Bryostatin 1 Induces Biphasic Activation of Protein Kinase D in Intact Cells*

(Received for publication, February 25, 1997, and in revised form, May 13, 1997)

Sharon A. Matthews Dagger , George R. Pettit § and Enrique Rozengurt Dagger

From the Dagger  Growth Regulation Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom, and the § Cancer Research Institute and Department of Chemistry, Arizona State University, Tempe, Arizona 85287

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Bryostatin 1 and phorbol esters are both potent activators of protein kinase C (PKC), although their specific biological effects can differ in many systems. Here, we report that bryostatin 1 activates protein kinase D (PKD), a novel serine/threonine protein kinase, in intact Swiss 3T3 cells and secondary mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct. The dose response of PKD activation induced by bryostatin 1 follows a striking biphasic pattern with maximal activation achieved at a concentration of 10 nM. Higher concentrations of bryostatin 1 (100 nM) reduced PKD activation induced by phorbol 12,13-dibutyrate to levels stimulated by bryostatin 1 alone. Bryostatin 1-induced PKD activation was markedly attenuated by treatment of cells with the PKC inhibitors bisindolylmaleimide I and Ro 31-8220. However, these agents did not inhibit PKD activity when added directly to in vitro kinase assays, suggesting that bryostatin 1 stimulates PKD activation through a PKC-dependent pathway in intact cells. Our results raise the possibility that activated PKD in intact cells could mediate some of the multiple biphasic biological responses induced by bryostatin 1.


INTRODUCTION

Bryostatin 1 is a natural macrocyclic lactone with potent antineoplastic properties in a variety of animal models (1-3) and has entered clinical trials as a potential therapeutic agent (4, 5). Bryostatin family members bind to and activate classic and novel isoforms of protein kinase C (PKC)1 (6-9), the major cellular targets of the tumor-promoting phorbol esters (10). Despite appearing to bind to the same cellular targets, the biological responses induced by bryostatin 1 frequently differ from those induced by phorbol esters. For example, many bryostatin 1-mediated effects have unusual characteristics such as biphasic dose-response relationships, delayed kinetics, and the ability to inhibit phorbol ester-induced responses (7, 11-16). The precise mechanism(s) by which bryostatin 1 induces these biological effects remain poorly understood.

The newly identified protein kinase D (PKD) is a mouse serine/threonine protein kinase with distinct structural features and enzymological properties (17-19). In particular, the catalytic domain of PKD, which is distantly related to Ca2+-regulated kinases, shows little homology to the highly conserved regions of the kinase domain of the PKC family. As a consequence of this, PKD does not phosphorylate a variety of known PKC substrates, indicating that PKD has a distinct substrate specificity (18, 19).

The amino-terminal region of PKD contains a putative transmembrane domain, two cysteine-rich zinc finger-like motifs, and a pleckstrin homology domain. Unlike all known PKC isoforms, PKD does not contain a pseudosubstrate motif upstream of the cysteine-rich region, and the sequence separating the cysteine-rich repeats of PKD (95 amino acids) is substantially longer than that of classical and novel PKCs (28 and 35 amino acids, respectively). Additionally, residues Ala-146, Ala-154, and Tyr-182 in the consensus cysteine-rich motif of PKD differ from those found in PKCs. However, both immunopurified PKD and a fusion protein containing the cysteine-rich region of PKD bind phorbol esters with high affinity, and PKD is directly stimulated in vitro by these agents, or by diacylglycerol, in the presence of phospholipids (18, 19). A human protein kinase called atypical PKCµ (20) with 92% homology to PKD is also stimulated in vitro by phorbol esters and phospholipids (21). These results indicate that PKD/PKCµ are phorbol ester/diacylglycerol-stimulated protein kinases. Recent studies have demonstrated a novel mechanism of activation of PKD. Specifically, treatment of intact cells with biologically active phorbol esters induces phosphorylation-dependent activation of PKD through a PKC-dependent pathway (22). PKD activity recovered from phorbol ester-stimulated cells can be measured by kinase assays in the absence of lipid activators. These results revealed an unsuspected connection between PKCs and PKD and suggested that PKD can function parallel to and/or downstream of PKC in signal transduction pathways.

The differences between the biological effects elicited by phorbol esters and bryostatin 1 in certain systems prompted us to examine whether bryostatin 1 regulates PKD activity in intact cells. Here we report that treatment with bryostatin 1 induces PKD activation in intact Swiss 3T3 cells and MEF and in COS-7 cells transfected with a PKD expression vector. A salient feature of our results is that bryostatin 1-mediated activation of PKD follows a striking biphasic dose-response relationship. PKD activation induced by treatment with PDB was inhibited by high concentrations of bryostatin 1. These results raise the possibility that PKD could mediate some of the multiple biological responses induced by bryostatin 1.


EXPERIMENTAL PROCEDURES

Cell Culture---Stock cultures of Swiss 3T3 cells were maintained in DMEM supplemented with 10% FBS in a humidified atmosphere containing 10% CO2 at 37 °C. For experimental purposes, cells were plated in 90-mm dishes at 6 × 105 cells/dish in DMEM containing 10% FBS and used after 6-8 days, when the cells were confluent and quiescent. COS-7 cells were plated in 90-mm dishes at 6 × 105 cells/dish in DMEM containing 10% FBS and used for transfection 1 day later. Secondary cultures of MEF were seeded in 90-mm dishes at 6 × 105 cells/dish in DMEM containing 10% FBS, switched down to 0.5% FBS after 3-4 days, and were used after 24 h when the cells were confluent and quiescent.

cDNA Expression Vectors and Transfection of COS-7 Cells

The PKD cDNA fragment spanning bases -125 to 3179 was inserted into the mammalian expression vector pcDNA3, as described (19). A kinase-deficient mutant (PKDK618M) was generated by site-directed mutagenesis using the Altered Sites II in vitro mutagenesis kit (Promega) and subcloned into pcDNA3 (pcDNA3-PKDK618M).

Exponentially growing COS-7 cells, 40-60% confluent, were transfected with the various plasmids using Lipofectin (Life Technologies, Inc.) according to the manufacturer's instructions. Briefly, 10 µg of DNA was used for 90-mm dishes. The DNA was diluted to 1 ml with Opti-MEM I (Life Technologies, Inc.) and then mixed with Lipofectin (20 µl) diluted to 1 ml with Opti-MEM I. After 15 min, the DNA-Lipofectin complex was diluted to 5 ml with Opti-MEM I, mixed gently, and overlaid onto rinsed (once with Opti-MEM I) COS-7 cells. The cultures were incubated at 37 °C for 6 h, and the medium was then replaced with fresh Opti-MEM I containing 10% FBS. The cells were used for experimental purposes 72 h later.

Immunoprecipitation

Cultured cells were washed three times in ice-cold phosphate-buffered saline and lysed in 50 mM Tris/HCl, pH 7.5, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM 4-(2'-aminoethyl)-benzenesulfonyl fluoride hydrochloride and 1% Triton X-100 (lysis buffer A). PKD was immunoprecipitated at 4 °C for 3 h with the PA-1 anti-peptide antiserum (1:100 dilution), raised against the synthetic peptide EEREMKALSERVSIL that corresponds to the COOH-terminal region of the predicted amino acid sequence of PKD, as described previously (18, 19). Immune complexes were recovered using protein A coupled to agarose.

Elution of PKD from Immunocomplexes

PKD immunocomplexes were washed once with lysis buffer A, twice with lysis buffer B (consisting of lysis buffer A minus Triton X-100), and once with kinase buffer (30 mM Tris/HCl, pH 7.5, 10 mM MgCl2, and 1 mM dithiothreitol). In some experiments PKD was then eluted from immunocomplexes by incubation with 0.75 mg/ml of the immunizing peptide in kinase buffer at 4 °C for 30 min.

Kinase Assay of PKD

PKD autophosphorylation was determined by in vitro kinase assays by mixing 30 µl of either immunocomplexes or eluted PKD with 5 µl of a phosphorylation mixture, consisting of kinase buffer (see above) containing 100 µM [gamma -32P]ATP final concentration (specific activity, 400-600 cpm/pmol). In vitro stimulation of eluted PKD was performed using 100 µg/ml PS and either 50 nM bryostatin 1 or 200 nM PDB, as described previously (19). After incubation at 30 °C for 10 min, the reactions were terminated by washing with 200 µl of cold kinase buffer and adding 2 × SDS-PAGE sample buffer (1 M Tris/HCl, pH 6.8, 0.1 mM Na3V04, 6% SDS, 0.5 M EDTA, pH 8, 4% 2-mercaptoethanol, and 10% glycerol), followed by SDS-PAGE analysis. The gels were dried, and the 110-kDa radioactive band corresponding to phosphorylated PKD was visualized by autoradiography. Autoradiographs were scanned in a LKB Ultrascan XL densitometer, and the labeled band was quantified using Ultrascan XL internal integrator.

Exogenous substrate phosphorylation by immunoprecipitated PKD was carried out by mixing 30 µl of the washed immunocomplexes with 5 µl of phosphorylation mixture (see above) containing 2.5 mg/ml syntide-2 (PLARTLSVAGLPGKK), a peptide based on phosphorylation site 2 of glycogen synthase (23, 24). After incubation at 30 °C for 10 min, the kinase reaction was stopped by adding 100 µl of 75 mM H3PO4 and then spotting 80 µl of supernatant onto P-81 phosphocellulose paper. Free [gamma -32P]ATP was separated from the labeled substrate by washing the P-81 paper four times (5 min each) in 75 mM H3PO4. The papers were dried, and radioactivity incorporated into syntide-2 was determined by Cerenkov counting. PKD autophosphorylation and syntide-2 phosphorylation by PKD in the absence or presence of activators were linear up to 10 min of incubation.

Kinase Assay of PKC

Purified PKC (5 ng) was assayed in the presence of 50 nM bryostatin 1, 100 µg/ml PS, and 1 mM CaCl2 in a phosphorylation mixture (see above) containing 2.5 mg/ml syntide-2. After incubation at 30 °C for 10 min, the kinase reaction was stopped by adding 100 µl of 75 mM H3PO4 and then spotting 80 µl of supernatant onto P-81 phosphocellulose paper. Free [gamma -32P]ATP was separated from the labeled substrate by washing the P-81 paper four times (5 min each) in 75 mM H3PO4. The papers were dried and radioactivity incorporated into syntide-2 determined by Cerenkov counting.

32Pi Labeling

Confluent and quiescent cultures of Swiss 3T3 cells were washed twice in phosphate-free DMEM and incubated at 37 °C with this medium containing 200 µCi/ml carrier-free 32Pi for 18 h. Cells were then stimulated with either bryostatin 1 or PDB, lysed, and immunoprecipitated with PA-1 antiserum. Samples were analyzed by SDS-PAGE and autoradiography.

Microcystin Treatment

Confluent and quiescent cultures of Swiss 3T3 cells were stimulated with 10 nM bryostatin 1 for 30 min and lysed. The lysates were then incubated for 30 min at 37 °C with either 1 µM microcystin or an equivalent volume of solvent. PKD was then immunoprecipitated and subjected to in vitro kinases assays, as described above.

Western Blot Analysis

For Western blot analysis of PKD, immunoprecipitates were washed three times with lysis buffer A (see above), extracted for 10 min at 95 °C in 2 × SDS-PAGE sample buffer, and analyzed by SDS-PAGE followed by transfer to Immobilon membranes. The transfer was carried out at 100 V, 0.4 A at 4 °C for 4 h using a Bio-Rad transfer apparatus in a buffer consisting of 200 mM glycine, 25 mM Tris, 0.01% SDS, and 20% CH3OH. Membranes were blocked using 5% non-fat dried milk in phosphate-buffered saline, pH 7.2, and incubated with PA-1 antiserum (1:500) at room temperature for 3 h 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 chemiluminesence (ECL) detection or protein A-I125 and autoradiography. In Fig. 1A, lower panel, lysates of bryostatin 1-treated cells were incubated at 37 °C for 30 min prior to PKD immunoprecipitation and Western blot analysis.


Fig. 1. Bryostatin 1, unlike phorbol esters, induces biphasic PKD activation in Swiss 3T3 cells. Panel A, confluent and quiescent cultures of Swiss 3T3 cells were incubated in the presence of increasing concentrations of bryostatin 1 for 30 min and lysed. Lysates were immunoprecipitated with PA-1 antiserum and subjected to in vitro kinase assays, SDS-PAGE, and autoradiography (IVK). PKD expression levels from parallel cultures treated with either increasing concentrations of bryostatin 1 or 200 nM PDB were determined by incubating lysates at 37 °C for 30 min prior to immunoprecipitation with the PA-1 antiserum and Western blot analysis (W. Blot) as described under "Experimental Procedures." Results shown are representative of three independent experiments. Panel B, to quantify levels of PKD autophosphorylation induced by increasing concentrations of bryostatin 1, autoradiograms were scanned using an LKB Ultrascan XL densitometer. The results shown are expressed as a percentage of the maximum response induced by bryostatin 1 and are the mean ± S.E. of three independent experiments. Panel C, confluent and quiescent cultures of Swiss 3T3 cells were stimulated with increasing concentrations of either PDB (2-1,000 nM) or 12-O-TPA (0.5-500 nM) for 10 min and lysed. PKD was immunoprecipitated and analyzed by in vitro kinase assays, SDS-PAGE, and autoradiography (IVK). Results shown are representative of two independent experiments.
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Materials

Bryostatin 1 was isolated from Bugula neritina as described previously (3). [gamma -32P]ATP (370 MBq/ml), 32Pi (10 mCi/ml), protein A-I125 (100 µCi/ml), and ECL reagents were from Amersham International (U. K.). PDB was obtained from Sigma. The inhibitors GF I and Ro 31-8220 were from LC Laboratories. Protein A-agarose was from Boehringer Mannheim. The MEK-1 inhibitor PD 098059 was the generous gift of Alan R. Saltiel, Department of Signal Transduction, Parke Davis Research Division, Ann Arbor, MI. Microcystin was from Sigma. Purified PKC from rat brain (containing alpha , beta , and gamma  isoforms) was from TCS Biologicals, U. K. Other items were from standard suppliers or as indicated in the text.


RESULTS

Treatment with Bryostatin 1 Induces PKD Activation in Intact Cells: Dose Response and Kinetics

To determine whether bryostatin 1 induces PKD activation in intact cells, quiescent cultures of Swiss 3T3 cells were treated with increasing concentrations (0.3-100 nM) of bryostatin 1 for 30 min, lysed, and the extracts immunoprecipitated with the PA-1 antiserum. The immunocomplexes were incubated with [gamma -32P]ATP and then analyzed by SDS-PAGE, autoradiography, and scanning densitometry to determine the level of PKD autophosphorylation. Stimulation of intact cells with bryostatin 1 induced a striking dose-dependent increase in PKD activity which was maintained during cell lysis and immunoprecipitation and which could be measured by autophosphorylation assays in the absence of further lipid activators (Fig. 1A, upper panel). Half-maximum and maximum stimulation were achieved at 1 nM and 5-10 nM, respectively (Fig. 1B). Maximal PKD activation induced by bryostatin 1 was comparable to that induced by 200 nM PDB in parallel cultures of Swiss 3T3 cells (Fig. 1A, upper panel). However, at higher concentrations of bryostatin 1 (>10 nM) PKD was activated to a lesser extent. This biphasic pattern of PKD activation was not the result of PKD degradation induced by high concentrations of bryostatin 1, as can be seen by Western blot analysis of PKD expression levels (Fig. 1A, lower panel). In contrast, neither PDB nor TPA induced biphasic activation of PKD. The maximum effects of PDB and TPA (achieved at 100 nM and 20 nM, respectively) were not decreased by increasing the concentration of these agents by 10- and 25-fold respectively (Fig. 1C). Thus, bryostatin 1, unlike phorbol esters, induces a biphasic activation of PKD in intact Swiss 3T3 cells.

A similar dose-dependent biphasic activation of PKD induced by bryostatin 1 (0.3-100 nM) was also seen in quiescent cultures of secondary MEF (Fig. 2) with a maximum response occurring at 10 nM. Thus, biphasic PKD activation in response to bryostatin 1 is not restricted to immortalized cell lines.


Fig. 2. Bryostatin 1 induces biphasic PKD activation in MEF. Confluent and quiescent cultures of MEF were treated with either increasing concentrations of bryostatin 1 as indicated for 30 min or with 200 nM PDB for 10 min, lysed, and the lysates immunoprecipitated with PA-1 antiserum. PKD activity, as shown by autophosphorylation, was determined by in vitro kinase assays followed by SDS-PAGE, autoradiography, and scanning densitometry (IVK). Results shown are the mean ± S.E. of three independent experiments. A representative autoradiogram is shown in the upper panel.
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The time course of PKD activation induced by 10 nM bryostatin 1 also contrasts with that stimulated by 200 nM PDB (Fig. 3A). Although PKD activation was maximal after 10 min of PDB stimulation, bryostatin 1-mediated PKD activation was delayed, with the maximal response only occurring after treatment for 30 min.


Fig. 3. Panel A, time course of PKD activation by bryostatin 1 and PDB. Confluent and quiescent cultures of Swiss 3T3 cells were incubated in the presence of either 10 nM bryostatin 1 or 200 nM PDB for various times as indicated and lysed. The lysates were immunoprecipitated with PA-1 antiserum and subjected to in vitro kinase assays, SDS-PAGE, and autoradiography. Autophosphorylation of PKD was quantified by scanning densitometry, and the results shown are expressed as a percentage of the maximum response induced by bryostatin 1 or PDB and are the mean ± S.E. of three independent experiments. Representative autoradiograms are shown (inset). Panel B, bryostatin 1 induces PKD activation measured by syntide-2 phosphorylation. Confluent and quiescent cultures of Swiss 3T3 cells were incubated in the presence of either increasing concentrations of bryostatin 1 for 30 min (solid bars), 200 nM PDB for 10 min (hatched bar), or an equivalent volume of solvent (open bar), lysed, and the lysates immunoprecipitated with PA-1 antiserum. PKD activity was measured in a syntide-2 phosphorylation assay as described under "Experimental Procedures." Results shown are expressed as the fold increase over control and are the mean ± S.E. of two independent experiments, each performed in duplicate.
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Subsequently, we determined whether bryostatin 1-mediated PKD activation could also be demonstrated using an exogenous substrate. The synthetic peptide syntide-2 (23, 24) has been identified as an efficient substrate for the catalytic domain of PKD (18) and for the full-length PKD (19). Therefore syntide-2 was chosen as a model exogenous substrate to assay PKD activity immunoprecipitated from lysates of Swiss 3T3 cells treated with increasing concentrations of bryostatin 1 (as indicated) or 200 nM PDB. As shown in Fig. 3B, a biphasic pattern of syntide-2 phosphorylation was seen in PKD immunoprecipitates from cells stimulated with bryostatin 1, which was comparable to the biphasic pattern of PKD autophosphorylation seen previously.

Bryostatin 1, at High concentrations, Antagonizes PKD Activation in Response to PDB

The biphasic bryostatin 1 dose-response curve of PKD activation in Swiss 3T3 cells prompted us to examine whether this agent, at high concentrations, could antagonize activation of PKD by PDB. To examine this possibility confluent and quiescent cultures of Swiss 3T3 cells were treated with either 200 nM PDB or with increasing concentrations of bryostatin 1 (1, 10, or 100 nM) in the absence or presence of 200 nM PDB for 30 min. The results presented in Fig. 4 show that activation of PKD by bryostatin 1 alone follows the biphasic pattern seen previously and that PDB-induced activation of PKD was not affected by the simultaneous addition of either 1 nM or 10 nM bryostatin 1. However, PKD activation induced by PDB in the presence of 100 nM bryostatin 1 was reduced to levels seen with 100 nM bryostatin 1 alone (Fig. 4). A similar antagonism of PKD activation was seen when cells were treated with 100 nM bryostatin 1 for 30 min prior to treatment with 200 nM PDB for 10 min (data not shown). These results demonstrate that treatment with high concentrations of bryostatin 1 antagonizes PKD activation induced by PDB.


Fig. 4. Effect of bryostatin 1 on PDB-induced PKD activation. Confluent and quiescent cultures of Swiss 3T3 cells were treated with various concentrations of bryostatin 1 for 30 min in the presence or absence of 200 nM PDB as indicated, lysed, and immunoprecipitated with PA-1 antiserum. Samples were then analyzed by in vitro kinase assays, SDS-PAGE, autoradiography, and scanning densitometry. The results shown are expressed as the percentage of PKD activation induced by PDB treatment alone and are the mean ± S.E. of three independent experiments. A representative autoradiogram is shown.
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Bryostatin 1 Stimulates PKD Activation in COS-7 Cells Transfected with a PKD Expression Construct

To confirm that the bryostatin 1-induced kinase activity measured was the result of activation of PKD rather than the presence of a coprecipitating protein kinase, we examined PKD autophosphorylation in COS-7 cells transfected with either a wild type PKD expression vector (pcDNA3-PKD) or with a kinase-defective PKD mutant (pcDNA3-PKDK618M) in which lysine 618 in the ATP binding site is replaced by methionine. Cells were treated with bryostatin 1 or PDB, lysed, and the immunoprecipitates subjected to in vitro kinase assays and to immunoblotting with the PA-1 antiserum. Bryostatin 1 treatment of COS-7 cells, like PDB, resulted in activation of wild type PKD (Fig. 5A, upper panel, left). In contrast, no inducible kinase activity was seen in COS-7 cells transfected with pcDNA3-PKDK618M (Fig. 5A, upper panel, right) despite similar PKD and PKDK618M expression levels (Fig. 5A, lower panels). Similarly, no kinase activity was detected in COS-7 cells transfected with the control vector pcDNA3 (data not shown). These results verified that the bryostatin 1-induced kinase activity measured in PKD immunoprecipitates was caused by the activation of PKD.


Fig. 5. Panel A, the kinase activity induced by bryostatin 1 in PA-1 immunocomplexes is PKD rather than a coimmunoprecipitating kinase. COS-7 cells were transfected with PKD expression vectors, either pcDNA3-PKD or pcDNA3-PKDK618M, as detailed under "Experimental Procedures." After 72 h, cultures were treated with 50 nM bryostatin 1 for 30 min (Bryo) or 200 nM PDB for 10 min, lysed, and PKD immunoprecipitated as described previously. Upper panels, PKD activity was determined by in vitro kinase assays followed by SDS-PAGE, autoradiography, and scanning densitometry (IVK). Lower panels, PKD and PKDK618M expression levels were determined by SDS-PAGE and Western blotting (W.BLOT). Results shown are representative of three independent experiments. Panel B, bryostatin 1 induces PKD phosphorylation in vivo. Confluent and quiescent Swiss 3T3 cells were incubated for 18 h with carrier-free 32Pi in phosphate-free medium and then stimulated with either 10 nM bryostatin 1 for 30 min or 200 nM PDB for 10 min (upper panel). In other experiments, 32Pi-labeled cells were stimulated with either 10 or 100 nM bryostatin 1 for 30 min (lower panel). Cells were lysed, immunoprecipitated with PA-1 antiserum, and the samples analyzed by SDS-PAGE and autoradiography. Results shown are representative of two independent experiments. Panel C, the protein phosphatase inhibitor microcystin prevents inactivation of PKD. Confluent and quiescent Swiss 3T3 cells were incubated with (+) or without (-) 10 nM bryostatin 1 for 30 min and lysed. Lysates were then treated with either 1 µM microcystin (M) or an equivalent volume of solvent for 30 min at 37 °C. PKD was then immunoprecipitated and subjected to autophosphorylation, SDS-PAGE, and autoradiography. Results shown are representative of three independent experiments.
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Bryostatin 1 Induces PKD Phosphorylation in Intact Swiss 3T3 Cells

The preceding experiments demonstrated that treatment with bryostatin 1 markedly increased the level of PKD autophosphorylation in "in vitro" kinase assays. We next examined whether bryostatin 1 induces PKD phosphorylation in intact cells. Confluent and quiescent cultures of Swiss 3T3 cells metabolically labeled with 32Pi were stimulated with 10 nM bryostatin 1 for 30 min or 200 nM PDB for 10 min. Cells were lysed, immunoprecipitated with PA-1 antiserum, and analyzed by SDS-PAGE and autoradiography. As shown in Fig. 5B, upper panel, 10 nM bryostatin 1 induced a 4.6-fold increase in the incorporation of 32Pi into PKD, which was comparable to that induced by 200 nM PDB. Interestingly, when cells were stimulated with 100 nM bryostatin 1, incorporation of 32Pi into PKD was 47% lower than after treatment of cells with 10 nM bryostatin 1 (Fig. 5B, lower panel). Thus, bryostatin 1 induces biphasic phosphorylation of PKD in intact cells.

To determine further the role of phosphorylation in maintaining the activated state of PKD, we examined whether endogenous protein phosphatases could reverse bryostatin 1-induced PKD activation. Lysates of Swiss 3T3 cells treated with bryostatin 1 were incubated at 37 °C for 30 min in the absence or presence of 1 µM microcystin, a potent inhibitor of protein phosphatases 1 and 2A, prior to PKD immunoprecipitation. As can be seen in Fig. 5C, incubation of the lysate in the absence of microcystin resulted in a marked decrease of PKD activity. Addition of this protein phosphatase inhibitor preserved substantial PKD activity (Fig. 5C).

Inhibitors of PKC Attenuate PKD Activation Induced by Bryostatin 1

We next examined potential signaling pathways leading to bryostatin 1-induced PKD activation in Swiss 3T3 cells. Inhibition of a variety of kinases, including p70S6K with rapamycin (20 nM), PI 3-kinase with wortmannin (50 nM), PKA with H-89 (60 µM), and p42MAPK/p44MAPK with the selective MEK-1 inhibitor PD 098059 (10 µM) did not affect PKD activation in response to bryostatin 1 (Fig. 6). Similarly, disruption of the actin cytoskeleton and inhibition of p125FAK tyrosine phosphorylation using cytochalasin D had no effect on subsequent PKD activation induced by bryostatin 1 (Fig. 6). However, treatment of Swiss 3T3 cells with GF I or Ro 31-8220, potent inhibitors of classic and novel isoforms of PKC (25, 26), did attenuate PKD activation induced by bryostatin 1 (Fig. 6).


Fig. 6. Bryostatin 1-induced PKD activation is attenuated by the PKC inhibitors GF I and Ro 31-8220. Confluent and quiescent cultures of Swiss 3T3 cells were treated with 3.5 µM GF I (GF 1), 2.5 µM Ro 31-8220 (Ro), 10 µM PD 098059 (PD), 20 nM rapamycin (RAP), 50 nM wortmannin (WOR), 60 µM H-89, 2.5 µM cytochalasin D (CYT D), or an equivalent volume of solvent for 1 h. Cells were then incubated in the absence (open bars) or presence of 10 nM bryostatin 1 (solid bars) for 30 min. The cultures were lysed, immunoprecipitated with PA-1 antiserum and PKD, subjected to in vitro kinase assays, SDS-PAGE, autoradiography, and scanning densitometry. Results shown are representative of two independent experiments and are expressed as a percentage of the maximal increase in PKD activity induced by bryostatin 1.
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We therefore investigated further the role of bryostatin 1-sensitive PKCs in the activation of PKD induced by treatment of intact cells with bryostatin 1. As shown in Fig. 7A, upper panels, pretreatment of Swiss 3T3 cells with increasing concentrations of either GF I or Ro 31-8220 led to a marked dose-dependent reduction in the subsequent activation of PKD elicited by bryostatin 1. In addition, we verified that GF I and Ro 31-8220 could also prevent PKD activation induced by PDB, in agreement with previous results (22). In striking contrast neither GF I nor Ro 31-8220 inhibited PKD activity, induced by treatment of intact cells with either bryostatin 1 or PDB, when added directly to in vitro kinase assays at concentrations identical to those used in intact cells (Fig. 7A, lower panels).


Fig. 7. Bryostatin 1 induces PKD activation through a PKC-dependent pathway. A: upper panels, confluent and quiescent cultures of Swiss 3T3 cells were pretreated with GF I, Ro 31-8220, or an equal volume of solvent (-) as indicated for 1 h. Cultures were subsequently treated with either 10 nM bryostatin 1 for 30 min or 200 nM PDB for 10 min, lysed, and PKD immunoprecipitated with PA-1 antiserum. PKD activity was determined by autophosphorylation followed by SDS-PAGE and autoradiography. Lower panels, Swiss 3T3 cells treated with either 10 nM bryostatin 1 for 30 min or 200 nM PDB for 10 min were lysed and PKD immunoprecipitated. PKD activity was determined by autophosphorylation carried out in the absence or presence of GF I, Ro 31-8220, or solvent added directly to the phosphorylation mixture, at concentrations identical to those used previously in intact cells. Samples were then analyzed by SDS-PAGE and autoradiography. Results shown are representative of three independent experiments. Panel B: COS-7 cells were transfected with pcDNA3-PKD and after 72 h lysed, and PKD was immunoprecipitated and eluted from the immunocomplexes as described previously. In vitro kinase assays were performed by incubating eluted PKD with 50 nM bryostatin 1 (Bryo), 100 µg/ml PS, and 3.5 µM GF I as indicated, followed by SDS-PAGE, autoradiography, and scanning densitometry. Results are expressed as a percentage of the maximum PKD activity induced by bryostatin 1 and PS and are the mean ± S.E. of three independent experiments. Inset, 5 ng of purified PKC (containing a mixture of alpha , beta , and gamma  isoforms) was activated by 50 nM bryostatin 1 and 100 µg/ml PS for 10 min in the absence (-) or presence (+) of 3.5 µM GF I in a syntide-2 phosphorylation assay, as described previously. A representative experiment is shown, expressed as the fold increase in syntide-2 phosphorylation (cpm × 104) over control levels.
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To verify that GF I does not prevent PKD stimulation induced by bryostatin 1 in vitro, cultures of COS-7 cells transfected with pcDNA3-PKD were lysed, and PKD was immunoprecipitated with the PA-1 antiserum. The enzyme was eluted from the immunocomplexes with the immunizing peptide, incubated with [gamma -32P]ATP in the absence or presence of PS and bryostatin 1, as indicated, followed by SDS-PAGE, autoradiography, and scanning densitometry. As shown in Fig. 7B, addition of 3.5 µM GF I to the incubation mixture did not prevent stimulation of PKD induced by bryostatin 1 and PS. In contrast, the activity of purified PKC, measured in parallel reactions, in the presence of bryostatin 1 and PS was strikingly inhibited by the inclusion of 3.5 µM GF I, as measured in a syntide-2 phosphorylation assay (Fig. 7B, inset). Taken together these results indicate that bryostatin 1-mediated PKD activation occurs through a PKC-dependent pathway in intact cells.


DISCUSSION

Bryostatin 1 binds to and activates PKC, but its biological effects differ greatly from other PKC activators such as the tumor-promoting phorbol esters (7, 11-15). These unexplained differences prompted us to examine the regulation of PKD activity by bryostatin 1 in intact cells.

The results presented here demonstrate, for the first time, that bryostatin 1 induces a striking activation of PKD in intact cultures of Swiss 3T3 cells, MEF, and in COS-7 cells transiently transfected with a PKD expression construct. Stimulation of intact cells with bryostatin 1 leads to the persistent activation of PKD which can be detected by in vitro kinase assays (as seen by autophosphorylation and syntide-2 phosphorylation) in the absence of any further lipid activators. Since the activated state of PKD induced by bryostatin 1 in intact cells is maintained during cell lysis and immunoprecipitation, it is likely that the enzyme is activated by a covalent modification. Here we demonstrate that bryostatin 1 stimulation of intact Swiss 3T3 cells markedly enhanced the incorporation of 32Pi into PKD in metabolically labeled cells. The role of phosphorylation in the regulation of PKD was also suggested by treatment of bryostatin 1-stimulated Swiss 3T3 cell lysates with the protein phosphatase 1 and 2A inhibitor microcystin, which partially prevented the inactivation of PKD by endogenous protein phosphatases.

The active state induced by bryostatin 1 stimulation of intact cells could be produced by an activating autophosphorylation step or by trans-phosphorylation mediated by an upstream protein kinase, for example, bryostatin 1-sensitive PKCs. Interestingly, bryostatin 1 has been shown to induce potent activation of PKCepsilon , as judged by translocation assays (11), and PKCepsilon has been implicated as one of the PKC isoforms that promote PKD activation (22). To distinguish between PKC-dependent and PKC-independent pathways of PKD activation we utilized inhibitors that discriminated between PKCs and PKD. We found that pretreatment of Swiss 3T3 cells with the PKC inhibitors GF I and Ro 31-8220 before stimulation with bryostatin 1 markedly attenuates PKD activation. Importantly, neither GF I nor Ro 31-8220 inhibits PKD activity when added directly in vitro. These results strongly suggest that persistent PKD activation induced by bryostatin 1 is mediated through a PKC-dependent pathway. Further experimental work will be required to elucidate whether PKCs directly phosphorylate and activate PKD or stimulate an intermediary kinase(s) that leads to activation of PKD.

Many biological responses induced by bryostatin 1 are characterized by a biphasic dose-response relationship, with a typical maximum effect occurring at ~10 nM, and suppression of phorbol ester-induced effects at higher concentrations. These include stimulation of growth of JB6 cells (13), sensitization of human cervical carcinoma cells to cis-diamminedichloroplatinum (II) (14), induction of cytokine secretion in mononuclear cells (15), and induction of c-Jun protein in NIH 3T3 cells (11). However, the mechanisms underlying these biphasic biological effects of bryostatin 1 remain poorly understood. A salient feature of the results presented here is that the dose-dependent activation of PKD induced by bryostatin 1 in Swiss 3T3 cells or MEF is clearly biphasic and reflects the biphasic pattern of PKD phosphorylation induced by bryostatin 1. Furthermore, bryostatin 1, at high concentrations (e.g.. 100 nM) reduced PKD activation in response to PDB to the same level achieved by treatment with bryostatin 1 alone. Interestingly, the biphasic activation of PKD in response to bryostatin 1 precedes most other biphasic biological responses induced by bryostatin 1. Our results, therefore, raise the intriguing possibility that at least some of the biphasic biological effects induced by bryostatin 1 in a variety of systems could be mediated via activated PKD, a proposition that warrants further experimental work.


FOOTNOTES

*   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.
   To whom correspondence should be addressed: Dagger Growth Regulation Laboratory, Imperial Cancer Research Fund, P. O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
1   The abbreviations used are: PKC, protein kinase C; PKD, protein kinase D; MEF, secondary mouse embryo fibroblasts; PDB, phorbol 12,13-dibutyrate; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PS, phosphatidylserine; PAGE, polyacrylamide gel electrophoresis; GF I, GF 1092030X or bisindolylmaleimide I; TPA, 12-O-tetradecanoylphorbol-13-acetate.

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