HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yuan, J. Articles by Rozengurt, E. Search for Related Content PubMed PubMed Citation Articles by Yuan, J. Articles by Rozengurt, E. Social Bookmarking                      What's this?

J Biol Chem, Vol. 275, Issue 3, 2157-2164, January 21, 2000

Activation of Protein Kinase D by Signaling through the  Subunit of the Heterotrimeric G Protein G_q^*

Jingzhen Yuan, Lee Slice, John H. Walsh, and Enrique Rozengurt^§

From the Department of Medicine, School of Medicine, the CURE Digestive Diseases Research Center, and Molecular Biology Institute, UCLA, Los Angeles, California 90095

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Protein kinase D (PKD/PKCµ) immunoprecipitated from COS-7 cells transiently transfected with a constitutively active  subunit of G_q (G_qQ209L) exhibited a marked increase in basal activity, which was not further enhanced by treatment of the cells with phorbol 12,13-dibutyrate. In contrast, transient transfection of COS-7 cells with activated G₁₂Q229L or G₁₃Q226L neither promoted PKD activation nor interfered with the increase of PKD activity induced by phorbol 12,13-dibutyrate. The addition of aluminum fluoride to cells co-transfected with PKD and wild type G_q induced a marked increase in PKD activity, which was comparable with that induced by expression of G_qQ209L. Treatment with the protein kinase C inhibitor GF I or Ro 31-8220 prevented the increase in PKD activity induced by aluminum fluoride. Expression of a COOH-terminal fragment of G_q that acts in a dominant negative fashion attenuated PKD activation in response to agonist stimulation of bombesin receptor. PKD activation in response to either G_q or bombesin was completely prevented by mutation of Ser⁷⁴⁴ and Ser⁷⁴⁸ to Ala in the kinase activation loop of PKD. Our results show that G_q activation is sufficient to stimulate sustained PKD activation via protein kinase C and indicate that the endogenous G_q mediates PKD activation in response to acute 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 of a wide range of biological responses, including changes in cell morphology, differentiation, and proliferation (1, 2). Molecular cloning has demonstrated the presence of multiple related 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 structural, enzymological and regulatory properties distinct from other members of the PKC family (8). For example, the catalytic domain of PKD is distantly related to Ca²⁺-regulated kinases, and the regulatory region of this kinase contains a putative trans-membrane domain, contains a pleckstrin homology domain that regulates enzyme activity (9, 10), and lacks a sequence with homology to a typical PKC autoinhibitory pseudosubstrate motif (6, 7). In particular, PKD is rapidly activated in intact cells through a mechanism that involves phosphorylation (8). 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 (9, 11-14). Several lines of evidence generated by using PKC-specific inhibitors and co-transfection of PKD with constitutively active PKC mutants suggest that PKD is activated by phosphorylation through a novel PKC-dependent signal transduction pathway in vivo (10-13). The residues Ser⁷⁴⁴ and Ser⁷⁴⁸ in the activation loop of PKD have been identified as critical phosphorylation sites in PKD activation induced by phorbol esters (15, 16).

Recently, we reported that PKD is rapidly activated by a variety of neuropeptide agonists, including bombesin, bradykinin, endothelin, and vasopressin, that signal through heptahelical receptors coupled to heterotrimeric G proteins (13, 14, 17). Although each of these receptors couples to G_q (18) and thereby to phospholipase C (PLC) (19), these data do not define G_q as a mediator of PKD activation, because these receptors also couple to other heterotrimeric G proteins including members of the G₁₂ family that have been recently implicated in pathways leading to PKC activation (20-23). In order to clarify the G protein pathways leading to PKD activation, we examined whether G_q-mediated signaling is sufficient to promote PKD activation in intact cells and whether endogenous G_q mediates PKD activation in response to bombesin receptor stimulation.

The results presented here demonstrate that either mutationally activated or aluminum fluoride-stimulated G_q induces striking PKD activation through a PKC-dependent pathway. Expression of a COOH-terminal fragment of G_q that acts in a dominant negative fashion attenuated PKD activation in response to agonist stimulation of bombesin receptor. PKD activation in response to either G_q or bombesin is completely prevented by mutation of Ser⁷⁴⁴ and Ser⁷⁴⁸ to Ala in the kinase activation loop of PKD. Our results indicate that G_q activation is sufficient to stimulate sustained PKD activation via PKC and show that the endogenous G_q mediates PKD activation in response to acute bombesin receptor stimulation.

