins (13 and q) and PKD were analyzed by Western blotting (W. Blot) aliquots of total cell lysates with antisera against 13, q, or PKD. The positions of immunoreactive G subunits at apparent Mr 43,000 and PKD at Mr 110,000 are indicated by the arrows to the left. D, COS-7 cells co-transfected with PKD and wild type Gq (PKD+q) or wild type G13 (PKD+13) were stimulated with 200 nM PDB for 10 min (as a maximum stimulation control) or with 2.5 µM aluminum fluoride (AlF) for 30 min and 1, 4, 8, or 24 h and lysed. The lysates were immunoprecipitated with PA-1 antiserum, and PKD activity in the immunocomplexes was determined by autophosphorylation. The curve graph shows the time course of the level of PKD autophosphorylation in these experiments quantified by scanning densitometry. The results expressed as a percentage of the maximum increase in phosphorylation.

Next, we used C. botulinum C3 toxin to determine whether PKD activation in response to G₁₃ signaling proceeds through a Rho-dependent pathway. As illustrated in Fig. 3, expression of C3 toxin blocked the increase in PKD activity produced by aluminum fluoride stimulation for the cells co-expressing PKD with G₁₃. In contrast, C3 toxin did not prevent the increase in PKD activity induced by treatment with aluminum fluoride of cells co-expressing PKD with G_q. These results strongly indicate that Rho mediates PKD activation induced by aluminum fluoride in G₁₃-transfected cells.

We reported previously (19) (and confirmed in this study) that PKD isolated from COS-7 expressing constitutively activated G₁₂ or G₁₃ did not exhibit increased catalytic activity. In view of the results shown in Fig. 3, we considered the possibility that chronic signaling leading to PKD activation by constitutively activated G₁₃ could be impaired. For example, G proteins including G₁₂ or G₁₃ have been shown to be phosphorylated by phorbol ester-sensitive PKC isoforms (57-59) leading to their desensitization (60). To determine whether PKD is activated in response to acute rather than chronic signaling through G₁₃, COS-7 cells were transfected with vectors encoding wild type G₁₃ or G_q, and after 72 h of incubation, the cultures were challenged with aluminum fluoride for various times (1-24 h). As shown in Fig. 3D, the increase in PKD activity induced by aluminum fluoride in cells transfected with G₁₃ declined gradually reaching almost base-line values after 24 h of continued exposure to aluminum fluoride. In contrast, PKD activation in response to aluminum fluoride-stimulated G_q remained undiminished even after 24 h of treatment. These results indicate that chronic stimulation of G₁₃ signaling results in desensitization of PKD activation mediated by this pathway.

The PKC Inhibitors GF I and Ro 31-8220 Prevent PKD Activation by Aluminum Fluoride in COS-7 Cells Transfected with G₁₃-- A number of recent studies have suggested a convergence between Rho- and PKC-mediated signaling in a variety of cell types (32-37). Consequently, we determined whether PKCs mediate PKD activation induced by G₁₃ activation, using inhibitors that discriminate between PKCs and PKD. COS-7 cells transiently transfected with wild type G₁₃ were treated for 1 h with the potent inhibitors of phorbol ester-sensitive isoforms of PKC GF I (also known as GF 109203X or bisindolylmaleimide I) or Ro 31-8220 (61, 62), prior to stimulation with 10 µM aluminum fluoride. As shown in Fig. 4A, exposure to either GF I or Ro 31-8220 potently blocked PKD activation induced by aluminum fluoride in G₁₃-transfected cells. In contrast, the compound GF V, which is structurally related to GF I but biologically inactive, did not affect PKD activation in response to aluminum fluoride in these cells. Previously, we demonstrated (9, 11) that either GF I or Ro 31-8220 does not inhibit PKD activity when added directly to the in vitro kinase assay at concentrations identical to those required to block PKD activation by aluminum fluoride in G₁₃-transfected COS-7 cells. Thus, the results shown in Fig. 4 imply that GF I and Ro 31-8220 do not inhibit PKD activity directly but interfere with G₁₃-mediated PKD activation in intact COS-7 cells by blocking PKC.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   PKD activation induced by aluminum fluoride-stimulated G13, RhoQL, or pOnco-Lbc (Lbc) is prevented by treatment with PKC inhibitors. Exponentially growing COS-7 cells were co-transfected with pcDNA3-PKD and pcDNA1-G13 or RhoQL or Lbc and used in the experiment 3 days after transfection. The transfected cultures were incubated with the selective PKC inhibitors GF 109230X (GF 1; 3.5 µM), Ro 31-8220 (Ro; 2.5 µM) or with other inhibitors including HA1077 (HA, 20 µM) and cytochalasin D (CytD, 2 µM) for 1 (for G13-transfected cells) or 2 h (for RhoQL- or Lbc-transfected cells), and control cells received an equivalent amount of solvent () or GF V (3.5 µM), an inactive analogue of GF 1. The cultures were subsequently left unstimulated (open bars) or stimulated (closed bars) with 10 µM aluminum fluoride (AlF) for 30 min and lysed. The lysates were immunoprecipitated with PA-1 antiserum, and PKD activity in the immunocomplexes was determined by autophosphorylation (in vitro kinase assay (IVK)) or by phosphorylation of syntide-2, as described under "Experimental Procedures." A and B, upper panels, the autoradiograms shown are representative of at least three independent in vitro kinase assay experiments with similar results; lower panels, syntide-2 phosphorylation in immune complexes. Results represent the mean ± S.E. from three independent experiments, each performed in duplicate.

To substantiate further that PKD activation induced through the G₁₃/Rho signaling pathway is mediated by PKC, we tested whether PKC inhibitors also inhibit PKD activation induced by either expression of pOnco-Lbc (Lbc) or RhoQL. COS-7 cells transiently transfected with wild type PKD and either Lbc or RhoQL were incubated with GF I/Ro 31-8220 or GF V. As shown in Fig. 4B, exposure to either GF I or Ro 31-8220 potently blocked PKD activation induced by expression of either Lbc or RhoQL. In contrast, addition of GF V did not affect PKD activation in response to Lbc or RhoQL.

The Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) family composed of ROCK (also known as Rho kinase or ROCK-II) and the closely related p160ROCK (also known as ROCK-I) has been identified as one of the downstream targets of Rho-GTP (63-65) that transduce Rho activation into cytoskeletal responses (66, 67). The ROCKs have also been implicated in PtdIns(4,5)P₂ generation (41) and PLD activation (43) which potentially could lead to Rhodependent PKC activation. The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077) has been recently identified as a potent inhibitor of ROCK (68). As shown in Fig. 4, A and B, treatment with 20 µM HA-1077 did not prevent PKD activation in response to aluminum fluoride-stimulated G₁₃, RhoQL, or Lbc. Similarly, disruption of the actin cytoskeletal organization in response to cytochalasin D did not interfere with PKD activation produced by aluminum fluoride-stimulated G₁₃, RhoQL, or Lbc. All these results indicate that stimulation of G₁₃/Rho signaling promotes PKD activation through a PKC-dependent but ROCK-independent pathway.

Substitution of Ser-744 and Ser-748 by Alanine Prevents PKD Activation in Response to G₁₃-- The residues Ser-744 and Ser-748 in the activation loop of PKD are required for PKC-dependent PKD activation induced by phorbol esters (14), oxidative stress (69), and bombesin (19). If Ser-744 and Ser-748 are target sites for activating phosphorylation events in response to G₁₃ signaling, their conversion to Ala should reduce or eliminate G₁₃-mediated activation of PKD. To test this possibility we used PKD mutants with single or double substitutions of these residues cloned in the expression vector pcDNA3 (i.e. PKD-S744A, PKD-S748A, or PKD-S744A/S748A). COS-7 cells, co-transfected with wild type PKD or PKD mutants and G₁₃, were treated with or without aluminum fluoride. PKD kinase activity was measured by either autophosphorylation or syntide-2 phosphorylation.

As shown in Fig. 5, PKD isolated from unstimulated cells had low catalytic activity that was markedly activated by aluminum fluoride-stimulated G₁₃. Single substitutions of either Ser-744 or Ser-748 by Ala resulted in PKD mutants that displayed reduced activity after stimulation (50% decrease in both single Ala mutants compared with stimulated wild type PKD). In contrast, substitution of both Ser-744 and Ser-748 for Ala in PKD completely blocked kinase activation induced by aluminum fluoride-stimulated G₁₃ (Fig. 5, A and B). In all cases, the protein expression levels of the transfected PKD mutants were comparable to that of wild type PKD, as shown by Western blot analysis (Fig. 5C). Thus, substitution of Ser-744 and Ser-748 in the activation loop of PKD by neutral non-phosphorylatable residues prevents the activation of this enzyme by aluminum fluoride-stimulated G₁₃ in vivo.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Substitution of Ser744 and Ser748 by alanine prevents PKD activation in response to G13. Exponentially growing COS-7 cells were co-transfected with pcDNA1 encoding wild type G13 (13) and wild type PKD (PKD) or different activation loop mutants PKD-S744A (S744A), PKD-S748A (S748A), PKD-S744A/S748A (S744A/S748A), or PKD-S744E/S748E (S744E/S748E). Three days after transfection, the cultures were unstimulated () or stimulated (+) with 10 µM aluminum fluoride ( AlF) for 30 min and lysed. The lysates were immunoprecipitated with PA-1 antiserum, and PKD activity was determined by in vitro kinase assay (IVK) as described under "Experimental Procedures." A, the autoradiogram of in vitro kinase assay shown is representative of at least three independent experiments. The position of autophosphorylated PKD at apparent Mr 110,000 is indicated by the arrow to the left; the bar graph shows the quantification of the level of PKD autophosphorylation in these experiments performed by scanning densitometry. The results expressed as a percentage of the maximum increase in phosphorylation are means ± S.E. of three independent experiments. B, syntide-2 phosphorylation in immune complexes. 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. C, levels of expression of wild type PKD and the different PKD mutants were analyzed by Western blotting (W. Blot) aliquots of total cell lysates with PKD antiserum.

As shown in Fig. 5, A and B, and in agreement with previous results (14), replacement of both serine residues with glutamic acid (PKD-S744E/S748E) markedly increased PKD basal activity. Interestingly, the activity of the PKD-S744E/S748E mutant was not significantly further enhanced by aluminum fluoride-stimulated G₁₃, suggesting that phosphorylation of these two sites induces maximal PKD activation in response to these pathways.

Role of Endogenous G₁₃ in Mediating PKD Activation in Response to Bombesin Receptor Activation-- The COOH terminus of G proteins plays a key role in their interaction with cognate receptors (70). Recently, peptides corresponding to this region of G_q or G_i have been shown to target the receptor-G protein interface in a selective manner and thereby block receptor-mediated PLC activation (71) and inwardly rectifying K⁺ channel activity (72, 73), respectively. For example, transient transfection of COS-7 cells with _1B-adrenergic receptors or M₁ muscarinic receptors and the COOH-terminal region of G_q attenuated inositol phosphate production in response to receptor activation (71).

In the present study, a dominant-negative strategy was also used to test the role of endogenous G₁₃ in bombesin receptor-mediated PKD activation. We generated chimeric fusion proteins between the COOH-terminal region of G₁₃ (referred as G₁₃CT) and GFP from Aequorea victoria, which forms an independent 30-kDa domain with inherent fluorescence (51). Initially, we verified that the GFP-G₁₃CT chimera is expressed in transiently transfected COS-7 cells as judged by Western blot analysis using antibodies directed against either GFP or the COOH-terminal region of G₁₃ (Fig. 6A). In addition, we also visualized the expression of the GFP-G₁₃CT chimera by examining GFP fluorescence in individual COS-7 cells (results not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 6.   The COOH-terminal region of G13 (13CT) prevents PKD activation in response to bombesin. Exponentially growing COS-7 cells were co-transfected with pcDNA3 encoding GFP (GFP) or GFP-13CT (GFP-13CT), BNR-pCD2 (BR) containing the cDNA encoding the bombesin/gastrin-releasing peptide receptor, and pcDNA3-PKD (PKD). Three days after transfection, the cultures were left unstimulated () or stimulated (+) with either 200 nM PDB or 10 nM bombesin (Bom) for 10 min and lysed. The lysates were immunoprecipitated with PA-1 antiserum, and PKD activity in the immunocomplexes was determined by autophosphorylation (in vitro kinase assay (IVK), B) as described under "Experimental Procedures." A, levels of expression of GFP-13CT were analyzed by Western blotting (W. Blot) aliquots of transfected cell lysates with either G13 or GFP antibodies, as indicated. B, the autoradiogram of in vitro kinase assay shown is representative of four independent experiments with similar results; the bar graph is quantification of the level of PKD phosphorylation performed by scanning densitometry. The results expressed as a percentage of the maximum increase in phosphorylation are mean ± S.E. of four independent experiments.

Next, we determined whether expression of GFP-G₁₃CT interferes with PKD activation via the bombesin receptor. COS-7 cells were co-transfected with PKD, bombesin receptor, and either GFP-G₁₃CT or GFP. After 72 h, the cells were challenged with either bombesin or PDB for 10 min and then lysed. PKD activity, after immunoprecipitation, was assayed by autophosphorylation. The results illustrated in Fig. 6B demonstrate that expression of GFP-G₁₃CT markedly attenuated the increase of PKD activity induced by bombesin. In contrast, expression of GFP-G₁₃CT did not interfere with PKD activation in response to PDB which directly stimulates PKC leading to PKD activation and therefore bypasses the receptor/G protein interaction. These results indicate that endogenous G₁₃ contributes to PKD activation in response to bombesin receptor activation.

Role of Endogenous G_q and Rho in Mediating PKD Activation in Response to Bombesin Receptor Activation-- In order to determine the contribution of Rho-dependent pathways to PKD activation in response to bombesin GPCR stimulation, we co-transfected COS-7 cells with expression vectors encoding bombesin GPCR and PKD with or without a C3 toxin expression vector. After 72 h, the cells were challenged with either bombesin or PDB for 10 min and lysed, and PKD activity, after immunoprecipitation, was assayed by autophosphorylation or syntide-2 phosphorylation. As illustrated in Fig. 7 (A and B), expression of C3 toxin attenuated the increase in PKD activity produced by bombesin but did not interfere with PKD activation promoted by PDB. These results indicate that maximal bombesin-induced PKD activation requires functional Rho.

View larger version (52K): [in this window] [in a new window]   Fig. 7.   PKD activation in response to bombesin is further abolished by the combination of Rho inhibitor C3 toxin with the COOH-terminal region of Gq (qCT). Exponentially growing COS-7 cells were co-transfected with pcDNA3-PKD (PKD), BNR-pCD2 (BR) containing the cDNA encoding the bombesin/gastrin-releasing peptide receptor, pEF-LacZ (pEF), or pEF-C3 (C3), and pcDNA3 encoding GFP (GFP) or GFP-qCT (GFP-qCT), as indicated. Three days after transfection, the cultures were left unstimulated () or stimulated (+) with either 200 nM PDB or 10 nM bombesin (Bom) for 10 min and lysed. The lysates were immunoprecipitated with PA-1 antiserum, and PKD activity in the immunocomplexes was determined by autophosphorylation (in vitro kinase assay (IVK), A) or by phosphorylation of syntide-2 (B) as described under "Experimental Procedures." A, the autoradiogram of in vitro kinase assay shown is representative of at least three independent experiments with similar results. The bar graph shows the quantification of the level of PKD autophosphorylation in these experiments performed by scanning densitometry. The results expressed as a percentage of the maximum increase in phosphorylation are mean ± S.E. of three independent experiments. B, syntide-2 phosphorylation in immune complexes. Results expressed as an increased fold in phosphorylation represent the mean ± S.E. from three experiments, each performed in duplicate. C, upper panel, levels of expression of GFP-qCT were analyzed by Western blotting (W. Blot) aliquots of transfected cell lysates with either Gq or GFP antibodies, as indicated; bottom panel, levels of expression of PKD were analyzed by Western blotting aliquots of total cell lysates with PKD antiserum.

Recently, we reported (19) that expression of a chimeric fusion protein between the COOH-terminal region of G_q (referred as G_qCT) and GFP attenuated bombesin-induced PKD activation. Here, we determined whether expression of C3 toxin together with GFP-G_qCT interferes with PKD activation induced by bombesin in an additive manner. COS-7 cells were co-transfected with PKD, bombesin receptor, and GFP-G_qCT with or without C3 toxin. In agreement with previous results, expression of GFP-G_qCT (but not GFP) attenuated the increase of PKD activity induced by bombesin (Fig. 7, A and B).

The salient feature of the results presented in Fig. 7 is that co-transfection of C3 toxin together with GFP-G_qCT almost abolished bombesin-induced PKD activation. In contrast, expression of GFP-G_qCT together with C3 toxin did not interfere with PKD activation in response to PDB which directly stimulates PKC. We verified that the GFP-G_qCT chimera is expressed in transiently transfected COS-7 cells as well as in cells co-transfected with GFP-G_qCT and C3 toxin, as judged by Western blot analysis using antibodies directed against either GFP or the COOH-terminal region of G_q (Fig. 7C, upper panel). In addition, we also confirmed that protein expression levels of PKD were comparable under all the experimental conditions used (Fig. 7C, lower panel). Taken together, the results presented in Figs. 6 and 7 indicate that endogenous G₁₃, G_q, and Rho mediate PKD activation in response to bombesin receptor activation.

Concluding Remarks-- Activation of a number of receptors that couple to heterotrimeric G proteins including bombesin, bradykinin, endothelin, vasopressin, and lysophosphatidic acid have been shown to stimulate PKD activation in a variety of cell types. Although each of these receptors activates G_q and G_q signaling stimulates PKD activity (Ref. 19 and results presented here), the kinetics and degree of PKD activation differ among the different receptors even in the same cell line. Some of the variation may be the result of receptors coupling to more than one G protein initiating additional signaling pathways that can also contribute to PKD activation. In this context, it is also relevant that expression of a COOH-terminal fragment of G_q that acts in a dominant-negative fashion attenuated (but did not eliminate) PKD activation in response to agonist stimulation of bombesin receptor (Ref. 19 and results presented here). These considerations raised the possibility that GPCRs promote PKD activation not only via G_q but also through other, as yet unidentified, G protein signaling pathways.

Many G_q-coupled receptors also interact with heterotrimeric G proteins of the G₁₂ family that are known to promote Rho activation via the novel guanine nucleotide exchange factor p115 GEF which directly links G₁₃ to Rho (26, 27). It is increasingly recognized that Rho and PKC signaling interact in a variety of cell types (see Introduction for references). In particular, recent studies demonstrated that Rho-GTP potently stimulates PKC activity in vitro (36) and that G_q and phospholipase C signaling are synergistic with Rho in vivo (37). Consequently, we examined in this study whether Rho- and G₁₃-mediated signaling can promote PKD activation in intact cells and whether endogenous Rho could mediate PKD activation in response to bombesin receptor stimulation.

Our results demonstrate that PKD immunoprecipitated from COS-7 cells transiently transfected with either a constitutively active mutant of Rho (RhoQ63L) or the Rho-specific guanine nucleotide exchange factor Lbc exhibited a marked increase in basal activity. These findings demonstrate, for the first time, that Rho-regulated signaling leads to PKD activation. Furthermore, addition of aluminum fluoride to cells co-transfected with PKD and wild type G₁₃ also induced a marked increase in PKD activity. Transfection of C. botulinum C3 toxin specifically blocked activation of PKD by RhoQ63L, Lbc, or by aluminum fluoride-stimulated G₁₃. In contrast, expression of the C3 toxin did not interfere with PKD activation induced through G_q. These results imply that, under our experimental conditions, the expression of the C3 toxin did not restrict the supply of PtdIns(4,5)P₂ necessary for PLC-mediated production of DAG. These results indicate that G₁₃ leading to Rho activation is a potential signaling pathway that mediates PKD activation.

PKD activation in response to either G₁₃ or RhoQL and pOnco-Lbc signaling is prevented by treatment with selective PKC inhibitors. PKD activation in response to G₁₃ signaling is also prevented by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Furthermore, aluminum fluoride-stimulated G₁₃ did not induce a further increase in PKD activity when Ser-744 and Ser-748 were mutated to Glu to mimic the phosphorylated residues. These data suggest that G₁₃ signaling, like bombesin receptor activation, G_q, and PDB, leads to PKD activation through phosphorylation of Ser-744/748 in the activation loop of PKD through a PKC-dependent pathway.

Dominant-negative strategies to uncouple heptahelical receptors from their cognate G proteins have received much attention, but only recently has it been shown that expression of the COOH-terminal region of G proteins can competitively inhibit receptor-G protein interaction (71, 73). For example, expression of the last 55 amino acids of G_q has been shown to target the receptor-G_q interface in a selective manner and thereby block receptor-mediated PLC activation in cultured cells and in transgenic mice (71). In agreement with these recent results, we reported previously (19) that expression of a COOH-terminal fragment of G_q that acts in a dominant-negative fashion attenuated (but did not eliminate) PKD activation in response to bombesin. In the present study, we demonstrate, for the first time, that expression of the COOH-terminal region of G₁₃ markedly reduces PKD activation in response to bombesin receptor stimulation. Furthermore, expression of C. botulinum C3 toxin also attenuated PKD activation in response to bombesin, implying that G₁₃/Rho signaling contributes to GPCR-induced PKD activation. Consistent with this notion, expression of the COOH-terminal region of G_q together with the C. botulinum C3 toxin, which inactivates Rho, virtually abolished PKD activation in response to bombesin receptor stimulation. In contrast, expression of the COOH-terminal region of G_q together with the C3 toxin did not interfere with PKD activation in response to PDB which bypasses receptor pathways and directly activates classic and novel PKCs. These results support a model in which bombesin GPCR stimulation induces PKD activation through both G_q and G₁₃/Rho signaling pathways.

In conclusion, our results demonstrate that G₁₃ contributes to PKD activation through a Rho- and PKC-dependent signaling pathway and indicate that PKD activation is mediated by both G_q and G₁₃ in response to acute bombesin receptor stimulation, as summarized in the scheme shown in Fig. 8. The findings presented here identify PKD as a novel downstream target in G₁₃ and Rho signaling.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Signal transduction pathways involved in PKD activation in response to bombesin stimulation. The scheme illustrates the molecular and pharmacological approaches used in this study. PKD activation in response to acute bombesin receptor signaling is mediated by both Gq and G13 through PKC. As shown in this pathway, PKD can be activated () by aluminum fluoride (AlF, stimulator of G13 or Gq) or pOnco-Lbc (Lbc, active form of GEF) or RhoQL (constitutively active mutant of Rho); PKD activation could be blocked (|) by COOH-terminal fragments of G13 (13CT) or Gq (qCT) (competitive inhibitors of receptor G protein interaction), by C3 toxin (C3, specific inhibitor of Rho) or GF1 or Ro 31-8220 (Ro) (selective inhibitors of PKC). Mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD prevents PKD activation through the G12 and Gq pathways. Rho kinase (ROCK) inhibitor, HA1077 (HA), and disruption of the actin cytoskeleton with cytochalasin D (CytD) do not interfere with PKD activation in response to bombesin. (Also see the text for details and abbreviations.)

	FOOTNOTES

^* This work was supported in part by National Institutes of Health 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 NRSA F32 CA84658-01A1 from the National Institutes of Health.

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

Published, JBC Papers in Press, August 15, 2001, DOI 10.1074/jbc.M105530200.

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; DAG, diacylglycerol; FBS, fetal bovine serum; G proteins, guanine nucleotide-binding regulatory proteins; GPCRs, G protein-coupled receptors; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; PDB, phorbol 12,13-dibutyrate; PKD, protein kinase D; PtdIns(4, 5)P₂, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES