©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression and Characterization of PKD, a Phorbol Ester and Diacylglycerol-stimulated Serine Protein Kinase (*)

(Received for publication, October 7, 1994; and in revised form, November 7, 1994)

Johan Van Lint (§) James Sinnett-Smith Enrique Rozengurt (¶)

From the Growth Regulation Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A novel protein kinase (named PKD) with an NH(2)-terminal region containing two cysteine-rich motifs has been expressed in COS-7 cells and identified as a receptor for phorbol esters. COS-7 cells transfected with a PKD cDNA construct (pcDNA3-PKD) exhibit a marked (4.8-fold) increase in [^3H]phorbol 12,13-dibutyrate binding. An antiserum raised against the COOH-terminal 15 amino acids of PKD specifically recognized a single 110-kDa band in PKD-transfected cells. PKD prepared by elution from immunoprecipitates with the immunizing peptide efficiently phosphorylated the synthetic peptide syntide-2. The enzyme only poorly phosphorylated a variant syntide-2 where arginine 4 has been replaced by an alanine. The addition of [^3H]phorbol 12,13-dibutyrate, 1-oleoyl-2-acetylglycerol, or 1,2-dioctanoyl-sn-glycerol in the presence of dioleoylphosphatidylserine stimulated the syntide-2 kinase activity of PKD in a synergistic fashion (4-6-fold). Furthermore, the autophosphorylation of PKD was strikingly stimulated by the same lipid activators (14-24-fold). Similar properties were found with PKD isolated from mouse lung. The substrate specificity of PKD is different from that of previously identified members of the protein kinase C family since it does not efficiently phosphorylate histone III-S, protamine sulfate, or a synthetic peptide based upon the conserved pseudosubstrate region of the protein kinase C family. Taken together, these data unambiguously establish PKD as a phorbol ester receptor and as a novel phospholipid/diacylglycerol-stimulated protein kinase.


INTRODUCTION

A rapid increase in the synthesis of lipid-derived second messengers is an important mechanism for transducing extracellular signals across the plasma membrane(1, 2) . The second messenger DAG, (^1)which is generated through alternative pathways(3, 4) , activates PKC, a major cellular target for the potent tumor-promoting phorbol esters(5, 6) . Molecular cloning has demonstrated the presence of multiple related PKC isoforms(1, 7) , i.e. classic PKCs (alpha, betaI, betaII, and ), novel PKCs (, , , and ), and atypical PKCs (, ), all of which possess a highly conserved catalytic domain. The regulatory domain of both classic and novel PKCs has a tandem repeat of zinc finger-like cysteine-rich motifs that confers phospholipid-dependent phorbol ester and DAG binding to these PKC isoforms(8, 9, 10, 11) . In contrast, atypical PKCs contain a single cysteine-rich motif, do not bind phorbol esters, and are not regulated by DAG(8, 12, 13, 14) . However, other proteins such as chimaerin(15) , UNC-13(16) , and Vav(17) , which possess a single cysteine-rich domain, bind DAG and phorbol esters. These studies emphasize the complexity of the signaling pathways initiated by DAG but do not exclude the possibility that other protein kinases, unrelated to the PKC family in their catalytic domain, could also play a role in mediating the cellular effects of DAG and phorbol esters.

Recently, we cloned a novel mouse serine protein kinase, named PKD, that consists of a putative regulatory and catalytic domain(18) . The NH(2)-terminal region of PKD contains a putative transmembrane domain, two cysteine-rich, zinc finger-like motifs, and a pleckstrin homology domain. Interestingly, the length of the sequence separating the cysteine-rich motifs in PKD (95 residues) is substantially longer than that of classic PKCs (28 amino acids) or novel PKCs (35 amino acids). Furthermore, two amino acids (Ala-146 and Ala-154) in the consensus of the cysteine-rich motif of PKD differ from those in PKCs. In contrast to all known PKCs including mammalian, Drosophila, or yeast isoforms, PKD does not contain sequences with homology to a typical PKC pseudosubstrate motif upstream of the cysteine-rich region.

The catalytic domain of PKD contains all 11 distinct subdomains characteristic of protein kinases(19) . Comparison of the deduced amino acid sequence of the catalytic domain of PKD with that of other protein kinases indicates that PKD is a distinct protein kinase that is distantly related to Ca-regulated kinases (^2)but does not belong to any of the protein kinase subfamilies(18) . (^3)In particular, the kinase subdomains of PKD show little similarity to the highly conserved regions of the kinase subdomains of the PKC family. For example, the motif XXDLKXX(N/D) in subdomain VI, which is important because it guides the peptide substrate into the correct orientation so that catalysis can occur(20) , is YRDLKLDN in all PKCs, which differs from that of PKD (HCDLKPEN) in every variable residue (X). The comparisons of the regulatory and catalytic domains of PKD with other kinases clearly establish PKD as a novel type of protein kinase and therefore it is important to elucidate the regulatory properties of this enzyme.

Johannes et al.(21) recently cloned a human protein kinase called atypical PKCµ with 92% homology to PKD (extending to 98% homology in the catalytic domain). In vitro phorbol ester binding studies and kinase assays with lysates of cells overexpressing PKCµ showed no increased phorbol ester binding and revealed phorbol ester-independent kinase activity. In contrast, our bacterial fusion protein encoding the zinc finger-like domains of PKD bound [^3H]PDBu with high affinity(18) . Hence it was necessary to establish whether PKD can serve as a cellular phorbol ester receptor in intact eukaryotic cells and whether the kinase activity of PKD is lipid regulated.

The results presented in this study indicate that PKD is a protein kinase with distinct substrate specificity that is markedly stimulated by DAG analogues and PDBu in a PS-dependent manner and serves as a novel receptor for PDBu in eukaryotic cells.


EXPERIMENTAL PROCEDURES

Materials

[-P]ATP (5000 Ci/mmol; 37 GBq = 1 mCi) was from Amersham International (United Kingdom). [^3H]PDBu (18.6 Ci/mmol) was obtained from DuPont NEN. PS, PDBu, diC8, and OAG were obtained from Sigma. Other items were from standard suppliers or as indicated in the text.

Cell Culture

Stock cultures of COS-7 cells were maintained in DMEM supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO(2) at 37 °C. For experimental purposes, cells were plated either in 33-mm Nunc dishes at 10^5 cells/dish or in 90-mm dishes at 9 times 10^5 cells/dish in DMEM containing 10% fetal bovine serum.

Immunoprecipitation

Cultured COS-7 cells were washed three times in ice-cold PBS and lysed in 50 mM Tris/HCl, pH 7.6, 2 mM EGTA, 2 mM EDTA, 2 mM dithiothreitol, protease inhibitors aprotinin (10 µg/ml), leupeptin (100 µg/ml), and pepstatin (0.7 µg/ml), and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and 1% Triton X-100 (lysis buffer A).

Mouse lungs were homogenized using a Polytron homogenizer (10 s at setting 17) in lysis buffer B (lysis buffer A without Triton X-100). After the homogenization, 1% Triton X-100 was added. Mouse lung and COS-7 cell lysates were clarified by centrifugation at 100,000 times g for 30 min at 4 °C.

The synthetic peptide EEREMKALSERVSIL corresponding to the COOH-terminal amino acid sequence of PKD was conjugated to keyhole limpet hemocyanin, and antisera were prepared as described previously (22) . Proteins were immunoprecipitated at 4 °C for 2 h with the PA-1 antipeptide antiserum (1:50 dilution) in the absence or the presence of the immunizing peptide (2 µg/µl antiserum). The immune complexes were recovered using protein A coupled to agarose.

Elution of PKD from Immunocomplexes

The immunoprecipitates prepared as described in the preceding section were washed once with lysis buffer A and twice with lysis buffer B. PKD was then eluted at 4 °C for 30 min by batchwise incubation of the immunoprecipitates with 0.5 mg/ml immunizing peptide in lysis buffer B (4 volumes of elution buffer/1 volume of protein A-agarose).

Western Blot Analysis

For Western blot analysis, immunoprecipitates were washed three times with lysis buffer A and extracted for 10 min at 95 °C in 2 times SDS-PAGE sample buffer (20 mM Tris/HCl, pH 6.8, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol) and analyzed by SDS-PAGE and by transfer to Immobilon membranes. In other cases samples for Western blot analysis were prepared by directly lysing cultures in 2 times sample buffer and boiling for 10 min. The transfer was carried out at 100 V, 0.4 A at 4 °C for 4 h using a Bio-Rad transfer apparatus. The transfer buffer consisted of 200 mM glycine, 25 mM Tris, 0.01% SDS, and 20% CH(3)OH. Membranes were blocked using 5% nonfat dried milk in PBS, pH 7.2, and incubated with PA-1 antiserum (1:500) at room temperature for 4 h in PBS containing 3% nonfat dried milk. Immunoreactive bands were visualized using I-protein A (0.1 µCi/ml) and autoradiography.

Preparation of Catalytic Domain Fusion Protein

A 1032-base pair fragment comprising the entire catalytic domain of PKD was inserted in-frame into the inducible bacterial expression vector pMAL-c2(23) . The maltose binding protein fusion protein expressed in Escherichia coli was purified by affinity purification through an amylose resin column (New England Biolabs Inc.).

Construction of cDNA Expression Vectors and COS-7 Cell Transfection

The PKD cDNA fragment spanning bases -125 to 3179 was assembled by ligating the MunI-ClaI fragment of the L16.1 clone to the MunI-ClaI digest of the L20.2 clone in Bluescript. The assembled fragment was then subcloned into the mammalian expression vector pcDNA3 between the XhoI and XbaI restriction sites. The correct ligation of the resulting construct, pcDNA3-PKD, was confirmed by DNA sequencing of both strands.

Exponentially growing COS-7 cells, 40-60% confluent, were transfected with either pcDNA-3 or pcDNA3-PKD using Lipofectin as described in detail by the manufacturer (Life Technologies, Inc.). Briefly, 2 or 12 µg of DNA were used for 35- or 90-mm dishes, respectively. The DNA was diluted to 100 µl with Opti-mem I medium (Life Technologies, Inc.) and then mixed with Lipofectin (6 or 36 µl) diluted to 100 µl with Opti-mem I medium. After 15 min, the DNA-Lipofectin complex was diluted to 2 or 10 ml with Opti-mem I medium, mixed gently, and overlaid onto rinsed (1 times with Opti-mem I) COS-7 cells. The cultures were then incubated at 37 °C for 6 h, and the medium was then replaced with fresh DMEM containing 10% fetal bovine serum. The cells were used for experimental purposes 72 h later. The level of PKD expression was determined by [^3H]PDBu binding and by Western blot analysis.

PDBu Binding to COS-7 Cells

[^3H]PDBu binding to intact COS-7 cells was performed as described previously(24) . Briefly, cultures of transfected and untransfected cells were washed twice with DMEM and incubated with binding medium (DMEM containing 1 mg/ml bovine serum albumin and 10 nM [^3H]PDBu) at 37 °C for 30 min. The cultures were then rapidly washed at 4 °C with PBS and lysed, and bound radioactivity was determined using a Beckman beta scintillation counter. Nonspecific binding was determined in the presence of 10 µM PDBu.

[^3H]PDBu Binding to PKD after Immunoprecipitation and Elution

PKD eluted from lysates of COS-7 cells transfected with pcDNA3-PKD was incubated for 30 min at 4 °C in 300 µl of a mixture containing 20 mM Tris, pH 7.4, 125 µg/ml PS, and various concentrations of [^3H]PDBu (1.25-100 nM). Nonspecific binding of [^3H]PDBu was measured in the presence of excess nonlabeled PDBu (10 µM).

Free [^3H]PDBu was separated from bound [^3H]PDBu by binding of PKD to polyethyleneimine-treated GF/F Whatman filters. The filters were washed rapidly three times with ice-cold 20 mM Tris, pH 7.4, and bound radioactivity was measured using liquid beta scintillation counting.

Kinase Assay of PKD after Immunoprecipitation and Elution

The kinase activity of the eluted PKD was determined by mixing 20 µl of the PKD preparation with 20 µl of a phosphorylation mixture containing 200 µM [-P]ATP (specific activity, 400-600 cpm/pmol), 30 mM Tris, pH 7.4, 30 mM MgCl(2), and 2.5 mg/ml syntide-2 (PLARTLSVAGLPGKK, a peptide based on the phosphorylation site two of glycogen synthase). In addition, PDBu, OAG, or diC8 was added in the absence or presence of 100 µg/ml PS as indicated. After 5 min of incubation, the kinase reaction was stopped by spotting 30 µl of the supernatant on P-81 phosphocellulose filter paper. The [-P]ATP was separated from the labeled substrates by washing the P-81 filter papers four times for 5 min in 75 mM H(3)PO(4). The papers were then dried, and the radioactivity was incorporated into syntide-2 determined by Cerenkov counting. Syntide-2 phosphorylation by PKD in the absence or presence of activators was linear up to 10 min of incubation.


RESULTS

Expression of PKD in COS-7 Cells and Characterization of the PA-1 Antiserum

To characterize PKD at the protein level in mammalian cells, we raised a polyclonal antiserum against the synthetic peptide EEREMKALSERVSIL that corresponds to the COOH-terminal region of the predicted amino acid sequence of PKD. Western blot analysis of an affinity-purified fusion protein containing the catalytic domain of PKD with the PA-1 antiserum revealed an immunoreactive band of 82 kDa, which corresponds to the calculated molecular mass of the fusion protein (Fig. 1A). The detection of this band was completely blocked by the immunizing synthetic peptide. These experiments verified the specificity of the PA-1 antiserum.


Figure 1: Expression of PKD in COS-7 cells and characterization of the PA-1 antiserum. A, Western blot analysis of the catalytic domain fusion protein. Various amounts (0.1-1 µg of protein) of purified catalytic domain fusion protein (calculated molecular mass is 82 kDa) were analyzed by SDS-PAGE and transferred to Immobilon membranes. Western blot analysis using the PA-1 antiserum in the absence or in the presence of immunizing peptide (1.0+) was carried out as described under ``Experimental Procedures.'' B, PKD protein expression in transfected COS-7 cells. COS-7 cells (lane 1) or COS-7 cells transfected with pcDNA3 (lane 2) or pcDNA3-PKD (lanes 3 and 4) were washed twice with PBS at 4 °C and solubilized in 2 times sample buffer. In each case 2 times 10^5 cells were used. Following SDS-PAGE and transfer to Immobilon membranes, Western blot analysis was carried out using PA-1 antiserum in the absence (lanes 1-3) or presence (lane 4) of the immunizing peptide. Similar results were obtained in 14 independent experiments. C, lysates from COS-7 cells (lane 5), COS-7 cells transfected with pcDNA3 (lane 6), or COS-7 cells transfected with pcDNA3-PKD (lanes 7-9) were immunoprecipitated with the PA-1 antiserum in the absence (lanes 5-7 and 9) or in the presence (lane 8) of the immunizing peptide. The resulting immunocomplexes were analyzed by Western blotting using the same antiserum (lanes 5-8). In lane 9, the immunoprecipitates were washed and incubated with the immunizing peptide (30 min at 4 °C). The resulting eluate was subjected to Western blot analysis with the PA-1 antiserum. Similar results were obtained in four independent experiments.



To examine PKD expression in COS-7 cells, an assembled DNA fragment corresponding to the complete sequence of PKD (spanning bases -125 to 3179) was inserted between the XhoI and XbaI sites of the mammalian expression vector pcDNA3, and the resultant plasmid (pcDNA3-PKD) was transiently transfected in COS-7 cells. Cultures of these cells were lysed, and the extracts were subjected to Western blot analysis using the PA-1 antiserum. As shown in Fig. 1B, PA-1 recognized a single band migrating with an apparent molecular mass of 110 kDa in COS-7 cells transfected with pcDNA3-PKD (lane 3). This band was not detected in lysates from COS-7 cells transfected with pcDNA3 or from untransfected cells or when the immunoblots were incubated with PA-1 antiserum in the presence of the immunizing peptide (lanes 1, 2, and 4). A band migrating with an identical molecular mass (110 kDa) was also obtained when lysates from COS-7 cells transfected with pcDNA3-PKD were immunoprecipitated with the PA-1 antiserum, and the immunoprecipitates were analyzed by Western blotting using the same antiserum (Fig. 1C, lane 7). The detection of the 110-kDa band was blocked by the inclusion of the immunizing peptide during the immunoprecipitation (lane 8). Incubation of PA-1 immunoprecipitates with the immunizing peptide eluted the 110-kDa band from the immunocomplexes (lane 9). Furthermore, no bands were detected in PA-1 immunoblots of PA-1 immunoprecipitates of lysates of either COS-7 cells (lane 5) or COS-7 cells transfected with pcDNA3 (lane 6) or when preimmune serum was used instead of the PA-1 antiserum (data not shown). In the absence of detergents, PKD was recovered in both soluble and particulate fractions of the pcDNA3-PKD-transfected COS-7 cells. These results clearly demonstrate the transient expression of PKD in COS-7 cells and substantiate the specificity of the PA-1 antiserum.

[^3H]PDBu Binding to COS-7 Cells Transfected with PKD

Next, we determined whether the expression of PKD in COS-7 cells confers increased phorbol ester binding to these cells. COS-7 cells were transfected with either pcDNA3 or pcDNA3-PKD. Cells transfected with pcDNA3-PKD showed a marked increase (4.8 ± 0.7-fold) in specific [^3H]PDBu binding as compared with that obtained with COS-7 cells transfected with the vector alone or with untransfected cells (Fig. 2, left panel). Addition of unlabeled PDBu inhibited [^3H]PDBu binding to COS-7 cells transfected with pcDNA3-PKD in a concentration-dependent manner (Fig. 2, right panel). These results indicate that PKD can serve as a novel phorbol ester receptor in intact cells.


Figure 2: Expression of PKD in COS-7 cells confers increased [^3H]PDBu binding. Left panel, control COS-7 cells (gray bars) or COS-7 cells transfected with either pcDNA3 vector alone (open bars) or with the pcDNA3-PKD (closed bars) all cultured in 90-mm dishes, were washed twice with DMEM and incubated at 37 °C with 6 ml of binding medium containing 10 nM [^3H]PDBu for 30 min. Cell-associated radioactivity was determined as described under ``Experimental Procedures.'' Nonspecific binding (hatched bars) was determined in the presence of 10 µM unlabeled PDBu. The values represent the mean ± S.E. of five independent experiments each performed in triplicate. Right panel, dose-dependent inhibition of [^3H]PDBu binding by unlabeled PDBu. COS-7 cells cultured in 35-mm dishes were transfected with either pcDNA3 (open circles) or pcDNA3-PKD (closed circles). After 72 h, the cultures were washed and incubated at 37 °C with 1 ml of binding medium for 30 min either in the absence or in the presence of increasing concentrations of unlabeled PDBu.



To measure the affinity of [^3H]PDBu binding to the full-length PKD, lysates of COS-7 cells transfected with pcDNA3-PKD were immunoprecipitated with the PA-1 antiserum, and PKD was eluted from the immunocomplexes by incubation with the immunizing peptide (Fig. 1C, lane 9). As shown in Fig. 3, the specific binding of [^3H]PDBu to the eluted PKD preparation was saturable; Scatchard analysis of the data revealed a K(d) of 2.2 nM. This value is comparable with the K(d) determined for PDBu binding to PKC(11) .


Figure 3: Analysis of [^3H]PDBu binding to immunopurified PKD. PKD eluted from lysates of COS-7 cells transfected with pcDNA3-PKD was incubated with various concentrations of [^3H]PDBu. Specific binding of [^3H]PDBu to PKD was measured as indicated under ``Experimental Procedures.'' Results are of a representative experiment, with each point determined in duplicate. The values are expressed as picomoles bound per assay. Inset, Scatchard analysis of [^3H]PDBu binding to PKD. B/F, bound/free.



PDBu Stimulates PKD in Synergy with PS

The preceding findings prompted an investigation of the effects of PDBu on PKD kinase activity. Previous experiments showed that the bacterially expressed catalytic domain of PKD efficiently phosphorylated the synthetic peptide syntide-2(18) . Therefore, we have chosen syntide-2 as a model substrate to assay the kinase activity of the full-length PKD expressed in COS-7 cells. Our initial measurements of PKD kinase activity in PA-1 immunoprecipitates gave variable results, probably due to the presence of interfering factors or to immobilization of the kinase on the protein A-agarose beads. In contrast, we found that elution of PKD from the PA-1 immunoprecipitates by the immunizing peptide produced consistent results.

To determine the effect of PS and PDBu on the kinase activity of PKD, lysates of COS-7 cells transfected with either pcDNA3 or pcDNA3-PKD were immunoprecipitated with the PA-1 antiserum. The syntide-2 kinase activity eluted from the resultant immunocomplexes was measured in the absence or presence of various effectors. As shown in Fig. 4, a marked increase in syntide-2 kinase activity was detected in the eluates of PA-1 immunoprecipitates of COS-7 cells transfected with pcDNA3-PKD as compared with those obtained from COS-7 cells transfected with pcDNA3 (closed barsversusopen bars, respectively). The immunoprecipitation of this activity with PA-1 was virtually abolished by addition of the immunizing synthetic peptide. Furthermore, preimmune serum failed to immunoprecipitate syntide-2 kinase activity from COS-7 cells transfected with pcDNA3 PKD (results not shown). Addition of PS (100 µg/ml) or PDBu (250 nM) singly caused only a small increase (1.7 ± 0.1- and 1.3 ± 0.1-fold, respectively) in the syntide-2 kinase activity obtained from pcDNA3-PKD-transfected cells. In contrast, the combination of PS (100 µg/ml) with PDBu (250 nM) caused synergistic stimulation of syntide-2 kinase activity (4.3 ± 0.2-fold, n = 3). The stimulation of syntide-2 kinase activity by PDBu in the presence of PS was dose-dependent; half-maximum stimulation was obtained at approximately 25 nM PDBu in the presence of PS (Fig. 4). Addition of Ca did not have any effect on PKD activity either in the absence or in the presence of PDBu and PS (Fig. 4, inset). Interestingly, the syntide-2 kinase activity of the bacterially expressed catalytic domain of PKD was not affected by PS, PDBu, or both, providing evidence that these effectors act through the regulatory domain of PKD (results not shown). Thus, PKD is a novel serine/threonine kinase that is directly stimulated by PDBu in a phospholipid-dependent manner.


Figure 4: Synergistic stimulation of kinase activity of PKD by PDBu and PS. COS-7 cells transfected with pcDNA3 (open bars) or with pcDNA3-PKD (closed bars) were lysed, and the lysates were incubated with the PA-1 antiserum. PKD was then eluted from the resultant immunoprecipitates and analyzed in a syntide-2 phosphorylation assay as described under ``Experimental Procedures.'' The specific activity of [-P]ATP was 450 cpm/pmol. Kinase activity was measured either in the absence(-) or presence (+) of 100 µg/ml PS and without or with 25, 250, or 500 nM PDBu as indicated. The values are the means ± S.E. (n = 4). Similar results were obtained in two independent experiments. Inset, effect of Ca on PKD activity. PKD syntide-2 kinase activity was measured in the absence (-) or in the presence (PS + PDBu) of 250 nM PDBu and 100 µg/ml PS either without (closed bars) or with (hatched bars) 0.5 mM CaCl(2). Similar results were obtained with 0.2 mM CaCl(2), but an inhibitory effect was noticed at 1 mM CaCl(2).



Diacylglycerol Analogs Stimulate PKD in Synergy with PS

It was important to determine whether, in addition to PDBu, PKD could also be stimulated by the second messenger DAG. Lysates of COS-7 cells transfected with either pcDNA3 or pcDNA3-PKD were immunoprecipitated with the PA-1 antiserum. The syntide-2 kinase activity eluted from the resultant immunocomplexes was measured in the absence or presence of the membrane-permeable DAG analogs OAG or diC8 with or without PS. As shown in Fig. 5, addition of 20 µM OAG and PS or 20 µM diC8 and PS synergistically stimulated PKD kinase activity (4.9 ± 0.3- and 5.7 ± 0.1-fold, respectively). Synergistic stimulation of PKD activity was also evident when OAG or diC8 was added at 2 µM instead of 20 µM (Fig. 5). In contrast, each of these lipids alone had only minor effects on the syntide-2 kinase activity of PKD.


Figure 5: Synergistic stimulation of kinase activity of PKD by OAG or diC8 in the presence of PS. COS-7 cells transfected with pcDNA3 (open bars) or with pcDNA3-PKD (closed bars) were lysed, and the lysates were immunoprecipitated with the PA-1 antiserum. PKD was then eluted from the immunoprecipitates and analyzed in a syntide-2 phosphorylation assay as described under ``Experimental Procedures.'' The specific activity of [-P]ATP was 490 cpm/pmol. Kinase activity was measured either in the absence(-) or presence (+) of PS at 100 µg/ml and without or with OAG or diC8, as indicated. The values are the means ± S.E. (n = 4). Similar results were obtained in two independent experiments.



Substrate Specificity of PKD

To examine the substrate specificity of the full-length PKD, we tested the phosphorylation of different protein and peptide substrates by PKD in the presence or absence of lipid cofactors. As shown in Table 1, of the various substrates tested, syntide-2 was phosphorylated most efficiently, whereas histone III-S and protamine sulfate were only poorly phosphorylated. To examine the molecular basis of PKD substrate specificity more closely, we synthesized a variant syntide-2 that contains an alanine instead of an arginine at position 4 (syntide-2-R4A). The poor phosphorylation of syntide-2-R4A indicates that PKD prefers substrates with basic residues upstream of the phosphorylatable serine. It is very likely that PKD has additional requirements for phosphorylation since histone III-S and protamine also contain serines that are preceded by arginines but were nevertheless not phosphorylated by PKD. Interestingly, a peptide based on the pseudosubstrate site of PKC (with an alanine replaced by a serine) was not phosphorylated by PKD, whereas this peptide is efficiently phosphorylated by all members of the PKC family(25, 26) . Taken together, these data suggest that PKD possesses a substrate specificity that is clearly different from previously identified members of the PKC family.



PKD Autophosphorylation Is Dependent on PDBu or DAG in the Presence of PS

Autophosphorylation, a common feature among protein kinases, can be part of the kinase activation process (27) and may also regulate the interaction of the kinase with other proteins(28) . PKD eluted from PA-1 immunoprecipitates of lysates of COS-7 cells transfected with pcDNA3-PKD was incubated with [-P]ATP in the absence or presence of lipid cofactors and then analyzed by SDS-PAGE to examine whether it undergoes autophosphorylation. As shown in Fig. 6, a single radioactive band was revealed after autoradiography. This band migrated with an apparent molecular mass of 110 kDa, which was identical to the molecular mass of the protein obtained in the eluate of PA-1 immunoprecipitates and visualized by Western blot analysis (Fig. 1C, lane 9). PKD autophosphorylation was dramatically stimulated by PS combined with PDBu, OAG, or diC8. Scanning densitometry of the autophosphorylating bands showed that the combination of PS with PDBu (200 nM), OAG (20 µM), or diC8 (20 µM) increased phosphorylation by 14-, 24-, or 19-fold, respectively. PKD incubated with PS, PDBu, OAG, or diC8 alone autophosphorylated only to a very small extent.


Figure 6: Synergistic stimulation of PKD autophosphorylation by PDBu, OAG, and diC8 in the presence of PS. COS-7 cells transfected with pcDNA3-PKD were lysed, and the lysates were incubated with the PA-1 antiserum. PKD was then eluted from immunoprecipitates and analyzed in an autophosphorylation assay as described under ``Experimental Procedures.'' Autophosphorylation was determined in the absence(-) or in the presence (+) of PS at 100 µg/ml. PDBu (20 and 200 nM), OAG (2 and 20 µM), and diC8 (2 and 20 µM) were added as indicated. Similar results were obtained in two independent experiments.



Properties of PKD from Mouse Lung

The preceding experiments characterized the lipid dependence of PKD expressed in COS-7 cells. To further substantiate these data, we have also investigated the properties of PKD extracted from an animal tissue. Our previous analysis of expression of PKD mRNA in mouse tissues revealed that lung shows a prominent expression of PKD mRNA(18) . Initial immunoblotting analysis of different animal tissues with the PA-1 antiserum verified that PKD has a wide tissue distribution and that mouse lung contains an abundant 110-kDa protein reacting with this antiserum in the absence but not in the presence of immunizing peptide. Lysates of mouse lung were incubated with the PA-1 antiserum in the absence or presence of the immunizing peptide, and the resulting immunocomplexes were treated with the immunizing peptide to elute PKD. As shown in Fig. 7, the ability of lung PKD to catalyze the phosphorylation of syntide-2 was stimulated by addition of PS in combination with either PDBu, OAG, or diC8. The detection of this activity was virtually abolished by the addition of the immunizing peptide during the immunoprecipitation. Furthermore, the autophosphorylation of the lung PKD was markedly stimulated (5-7-fold) by the addition of PDBu, OAG, or diC8 in the presence of PS (Fig. 7, lower panel).


Figure 7: Kinase activity and autophosphorylation of mouse lung PKD. Mouse lungs were homogenized, and the lysates were incubated with the PA-1 antiserum. PKD was then eluted from immunoprecipitates by incubation with the immunizing peptide and analyzed in a syntide-2 kinase assay (upper panel) and in an autophosphorylation assay (lower panel) as described under ``Experimental Procedures.'' The specific activity of the [-P]ATP was 540 cpm/pmol. The kinase assay and the autophosphorylation assay were performed in the absence(-) or presence of 100 µg/ml PS (+) and without or with 200 nM PDBu, 20 µM OAG, and 20 µM diC8, as indicated.




DISCUSSION

The results presented here indicate that PKD is a protein kinase with distinct substrate specificity that is stimulated by phorbol ester or diacylglycerols in a phospholipid-dependent manner and can serve as a receptor for PDBu in intact cells. The following lines of evidence support our conclusion. 1) COS-7 cells transfected with pcDNA3-PKD displayed a marked increase in high affinity PDBu binding. 2) The kinase activity of PKD, as measured with the substrate syntide-2, was regulated by lipids. The addition of PDBu, OAG, or diC8 in combination with PS markedly stimulated the kinase activity of PKD in a synergistic fashion. 3) The same lipid effector combinations strikingly increased the autophosphorylation of PKD in a synergistic fashion. In agreement with these findings, we have previously demonstrated that the bacterially expressed NH(2)-terminal region of PKD, which contains two cysteine-rich zinc finger-like domains bound [^3H]PDBu in a specific fashion(18) . To further substantiate the validity of our conclusions we also isolated PKD from mouse lung tissue. We demonstrate for the first time the existence of PKD activity in an animal tissue and show that its regulatory characteristics are similar to those of recombinant PKD expressed in COS-7 cells.

Johannes et al.(21) recently cloned a human protein kinase termed PKCµ with 92% homology to PKD. While it is highly likely that the two kinases are functional homologs, Johannes et al.(21) failed to demonstrate any phorbol ester binding and regulation and consequently concluded that this enzyme is an atypical PKC. These authors suggested that two amino acid substitutions in the cysteine-rich region could be responsible for these results. In addition, they suggest that the spacing of the cysteine-rich motifs by 87 amino acids (as opposed to 28-35 amino acids in phorbol ester-binding PKCs) could result in an inappropriate conformation for efficient PDBu binding. In contrast, our results provide compelling evidence indicating that PKD is a novel target for phorbol esters as well as for DAG.

The structural differences between the catalytic domains of PKD and PKCs (see the Introduction) suggested that these proteins could have important functional differences. We examined the ability of full-length PKD to phosphorylate a variety of potential substrates. These studies revealed that PKD preferentially phosphorylated the synthetic peptide syntide-2. The poor phosphorylation of a variant syntide-2 with arginine-4 replaced by an alanine indicates that the presence of a basic amino acid upstream of the phosphorylatable serine seems to be essential for efficient phosphorylation. In contrast, PKD did not efficiently catalyze phosphorylation of a number of substrates utilized by PKCs such as protamine sulfate, histone III-S, and a peptide substrate based on the sequence of the pseudosubstrate region of PKC. Importantly, the PKC peptide is very efficiently phosphorylated by classic, novel, and atypical PKCs(25, 26) . Although PKCs alpha, beta, and utilize histone as a substrate more effectively than the novel PKCs(29, 30) , it has become apparent that this difference is not an intrinsic property of the catalytic domains but is conferred by the influence of the regulatory domain. Indeed, novel PKCs such as and that have been rendered constitutively active by proteolysis or mutation phosphorylate histone as efficiently as the classic PKCs(31, 32, 33) . In this context, the inability of both full-length PKD and of the bacterially expressed catalytic domain of PKD (18) to efficiently phosphorylate histone clearly emphasizes the functional differences between PKD and PKCs.

Taken together, our results indicate that PKD is not an atypical PKC since we clearly demonstrate that PKD binds phorbol esters and that the activity of this enzyme is markedly stimulated by PDBu, OAG, or diC8 in the presence of PS. Moreover, we found fundamental differences in both substrate specificity and catalytic domain protein structure between PKD and PKCs. In addition, PKD can be distinguished from PKCs by other important structural features. 1) PKD does not contain a typical PKC pseudosubstrate motif, which is present upstream of the cysteine-rich region in all PKCs. 2) The NH(2) terminus of PKD possesses a highly hydrophobic stretch of amino acids, suggesting a transmembrane domain, which is not found in any of the PKCs. 3) PKD contains a pleckstrin homology domain inserted between the cysteine-rich motifs and the catalytic domain. Pleckstrin homology domains have recently been identified in a variety of intracellular signaling and cytoskeletal proteins but are not present in PKCs(34) . We conclude that PKD cannot be classified in any of the PKC subfamilies.

Phorbol esters act as potent tumor promoters and induce a variety of responses in many cultured cell types including effects on ionic channels, second messenger production, cell-cell communication, membrane transport, protein phosphorylation, and cellular growth, morphology, differentiation, and transformation(1, 5, 6, 35, 36) . The identification of another target for phorbol esters, PKD, which is expressed in many organs and tissues as well as in cultured cells (18) ^3 raises the possibility that some of the actions of phorbol esters could be mediated partially or exclusively by PKD.

One of the earliest responses of many cell types to extracellular stimuli is an increase in the synthesis of DAG, the physiological second messenger generated through multiple pathways(3, 4) . The results presented here demonstrate, for the first time, that the protein kinase activity of PKD is synergistically stimulated by diacylglycerol analogs in combination with PS. Thus, PKD could be a novel component in the signal transduction of many growth factors, regulatory peptides, and cytokines that elevate DAG in their target cells.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Postdoctoral fellow of the European Molecular Biology Organization.

To whom correspondence should be addressed.

(^1)
The abbreviations used are: DAG, 1,2-diacylglycerol; PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate; diC8, 1,2-dioctanoyl-sn-glycerol; OAG, 1-oleoyl-2-acetylglycerol; PS, dioleoylphosphatidylserine; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PKD, protein kinase D.

(^2)
The rank order of homologies between the catalytic domain of PKD and that of some other kinases is: myosin light chain kinase of Dictyostelium (41%) > Ca/calmodulin-dependent protein kinase type II > Ca/calmodulin-dependent protein kinase type IV > cAMP-dependent protein kinase > phosphorylase b kinase.

(^3)
J. Van Lint, A. Valverde, J. Sinnett-Smith, and E. Rozengurt, unpublished results.


ACKNOWLEDGEMENTS

We thank Simon Broad for expert technical assistance.


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