	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% fetal bovine serum at 37 °C in a humidified atmosphere containing 10% CO₂. For experimental dishes, cells were subcultured at 6 × 10⁴ cells/ml in 6-cm (5-ml) or 10-cm (10-ml) dishes on the day prior to transfections. All transfections and cotransfections were carried out with equivalent amounts of DNA (6 µg/6-cm dish, 12 µg/10-cm dish). Transfections were carried out in Opti-MEM (Life Technologies, Inc.) using Lipofectin (Life Technologies) 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 fetal bovine serum for 5-6 h or overnight, and then fetal bovine serum (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 constitutively active mutant murine G_qQ209L (G_qQL) and wild type G_q (G_qwt) subunit cDNAs in the eukaryotic expression vector pcDNA-1 (Invitrogen) (24) were obtained from the American Type Tissue Collection (Manassas, VA). The murine G₁₂ subunit cDNAs in pcDNA-1 were gifts from Dr. H. R. Bourne (University of California at San Francisco) and included the constitutively active mutants G₁₂-Q229L and G₁₃-Q226L (G₁₂QL and G₁₃QL) (25). The constructs pcDNA3-PKD, encoding PKD (26); pcDNA3-PKD/K618M, encoding kinase-deficient mutant PKD (11); and pcDNA3-PKD mutants encoding PKD with site-specific mutations within the activation loop in the catalytic domain including the single mutants (PKD-S744A, PKD-S748A, and PKD-D733A) and double or triple mutants (PKD-S744/S748A, PKD-S744/S748E, and PKD-D733A/S744/S748E) have been described previously (15). BNR-pCD2, containing the cDNA encoding the bombesin/GRP receptor, was kindly provided by Dr. Jim Battey (Laboratory of Molecular Biology, NIDCD, National Institutes of Health, Bethesda, MD).

Polymerase chain reaction was used to generate DNA encoding for the carboxyl-terminal region of G_q (residues 305-359) using the murine G_q cDNA as a template with sense (5'-GCTCAAGCTTCGGCTCGAGAATTCATCCTGAAAATG-3') and antisense (5'-GGTGGATCCTTAGACCAGATTGTACTCCTTCAG-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 into pGFP-C1 (CLONTECH, Inc., La Jolla, CA) such that the resulting fusion protein produced by this plasmid would be a hybrid GFP containing G_q (residues 305-359) 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)-bengenesulfonyl 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, 26). 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 the 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 the addition of 2× SDS-PAGE sample buffer (200 mM Tris/HCl, pH 6.8, 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% gels (for G proteins), proteins were transferred to Immobilon-P membranes (Millipore Corp.), as described previously (11, 27) and blocked by overnight incubation with 5% nonfat dried milk in phosphate-buffered saline, pH 7.2. Membranes were incubated at room temperature for 3 h with antisera specifically recognizing either PKD, the different G proteins (G_q, G₁₂, or G₁₃), or GFP at a 1:250-1:500 dilution in phosphate-buffered saline containing 3% nonfat 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, which 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 NH₂-terminal cysteine added for coupling) cross-linked to keyhole limpet hemocyanin with the heterobifunctional reagent sulfosuccinimidyl 4-(p-maleimidophenyl) butyrate, as described (28). The antibody for GFP was raised in rabbits to a GST-GFP fusion protein, as recently described (29).

Materials-- [-³²P]ATP (370 MBq/ml), ³²P_i (10 mCi/ml), ¹²⁵I-labeled protein A (15 mCi/ml), horseradish peroxidase-conjugated donkey anti-rabbit Ig, enhanced chemiluminescence reagents, and glutathione-Sepharose were from Amersham Pharmacia Biotech. Protein A-agarose and Pefabloc were from Roche Molecular Biochemicals. Rabbit anti-G₁₂ was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Opti-MEM and Lipofectin were from Life Technologies. GF I was obtained from Sigma. Ro 31-8220 was from Calbiochem. All other reagents were from standard suppliers or as described and were the highest grade commercially available.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

G_qQL Induces PKD Activation in COS-7 Cells-- Mutations in the catalytic domain of G subunits that inhibit their intrinsic GTPase activity are known to convert these proteins into constitutively active  subunits (30). To examine the effects of G subunits on PKD activation, COS-7 cells were transiently co-transfected with expression plasmids encoding wild type PKD and constitutively active G mutants G_qQ209L, G₁₂Q229L, and G₁₃Q226L, which are deficient in GTPase activity (24, 25, 31). PKD was immunoprecipitated from the lysates of transfected cells, 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.

As shown in Fig. 1A, PKD isolated from unstimulated COS-7 cells had low catalytic activity that was markedly activated by PDB stimulation of intact cells (~10-fold increase). In contrast, PKD immunoprecipitated from COS-7 cells overexpressing constitutively active mutant G_qQL exhibited a marked increase in basal activity, which was not further enhanced by treatment of the cells with PDB. Transient transfection of COS-7 cells with activated G₁₂QL or G₁₃QL expression plasmids neither promoted PKD activation nor interfered with the increase of PKD activity induced by PDB. Similarly, overexpression of wild-type G_q (Fig. 2) or G₁₂ and G₁₃ (results not shown) in COS-7 cells did not induce PKD activation. Western blot analysis confirmed that the cells transfected with the G_qQL, G₁₂QL, or G₁₃QL expression plasmids overexpressed these G subunits and verified the expression of PKD under all these conditions (Fig. 1B). These results suggest that PKD activation in response to G_qQL expression is specific for the activated state of this G subunit.

View larger version (48K): [in this window] [in a new window]   Fig. 1.   The constitutively activated mutant GqQ209L (qQL) induces PKD activation in COS-7 cells. Exponentially growing COS-7 cells were co-transfected with pcDNA3-PKD (PKD) and pcDNA1 or pcDNA1 encoding the constitutively activated mutant of Gq (qQL), constitutively activated mutant of G12 (12QL), or constitutively active mutant of G13 (13QL). The control cells were transfected with pcDNA3 and pcDNA1. Three days after transfection, the cultures were incubated for 10 min in the absence () or presence (+) of 200 nM PDB and 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 an apparent Mr of 110,000 is indicated by the arrow to the left. Similar results were obtained in five 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 a percentage of the maximum increase in phosphorylation are means ± S.E. (n = 5). B, levels of expression of G subunits and PKD were analyzed by Western blotting (W. Blot) aliquots of total cell lysates with antisera against Gq, G12, G13, or PKD. The positions of immunoactive GqQL, G12QL, and G13QL at apparent Mr of 43,000 and PKD at apparent Mr of 110,000 are indicated by the arrows to the left.

View larger version (47K): [in this window] [in a new window]   Fig. 2.   Aluminum fluoride induces PKD activation in COS-7 cells transfected with wild type Gq protein. Exponentially growing COS-7 cells were co-transfected with pcDNA3-PKD (PKD) and pcDNA1 or pcDNA1 encoding wild type Gq (qwt). The control cells were transfected with pcDNA3 and pcDNA1. Three days after transfection, the cultures were unstimulated () or stimulated with 200 nM PDB for 10 min or with 10 µM aluminum fluoride (10 mM NaF, 10 µM AlCl3) (AlF4) 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, IVK) or by phosphorylation of the synthetic peptide syntide-2 (B), as described under "Experimental Procedures." A, upper panel, the autoradiogram shown is representative of at least three independent experiments. The position of autophosphorylated PKD at apparent Mr of 110,000 is indicated by the arrow to the left; lower panel, levels of expression of wild type Gq subunit (qwt) were analyzed by Western blotting (W. Blot) aliquots of total cell lysates with antiserum against Gq. The position of immunoreactive wild type Gq at apparent Mr of 43,000 is indicated by the arrow to the left. B, syntide-2 phosphorylation in immune complexes. The figures represent the mean ± S.E. obtained from three independent experiments, each performed in duplicate.

Aluminum Fluoride Stimulates PKD Activation in COS-7 Cells Transfected with Wild Type G_q-- An increase in PKD activity in response to expression of constitutive active G_q could be mediated by mechanisms arising from long term activation of G protein-regulated pathways, e.g. secreted factors that activate cellular receptors in an autocrine manner or alteration in the levels of regulators of G protein function. To assess this possibility, we examined G protein signaling in an acutely regulated system.

Aluminum fluoride activates heterotrimeric G proteins due to its ability to mimic the -phosphoryl group of GTP when complexed with the GDP-bound  subunit (32). We transiently transfected COS-7 cells with vector or wild type G_q (rather than the constitutively active form) and then stimulated the cells with either 10 µM aluminum fluoride or PDB, as a positive control. PKD activity in immunocomplexes was determined by autophosphorylation or by phosphorylation of syntide-2 (33, 34), a synthetic peptide previously demonstrated to be an excellent substrate for PKD (6). As shown in Fig. 2, the addition of aluminum fluoride to cells co-transfected with PKD and wild type G_q induced a marked increase in PKD activity, which was comparable with that induced by expression of G_qQL. Western blot analysis confirmed that the cells transfected with the G_q expression plasmid overexpressed this G subunit (Fig. 2A). In contrast, the addition of aluminum fluoride to cells transfected with PKD in the absence of G_q failed to induce any significant increase in PKD activity. Thus, acute stimulation of G_q by aluminum fluoride substantiated the conclusion that G_q activation leads to PKD activation.

To verify that the kinase activity induced by either expression of constitutively activated G_q or by treatment with aluminum fluoride of cells transfected with wild type G_q was due to PKD rather than to the presence of a co-precipitating protein kinase, we examined G_q-induced PKD activation in cells transfected with wild type PKD or with a kinase-deficient PKD mutant (PKD K618M) in which lysine 618 in the ATP binding site is substituted by methionine (11). Fig. 3 shows that expression of constitutively activated G_q or treatment with aluminum fluoride did not induce detectable kinase activity when COS-7 cells were transfected with PKD K618M, as judged by autophosphorylation or by syntide-2 phosphorylation assays. Western blot analysis verified that the expression levels of wild type PKD and PKD K618M were similar and illustrated that stimulation with aluminum fluoride or expression of constitutively activated G_q induced a mobility shift of wild type (but not kinase-deficient) PKD (Fig. 3A). These results demonstrate that the G_q-induced kinase activity measured in PKD immunoprecipitates is due to the activation of PKD rather than to co-immunoprecipitating kinases.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   GqQL and aluminum fluoride-stimulated Gq do not induce kinase activity in immunocomplexes of PKD kinase-deficient mutant (PKDK618M). Exponentially growing COS-7 cells were cotransfected with either pcDNA3-PKD (PKD) or pcDNA3-PKDK618M (PKDK618M) and pcDNA1-GqQL (qQL) or pcDNA1-Gq wild type (qwt). The control cells were transfected with pcDNA3 and pcDNA1. Three days after transfection, the cultures were left unstimulated () or stimulated with 10 µM aluminum fluoride (AlF4) for 30 min and lysed. The lysates were immunoprecipitated with PA-1 antiserum, and PKD activity in the immunocomplexes was determined by autophosphorylation. (A, IVK) or by phosphorylation of the synthetic peptide syntide-2 (B), as described under "Experimental Procedures." A, upper panel, the autoradiogram shown is representative of at least three independent experiments; lower panel, levels of expression of PKD and PKDK618M were analyzed by Western blotting (W. Blot) aliquots of total cell lysates with PA-1 antiserum. B, syntide-2 phosphorylation in immune complexes. Results represent the mean ± S.E. from four independent experiments, each performed in duplicate.

Aluminum Fluoride Stimulates PKD Phosphorylation in COS-7 Cells Transfected with Wild Type G_q-- The preceding experiments demonstrated that mutationally activated or aluminum fluoride-stimulated G_q increased PKD autophosphorylation in in vitro kinase assays. We next examined whether G_q activation induces PKD phosphorylation in intact cells. COS-7 cells transfected with PKD, PKD, and G_q or vectors (pcDNA3 and pcDNA1, as indicated in Fig. 4) were metabolically labeled with ³²P_i and then stimulated with 10 µM aluminum fluoride or 200 nM PDB. Cells were lysed, and PKD was immunoprecipitated with PA-1 antiserum and analyzed by SDS-PAGE and autoradiography. As shown in Fig. 4, aluminum fluoride induced PKD phosphorylation in COS-7 cells transfected with PKD and G_q but did not produce any detectable effect in cells transfected with PKD alone. As a control, we verified in parallel cultures that PDB induced PKD phosphorylation in cells transfected with PKD either with or without G_q. These results indicate that stimulation of G_q by aluminum fluoride induces PKD phosphorylation in intact cells.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Aluminum fluoride stimulates PKD phosphorylation in COS-7 cells transfected with wild type Gq protein. Exponentially growing COS-7 cells were co-transfected with pcDNA3-PKD (PKD) and pcDNA1 or pcDNA1 encoding wild type Gq (qwt). The control cells were transfected with pcDNA3 and pcDNA1. Three days after transfection, cells were washed twice with Pi-free Dulbecco's modified Eagle's medium, incubated in this medium for 30 min, and metabolically labeled with carrier-free 32Pi (200 µCi/ml) for 5 h. At the end of this labeling period, the cultures were left untreated () or stimulated (+) with either 200 nM PDB for 10 min or with 10 µM aluminum fluoride (AlF4) for 30 min and lysed. The lysates were immunoprecipitated with PA-1 antiserum, and the immune complexes were washed with lysis buffer and eluted in 2× SDS-PAGE sample buffer by boiling for 10 min. The supernatants were analyzed by SDS-PAGE and autoradiography as described under "Experimental Procedures." The Mr 110,000 band corresponding to phosphorylated PKD is indicated by the arrow. The autoradiogram shown (upper panel) is representative of three independent experiments. Quantification of the level of PKD phosphorylation was performed by scanning densitometry (lower panel). The results expressed as a percentage of the maximum increase in phosphorylation are means ± S.E. of three independent experiments.

The PKC Inhibitors GF I and Ro 31-8220 Prevent PKD Activation by Aluminum Fluoride in COS-7 Cells Transfected with G_q-- Next, we determined whether PKCs mediate PKD activation induced by G_q activation using inhibitors that discriminate between PKCs and PKD (11, 13). COS-7 cells transiently co-transfected with wild type G_q and PKD were treated for 1 h with the PKC inhibitors GF I (also known as GF 109203X or bisindolylmaleimide I) and Ro 31-8220 (35, 36) prior to stimulation with 10 µM aluminum fluoride or PDB. Treatment with either GF I or Ro 31-8220 prevented the increase in PKD activity induced by aluminum fluoride in G_q-transfected COS-7 cells, as shown by autophosphorylation (Fig. 5A) or syntide-2 phosphorylation assays (Fig. 5B). In contrast, GFV, which is structurally related to GF I but biologically inactive, did not affect PKD activation in response to either aluminum fluoride or PDB. Previously, we demonstrated that GF I and Ro 31-8220 do not directly inhibit PKD activity when added to the in vitro kinase assay at concentrations identical to those required to block PKD activation by aluminum fluoride in G_q-transfected COS-7 cells (11, 13). Thus, the results shown in Fig. 5 imply that G_q-mediated PKD activation in intact COS-7 cells is mediated by PKC.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   PKC inhibitors block PKD activation induced by aluminum fluoride in COS-7 cells transfected with wild type Gq protein. Exponentially growing COS-7 cells were co-transfected with pcDNA3-PKD and pcDNA1-Gqwt. Three days after transfection, the cultures were incubated with the selective PKC inhibitors GF 109230X (+GF 1; 3.5 µM) and Ro 31-8220 (+Ro; 2.5 µM) for 1 h, and control cells received an equivalent amount of solvent () or GF V (+GF V; 3.5 µM), an inactive analog of GF1. The cells were subsequently unstimulated () or stimulated (+) with 10 µM aluminum fluoride (AlF4) for 30 min and lysed. The lysates were immunoprecipitated with PA-1 antiserum, and PKD activity in the immunocomplexes was determined by autophosphorylation (A, IVK) or by phosphorylation of syntide-2 (B), as described under "Experimental Procedures." A, the autoradiogram shown is representative of at least three independent experiments with similar results. B, syntide-2 phosphorylation in immune complexes. Results represent the mean ± S.E. from three independent experiments, each performed in duplicate.

Substitution of Ser⁷⁴⁴ and Ser⁷⁴⁸ by Alanine Prevents PKD Activation in Response to G_q-- A critical aspect in the regulation of protein kinases that function in signaling cascades is the phosphorylation of activating residues located in a region spanning the highly conserved sequences DFG (in kinase subdomain VII) and APE (in kinase subdomain VIII) of the kinase catalytic domain termed the "activation loop" or "activation segment" (37, 38). Recently, we identified Ser⁷⁴⁴ and Ser⁷⁴⁸ as activating residues in the activation loop of PKD and demonstrated that these residues are phosphorylated in intact cells in response to PDB stimulation (15). Here, we examined whether these residues are also important in PKD activation in response to G_q activation or bombesin receptor stimulation.

If Ser⁷⁴⁴ and Ser⁷⁴⁸ are critical target sites for activating phosphorylation(s) events in response to G_q, their conversion to alanine should reduce or eliminate G_q-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 either G_qQL (Fig. 6A, upper panel) or G_q (Fig. 6A, lower panel), were treated with or without aluminum fluoride or PDB and lysed. PKD was immunoprecipitated from the extracts with the PA-1 antibody. The immunocomplexes were incubated with [-³²P]ATP and analyzed by SDS-PAGE and autoradiography to determine the level of PKD activity by autophosphorylation.

View larger version (63K): [in this window] [in a new window]   Fig. 6.   Substitution of Ser744 and Ser748 by alanine prevents PKD activation in response to Gq. Exponentially growing COS-7 cells were co-transfected with pcDNA1 encoding constitutively active mutant Gq (qQL) or wild type Gq (qwt) and wild type PKD (PKD) or different activation loop mutant PKD-S744A (S744A), PKD-S748A (S748A), PKD-S744A/PKD-S748A (S744A/S748A), or PKD-S744E/PKD-S748E (S744E/S748E). Three days after transfection, the cultures were unstimulated () or stimulated with 200 nM PDB for 10 min or with 10 µM aluminum fluoride (AlF4) 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. B, 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 PA-1 antiserum.

As shown in Fig. 6, PKD isolated from unstimulated cells transfected with G_q had low catalytic activity that was markedly activated to the same degree by G_qQL, aluminum fluoride-stimulated G_q, or PDB. Substitution of both Ser⁷⁴⁴ and Ser⁷⁴⁸ for Ala in PKD completely blocked kinase activation induced by either mutationally activated or aluminum fluoride-stimulated G_q. Single substitutions of either Ser⁷⁴⁴ or Ser⁷⁴⁸ for 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 all cases, the protein expression levels of the transfected PKD mutants were comparable with that of wild type PKD, as shown by Western blot analysis (Fig. 6B). Thus, alanine substitution of Ser⁷⁴⁴ and Ser⁷⁴⁸ in the activation loop of PKD prevents the activation of this enzyme by G_q in vivo.

Some protein kinases that are activated by phosphorylation in the activation loop can be rendered constitutively active by substitution of the phosphorylated residue(s) for glutamic acid (39). As shown in Fig. 6A and in agreement with recent results (15), replacement of both serine residues with glutamic acid (PKD-S744E/S748E) markedly increased basal activity. Interestingly, the activity of the PKD-S744E/S748E mutant was not further increased by mutationally activated G_q, aluminum fluoride-stimulated G_q, or PDB, suggesting that phosphorylation of these two sites induces maximal PKD activation in response to these pathways. Western blot analysis verified that the expression levels of wild type PKD and constitutive activated PKD mutants were similar (Fig. 6B).

Substitution of Ser⁷⁴⁴ and Ser⁷⁴⁸ by Alanine Prevents PKD Activation in Response to Bombesin-- Bombesin and its mammalian counterpart gastrin-releasing peptide bind to a heptahelical receptor (40, 41) that couples to G_q with high affinity (42, 43) and induces a complex array of early signaling events (44). Previously, we demonstrated that bombesin induces a rapid increase in PKD activity in Swiss 3T3 cells (13). Here, we examined whether bombesin-induced PKD activation requires the phosphorylation of Ser⁷⁴⁴/Ser⁷⁴⁸ in the activation loop.

COS-7 cells transiently co-transfected with bombesin receptor and wild type PKD or PKD-S744A, PKD-S748A, or PKD-S744A/S748A were treated with or without bombesin or PDB for 10 min and then lysed. PKD activity in the immune complexes was measured by autophosphorylation. As shown in Fig. 7, treatment with bombesin for 10 min induced a marked increase in kinase activity in COS-7 cells co-transfected with wild type PKD and bombesin receptor. The increase in PKD activity induced by bombesin was completely abolished by mutation of Ser⁷⁴⁴ and Ser⁷⁴⁸ to Ala. Single substitutions of either Ser⁷⁴⁴ or Ser⁷⁴⁸ for Ala resulted in PKD mutants that displayed reduced activity after bombesin stimulation. Thus, substitution of Ser⁷⁴⁴ and Ser⁷⁴⁸ in the activation loop of PKD by alanine prevents the activation of this enzyme by either bombesin, G_q, or PDB in vivo.

View larger version (55K): [in this window] [in a new window]   Fig. 7.   Substitution Ser744 and Ser748 by alanine prevents PKD activation in response to bombesin. Exponentially growing COS-7 cells were co-transfected with BNR-pCD2 containing the cDNA encoding the bombesin/GRP receptor (BR) and wild type PKD (PKD) or different activation loop mutants PKD-S744A (S744A), PKD-S748A (S748A), PKD-S744A/PKD-S748A (S744A/S748A), or PKD-S744E/PKD-S748E (S744E/S748E) or a kinase dead PKD-D733A (D733A) or a triple mutant PKD-D733A/S744E/S748E (D733A/S744E/S748E). Three days after transfection, the cultures were unstimulated () or stimulated with 200 nM PDB or with 10 nM bombesin (Bom) for 10 min and lysed. The lysates were immunoprecipitated with PA-1 antiserum, and PKD activities were determined by in vitro kinase assay (IVK) as described under "Experimental Procedures." A, the autoradiogram of in vitro kinase assay shown is representative of three independent experiments with similar results. B, 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 PA-1 antiserum.

The results presented in Fig. 7 also show that the high constitutive kinase activity of the PKD-S744E/S748E mutant was not significantly further stimulated by bombesin, suggesting that phosphorylation of these two sites induces maximal PKD activation in response to this neuropeptide. The lack of either basal activity or bombesin-induced activation in PKD immunoprecipitates from COS-7 cells transfected with either PKD or PKD-S744E/S748E carrying the kinase-inactivating D733A mutation (45) indicates that the kinase activity measured is due to the activation of PKD rather than to co-immunoprecipitating protein kinases (Fig. 7A). In all cases, the protein expression levels of the transfected PKD mutants were comparable with that of wild type PKD, as shown by Western blot analysis (Fig. 7B).

Role of Endogenous G_q 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 (46). 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 (47) and inwardly rectifying K⁺ channel activity (48, 49), 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 (47).

In the present study, a dominant negative strategy was also used to test the role of endogenous G_q in bombesin receptor-mediated PKD activation. We generated chimeric fusion proteins between the COOH-terminal region of G_q (referred to as G_qCT) and GFP from Aequorea victoria, which forms an independent 30-kDa domain with inherent fluorescence (50). Initially, we verified that the GFP-G_qCT 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 G_q (Fig. 8A). In addition, we also visualized the expression of the GFP-G_qCT chimera by examining GFP fluorescence in individual COS-7 cells (results not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 8.   The COOH-terminal regions of Gq (qCT) prevents PKD activation in response to bombesin. Exponentially growing COS-7 cells were co-transfected with pcDNA3 encoding GFP (GFP) or GFP-qCT (GFP-qCT), BNR-pCD2 (BR) containing the cDNA encoding the bombesin/GRP receptor, and pcDNA-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 (IVK, B) or by phosphorylation of syntide-2 (C) as described under "Experimental Procedures." A, 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. B, upper panel, the autoradiogram of in vitro kinase assay shown is representative of five independent experiments with similar results; middle panel, quantification of the level of PKD phosphorylation was performed by scanning densitometry. The results expressed as a percentage of the maximum increase in phosphorylation are mean ± S.E. of five independent experiments; bottom panel, levels of expression of PKD were analyzed by Western blotting (W. Blot) aliquots of total cell lysates with PA-1 antiserum. C, syntide-2 phosphorylation in immune complexes. Results represent the mean ± S.E. from three experiments, each performed in duplicate.

Next, we determined whether expression of GFP-G_qCT interferes with PKD activation via the bombesin receptor. COS-7 cells were co-transfected with PKD, bombesin receptor, and either GFP-G_qCT 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 or syntide-2 phosphorylation. The results illustrated in Fig. 8 (B and C) demonstrate that expression of GFP-G_qCT markedly attenuated the increase of PKD activity induced by bombesin. In contrast, expression of GFP-G_qCT did not interfere with PKD activation in response to PDB, which directly stimulates PKC leading to PKD activation and therefore bypasses the receptor/G_q interaction. These results indicate that endogenous G_q mediates PKD activation in response to bombesin receptor activation.

Conclusion-- PKD/PKCµ is a serine/threonine protein kinase with distinct structural, enzymological, and regulatory properties. Recently, activation of a number of receptors that couple to heterotrimeric G proteins, including those for bombesin, bradykinin, endothelin, and vasopressin, has been shown to stimulate PKD activation in a variety of cell types. Here, we examined the mechanism(s) by which G protein-coupled receptors lead to PKD activation.

It is generally thought that G_q stimulation of the  isoforms of PLC catalyzes the production of inositol 1,4,5-trisphosphate that triggers the release of Ca²⁺ from internal stores and DAG that directly activates the classic and novel isoforms of PKC (reviewed in Ref. 19). Accordingly, it is well established that constitutively activated forms of G_q stimulate the  isoforms of PLC in vitro and induce persistent stimulation of inositol phosphate production in intact cells (19). In contrast, the other important arm of this bifurcating signaling pathway, namely the production of DAG and the activation of PKC, has been less frequently measured. In this context, it is relevant that DAG, unlike inositol 1,4,5-trisphosphate, can be generated through routes other than phosphoinositide hydrolysis mediated by G_q-stimulated PLC (51) and that G_q-coupled receptors also interact with other heterotrimeric G proteins including members of the G₁₂ family that have been recently implicated in pathways leading to PKC activation (20-22). It is also of interest that expression of active G_q did not induce persistent activation of mitogen-activated protein kinases in either NIH 3T3 cells (52) or PC12 cells (53), suggesting that chronic G_q-PLC activation could lead to PKC down-regulation. Consequently, we examined whether G_q-mediated signaling is sufficient to promote PKD activation in intact cells and whether endogenous G_q mediates PKD activation in response to bombesin receptor stimulation.

Our results demonstrate that either mutationally activated or aluminum fluoride-stimulated G_q induces striking PKD activation through a PKC-dependent pathway. PKD activation in response to bombesin receptor stimulation, G_q, or PDB is completely prevented by mutation of Ser⁷⁴⁴ and Ser⁷⁴⁸ to Ala in the kinase activation loop of PKD. Furthermore, none of these stimuli induced a further increase in PKD activity when Ser⁷⁴⁴ and Ser⁷⁴⁸ were mutated to Glu to mimic the phosphorylated residues. These data indicate that bombesin receptor activation, G_q stimulation, and PDB lead to PKD activation through the same mechanism, namely phosphorylation of Ser⁷⁴⁴/Ser⁷⁴⁸ in the activation loop of PKD.

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 (47, 49). 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 (47). Here, we pursued a similar strategy and demonstrate, for the first time, that expression of a chimeric fusion protein consisting of the COOH-terminal region of G_q and GFP attenuated PKD activation in response to agonist stimulation of bombesin receptor but not in response to PDB, which bypasses the receptor. GFP conjugates with COOH-terminal peptides of G proteins may provide a useful approach to monitor the expression of competing (dominant negative) G protein peptides in intact cells.

Expression of constitutively active G_q is known to induce a variety of biological responses including transformation (54, 55), differentiation (55), and apoptosis (56). G_q signaling is of great interest in the development and decompensation of cardiac hypertrophy (57-59). Consequently, there is a renewed interest in identifying downstream targets that are persistently activated by expression of activated G_q (60). Our results indicate that G_q activation is sufficient to stimulate sustained PKD activation via PKC and show that the endogenous G_q mediates PKD activation in response to acute bombesin receptor stimulation.

	ACKNOWLEDGEMENTS

We thank Dr. Henry R. Bourne for the kind gifts of the expression constructs for G₁₂-Q229L and G₁₃-Q226L and Dr. R. Waldron and J. Sinnett-Smith for discussions.

	FOOTNOTES

^* This work was supported by National Institutes of Health (NIH) Grant DK 55003 (to E. R.).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 NIH Grant T32 DK07180.

^§ To whom all 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

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; PKD, protein kinase D; DAG, diacylglycerol; G proteins, guanine nucleotide-binding regulatory proteins; G_qQL, constitutively active mutant murine G_q-Q209L; G₁₂-QL constitutively active mutant G₁₂-Q229L, G₁₃-QL constitutively active mutant G₁₃-Q226L; G_qwt, wild type G_q; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; PDB, phorbol 12,13-dibutyrate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES