Molecular Cloning and Characterization of the Human Protein Kinase D2

A NOVEL MEMBER OF THE PROTEIN KINASE D FAMILY OF SERINE THREONINE KINASES*

Sabine SturanyDagger , Johan Van Lint§, Friedericke MüllerDagger , Monika Wilda||, Horst Hameister||, Michael Höcker**, Andreas BreyDagger , Ulrike GernDagger , Jackie Vandenheede§, Thomas GressDagger , Guido AdlerDagger , and Thomas SeufferleinDagger DaggerDagger

From the Dagger  Abteilung Innere Medizin I and || Institut für Humangenetik, Medizinische Fakultät der Universität, Ulm, Germany, the § Afdeling Biochemie, Katholieke Universiteit Leuven, Belgium, and the ** Medizinische Klinik mit Schwerpunkt Gastroenterologie und Hepatologie, Universitätsklinikum Charité, Campus Virchow-Klinikum, Humboldt Universität, Berlin, Germany

Received for publication, September 22, 2000, and in revised form, October 29, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have isolated the full-length cDNA of a novel human serine threonine protein kinase gene. The deduced protein sequence contains two cysteine-rich motifs at the N terminus, a pleckstrin homology domain, and a catalytic domain containing all the characteristic sequence motifs of serine protein kinases. It exhibits the strongest homology to the serine threonine protein kinases PKD/PKCµ and PKCnu , particularly in the duplex zinc finger-like cysteine-rich motif, in the pleckstrin homology domain and in the protein kinase domain. In contrast, it shows only a low degree of sequence similarity to other members of the PKC family. Therefore, the new protein has been termed protein kinase D2 (PKD2). The mRNA of PKD2 is widely expressed in human and murine tissues. It encodes a protein with a molecular mass of 105 kDa in SDS-polyacrylamide gel electrophoresis, which is expressed in various human cell lines, including HL60 cells, which do not express PKCµ. In vivo phorbol ester binding studies demonstrated a concentration-dependent binding of [3H]phorbol 12,13-dibutyrate to PKD2. The addition of phorbol 12,13-dibutyrate in the presence of dioleoylphosphatidylserine stimulated the autophosphorylation of PKD2 in a synergistic fashion. Phorbol esters also stimulated autophosphorylation of PKD2 in intact cells. PKD2 activated by phorbol esters efficiently phosphorylated the exogenous substrate histone H1. In addition, we could identify the C-terminal Ser876 residue as an in vivo phosphorylation site within PKD2. Phosphorylation of Ser876 of PKD2 correlated with the activation status of the kinase. Finally, gastrin was found to be a physiological activator of PKD2 in human AGS-B cells stably transfected with the CCKB/gastrin receptor. Thus, PKD2 is a novel phorbol ester- and growth factor-stimulated protein kinase.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The PKC1 family of serine threonine kinases is implicated in the regulation of fundamental biological phenomena such as growth, differentiation, transformation, and apoptosis. Molecular cloning has demonstrated the presence of multiple related PKC isoforms, i.e. classical PKCs (cPKCs: alpha , beta 1, beta 2, and gamma ), novel PKCs (nPKCs: delta , epsilon , eta , and theta ), and atypical PKCs (aPKCs: zeta  and lambda ), all of which possess a highly conserved catalytic domain. The regulatory domain of both classical 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. In contrast, atypical PKCs contain a single cysteine-rich motif, do not bind phorbol esters, and are not regulated by DAG. Another phorbol ester and DAG-stimulated protein kinase containing a duplex cysteine-rich zinc-finger-like motif has been cloned from a murine cDNA library and termed protein kinase D (PKD) (1). Its human isotype, termed PKCµ (2), was initially regarded as a member of the aPKC subfamily (2). However, examination of the catalytic domain of PKD/PKCµ revealed that the kinase subdomains show little similarity to the highly conserved regions of the kinase subdomains of the PKC family but are related to the calmodulin-dependent protein kinase II-like protein kinases. Furthermore, both the substrate specificity of PKD/PKCµ (1, 3) and their sensitivity to inhibitors (4) are unlike those of the PKC family. In addition, these proteins contain a pleckstrin homology domain located between the duplex zinc-finger like motif and the kinase domain but lack sequences with homology to a typical PKC pseudosubstrate motif upstream of the cysteine-rich region. Interestingly, PKD is itself regulated by novel PKCeta and PKCepsilon (5, 6). Thus, these kinases are more distantly related to the PKC family and may constitute a distinct family of serine threonine kinases (7, 8). Recently, the sequence of a third member of this group has been reported, which has been termed PKCnu (9).

Here we report the cloning of a novel protein kinase termed PKD2. The PKD2 mRNA is widely expressed and encodes an 878-amino acid protein that is most highly related to human PKCµ (69% identity). It contains a duplex zinc-finger-like motif, a pleckstrin homology domain, and a catalytic domain exhibiting all the characteristic sequence motifs of serine protein kinases. The catalytic domain exhibits the highest similarity to PKD/PKCµ (92% identity). The deduced amino acid sequence encodes a protein with a molecular mass of 105 kDa in SDS-PAGE. PKD2 serves as a novel receptor for PDBu in eukaryotic cells and is activated in vitro by phorbol esters in a PS-dependent manner. In addition, PDBu stimulated the autokinase activity of PKD2. Moreover, activated PKD2 efficiently phosphorylated histone H1 in vitro. We identified the C-terminal Ser876 residue as an in vivo phosphorylation site within active PKD2. Phosphorylation of Ser876 correlated well with the activation status of the kinase. Finally, gastrin was found to be a potent physiological activator of PKD2 in human AGS-B cells stably transfected with the CCKB receptor. Thus, PKD2 is a novel phorbol ester- and growth factor-activated protein kinase and therefore a novel component in the signal transduction pathways induced by phorbol esters and the CCKB/gastrin receptor.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [gamma -32P]ATP (5000 Ci/mmol; 37 GBq = 1 mCi) and [3H]PDBu (18.6 Ci/mmol) were from Amersham Pharmacia Biotech. The polyclonal anti-PKCµ antibodies directed against the C or the N terminus of PKCµ were from Santa Cruz Biotechnology Inc. (antibodies D-20 and N-20, respectively). All other reagents were from standard suppliers as indicated in the text.

Cell Culture-- Stock cultures of HEK293, HL60, and AGS-B cells were maintained in DMEM supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO2 at 37 °C. AGS-B cells are AGS human gastric cancer cells (American Type Culture Collection) stably transfected with the expression construct CCKB-pcDNAI-neo comprising the full-length coding region of the human CCKB/gastrin receptor and the neomycin resistance gene as described previously (10). Human umbilical vein endothelial cells (HUVEC) were isolated from anonymous pathological specimens by collagenase digestion with the use of established techniques as previously described (11).

Cloning of PKD2 and Construction of cDNA Expression Vectors and the PKD2-S876A Mutant-- A 1.672-kb EST clone (Xs220) was used for 5'-RACE using a 5'-RACE system for rapid amplification of cDNA ends (version 2.0, Life Technologies Inc.). Several rounds of RACE yielded overlapping clones resulting in a 5'-extension of the Xs220 clone by 805 bp. We did not achieve any further 5'-extension using RACE. Therefore, a human pancreatic ductal library was used for hybridization with the composite clone resulting from Xs220 and the sequence obtained in the 5'-extensions. One of the clones obtained in this screen yielded a 3583-bp cDNA with a putative initiation codon at position 724. To generate a FLAG-PKD2 construct the ATG in full-length PKD2 was destroyed by PCR. Using degenerate (underlined) primers (sense: 5'-gcgaattcgccaccgccccctctt-3', antisense: 5'-ggccctggcgcactaggccgaaga-3'), a 450-bp fragment was generated. This fragment was cloned together with the remaining PKD2 cDNA in-frame into the FLAG-pcDNA3 vector. A single point mutation at the Ser876 residue was generated using a polymerase chain reaction-based technique. Briefly, a polymerase chain reaction fragment containing mutant nucleotides (underlined) encoding a serine to alanine substitution at the 876 site was obtained using the following oligonucleotides: forward primer, 5'-gggctggcggagcgcatcgctgtt-3'; reverse primer, 5'-ccagtttgggcaggaagccacttt-3'. The resulting polymerase chain reaction fragment was digested with BamHI/BssHII and used to replace the original pcDNA3-FLAG-PKD2 BamHI/BssHII fragment. All constructs were verified by restriction enzyme digestion and DNA sequencing.

HEK293 Cell Transfection-- Exponentially growing HEK293 cells (5 × 104 cells/35-mm dish) were transfected with either pcDNA3-FLAG, pcDNA3-FLAG-PKD2, or pcDNA3-FLAG-PKD2-S876A using Fugene as described in detail by the manufacturer (Roche Molecular Biochemicals). Briefly, 0.5 µg of DNA was used for each 35-mm dish. Fugene was diluted with 100 µl of DMEM and, after 5 min, added to the DNA. After another 15 min of incubation with gentle mixing, the DNA·Fugene complex in DMEM was added to HEK293 cells in DMEM/10% fetal bovine serum. The cells were used for experimental purposes 48 h later.

Northern (RNA) Blot Analysis-- Northern blot analysis was performed using a [alpha -32P]dCTP-labeled 737-bp EcoRI/BstZ17I PKD2 cDNA fragment corresponding to bases 1-740 of human PKD2 according to standard procedures. RNA from several primary human tissues was analyzed with commercially available poly(A)+-RNA blots (CLONTECH).

cDNA Synthesis and in Situ Hybridizations-- cDNA synthesis for PCR analysis was performed according to the manufacturer's protocol using RNA prepared from the murine T-lymphocyte cell line EL4, which was obtained from the ATCC. RT-PCR was performed using the sense primer 5'-ctgccctgcacggctgaaga-3' corresponding to bases 643-662 and the reverse primer 5'-cagggaagatctggtagaca-3' corresponding to bases 1641-1660 of human PKD2. The RT-PCR yielded a 1.023-kb fragment encoding the second cysteine-rich domain and the PH domain of murine PKD2. This fragment shared 885 identical bases with human PKD2 (87% identity). Embryos were fixed overnight with 4% paraformaldehyde in PBS at 4 °C and were subsequently prepared for cryostat sectioning. 10-µm sections were mounted on slides. The 1.023-kb murine PKD2 fragment was used to prepare sense and antisense riboprobes. RNA probes for each cDNA strand were generated by in vitro transcription using the Sp6/T7 in vitro transcription kit (Roche Molecular Biochemicals) and [35S]rUTP (Amersham Pharmacia Biotech) as radioactive label. Labeled RNA (80,000 cpm/µl) was added to the hybridization mix consisting of 50% formamide, 10% dextran sulfate, 0.3 M NaCl, 10 mM Tris, 10 mM sodium phosphate, pH 6.8, 20 mM dithiothreitol (DTT), 0.1% Triton X-100, and 0.1 mg/ml Escherichia coli RNA. Hybridization was performed overnight at 54 °C. The slides were washed at the same temperature in hybridization salt solution. After RNase A digestion, washing was continued in 0.2× and 0.1× SSC, and the slides were dehydrated at increasing ethanol concentrations and dried for autoradiography. The slides were coated with Ilford K5 emulsion and exposed for 14-28 days in the dark. After development, the slides were counterstained with Giemsa and photographed with bright and dark field illumination.

In Vitro Transcription and Translation-- In vitro transcription and translation were performed using the TNT quick coupled transcription/translation system according to the manufacturer's instructions (Promega Corp.). T7 RNA polymerase was used for transcription of PKD2 from the T7 promoter in pcDNA3, obtaining a sense transcript of PKD2. [35S]Methionine-labeled proteins were subjected to reducing SDS-PAGE and further developed by autoradiography.

PDBu Binding to HEK293 Cells-- For determination of [3H]PDBu binding to intact HEK293 cells, cultures of HEK293 cells transfected with either pcDNA3-FLAG or pcDNA3-FLAG-PKD2 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 1 µM PDBu.

PKD2 Antibodies, Western Blotting, and Immunoprecipitations-- The synthetic peptide GLPTDRDLGGACPPQD corresponding to amino acids 849-864 of PKD2 and the synthetic phosphopeptide AERISpVL corresponding to amino acids 872-878 of human PKD2, respectively, were conjugated to keyhole limpet hemocyanin, and antisera were prepared in rabbits. The polyclonal PKD2 antibody directed against amino acids 849-864 of human PKD2 was subsequently subjected to affinity purification. For detection of PKD2 protein, membranes were blocked using 3% nonfat dry milk in phosphate-buffered saline (pH 7.2) and incubated for 2 h with anti-FLAG mAb (1:440 = 5 µg/ml, Sigma), polyclonal PKD2 antibody (1:200), or polyclonal pSer876 antibody (1:2000) in phosphate-buffered saline containing 3% nonfat dry milk. Immunoreactive bands were visualized using horseradish peroxidase-conjugated anti-mouse (for the anti-FLAG antibody) or anti-rabbit IgG (for the anti-PKD2 or pSer876 antibodies) and subsequent ECL. For immunoprecipitations, HEK293 cells on 35-mm dishes transfected with various plasmids as described in the figure legends were washed twice in ice-cold PBS and lysed in a solution containing 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), pepstatin (0.7 µg/ml), 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and 1% Triton X-100 (lysis buffer I). Proteins were immunoprecipitated with the PKD2 antiserum (1:100 dilution), the pSer876 PKD2 antiserum (1:200), or with the monoclonal FLAG antibody precoupled to Sepharose (Sigma) in the absence or presence of the immunizing peptide (2 µg/µl antiserum). The immune complexes were recovered using protein A-Sepharose where appropriate. For the immunoprecipitations of endogenous PKD2 we used 1 mg of protein/condition.

Elution of PKD2 from Immunocomplexes-- Immunoprecipitates were prepared as described above and washed once with lysis buffer I and twice with lysis buffer II (lysis buffer I without Triton X-100). 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 II (elution buffer; 4 volumes of elution buffer/1 volume of protein A-Sepharose).

In Vitro Kinase Assay-- For experiments investigating the effect of PS and PDB on in vitro kinase activity, PKD2 was eluted from the immunocomplexes as described in the preceding section and 20 µl of the eluate was mixed with 20 µl of phosphorylation mix containing 100 µM [gamma -32P]ATP (specific activity, 400-600 cpm/pmol), 30 mM Tris, pH 7.4, 30 mM MgCl2, 1 mM DTT, and 250 nM PDBu and/or 100 µg/ml PS. The mixture was subsequently incubated for 10 min at 30 °C. Reactions were terminated by adding an equal amount of 2× SDS-PAGE sample buffer (1 M Tris-HCl, pH 6.8, 0.1 mM Na3VO4, 6% sodium dodecyl sulfate, 0.5 M EDTA pH 8.0, 4% 2-mercaptoethanol, 10% glycerol) and were further analyzed by SDS-PAGE. To examine autokinase activity and histone or aldolase phosphorylation, PKD2 was not eluted. Immune complexes were washed twice with lysis buffer I, twice with lysis buffer II, and twice with kinase buffer (30 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM DTT) and then resuspended in 30 µl of kinase buffer containing 0.5 mg/ml histone H1 or 0.5 mg/ml aldolase and 100 µM [gamma -32P]ATP. Reactions were incubated for 10 min at 30 °C, terminated by adding an equal amount of 2× SDS-PAGE sample buffer and further analyzed by SDS-PAGE.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Structural Analysis of PKD2-- We screened human EST data bases using degenerate oligonucleotide sequences based on the amino acid sequence conserved in the C terminus of serine kinases. A clone was identified that contained a novel human sequence. This EST clone contained 1661 bp of coding region and 11 bp of 3'-untranslated region. Using 5'-rapid amplification of cDNA ends to isolate overlapping 5'-fragments of this novel cDNA, the EST clone could be extended by a further 805 bp. The composite clone was then used to screen a human ductal pancreatic library. A cDNA clone was isolated that encoded a new member of the serine threonine protein kinase family. The open reading frame of the clone encompasses 2634 nucleotides. Downstream of the first in-frame ATG, a purine (G) is located at position +4. This is in accordance with the Kozak predictions for an initiation codon (12). Upstream of this first ATG the 5'-untranslated sequence contains three in-frame stop codons (data not shown).2 The deduced amino acid sequence indicated that this kinase is a protein of 878 amino acids with a predicted molecular mass of 97 kDa (Fig. 1A). Data base search revealed that the kinase has the highest homology to PKD/PKCµ and PKCnu sharing 69 and 64% identical amino acids with PKD/PKCµ and PKCnu , respectively (Fig. 1B and Table I). Therefore, we designated the novel protein PKD2 for protein kinase D2. Hydropathy analysis revealed a hydrophobic area at the N terminus of PKD2, which could correspond to a potential transmembrane region. Located downstream from amino acid 138, PKD2 contains two characteristic cysteine-rich motifs (His-X12-Cys-X2-Cys-X10-14-Cys-X4-His-X2-Cys-X7-Cys), which form a complex with the heavy metal ions zinc and cadmium (13) and are responsible for phorbol ester binding (14, 15). These duplex zinc-finger-like repeats are also found in all members of the PKC family except PKCzeta and PKClambda /iota , in which only a single domain has been identified. At the level of the amino acids, the first zinc-finger-like motif of PKD2 shows 92 and 84% identity, and the second zinc-finger 88 and 88% identity to the corresponding domains in PKD/PKCµ and PKCnu , respectively.



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Fig. 1.   A, predicted amino acid sequence of human PKD2. The cysteine-rich domains (CRD), the PH domain and the kinase domain are in bold. B, the predicted amino acid sequence of human PKD2 was aligned with human PKCµ (NM 002742), murine PKD (NM 008858), and human PKCnu (NM 005813) amino acid sequences using the Align PPC/MacMolly Tetra program. Identical amino acids are denoted in boldface.


                              
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Table I
The percentage of amino acid identity between human PKD2 and the other PKD family members, PKD/PKCµ (mouse/human), and human PKCnu
Identity calculations were performed using the Align PPC/MacMolly Tetra program.

In contrast, we found no homology to the second conserved domain (C2) common to all classical PKCs, the activation of which is Ca2+-dependent. Interestingly, the two cysteine-rich domains in PKD2 are separated by 76 amino acids. This is in contrast to a 15- and a 22-amino acid spacer in cPKCs and nPKCs but similar to the 95-amino acid spacer in PKD (7). The tandemly repeated cysteine-rich domains are followed by a region of ~113 amino acids that exhibits the characteristics of a PH domain, which is also found in PKD/PKCµ and PKCnu (2, 7, 9) but cannot be detected in other PKCs. PKD2 shares 65 and 58% of the amino acids in this domain with PKD/PKCµ and PKCnu , respectively (Fig. 1B and Table I).

Comparison of the putative kinase domain of PKD2 to those of other kinases (Fig. 1B) revealed the expected identity of those amino acids that are invariant among known kinase family members. Thus, the ATP binding consensus sequence Gly-X-Gly-X2-Gly-X16-Lys, where X represents any amino acid, is conserved in the kinase domain of PKD2 (amino acids 558-580). Furthermore, the invariant aspartate essential for kinase activity is located at the predicted position within the conserved motif HCDLXXXN (amino acids 672-678). In PKD2 the XXX motif is KPE, which is found most often in serine threonine kinases and not AAR or RAA, which are typical of tyrosine kinases (16). The amino acid sequence of the PKD2 kinase domain is very closely related to that of PKD/PKCµ (92%) and PKCnu (91%) but not to any other member of the PKC family (Table I). Of all other members of the PKC family, PKCbeta exhibited the highest similarity to PKD2 with only 27 and 34% identical amino acids in the kinase domain and the first cysteine-rich domain, respectively.

Expression of PKD2 in Various Tissues-- Northern blot analysis of human poly(A)+-RNA from various human tissues using an 807-kb fragment comprising the 5'-end, the first zinc-finger-like motif, and partly the second zinc-finger-like motif identified a 4-kb transcript in several human tissues indicating that the cloned cDNA is full-length and that PKD2 is widely distributed (Fig. 2A). High steady-state levels were detected in human pancreas, heart, lung, smooth muscle, and brain. Interestingly, in contrast to PKCµ (2), high levels of PKD2 mRNA could not be detected in kidney and liver suggesting that the different members of the PKD family exhibit some degree of tissue-specific expression. Overall, constitutive mRNA expression of PKD2 in normal tissues appeared rather low. Typically, a signal could only be detected after a longer exposure (>4 days) of the blots. In some tissues, an additional transcript of about 2 kb was detected, which might be a result of alternative splicing.



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Fig. 2.   A. Northern blot analysis of the PKD2 gene expression in various human tissues (P = pancreas, K = kidney, SM = smooth muscle, Li = liver, Lu = lung, Pl = placenta, B = brain, H = heart). B, in situ localization of the murine PKD2 transcripts in colon, lung, and testis of adult mice. Sections were hybridized with antisense murine PKD2 riboprobes and photographed under brightfield (left) and dark-field (right) illumination. The microphotographs of colon (Co) and lung (Lu) were taken at 50× magnification, the microphotograph of the testis (Te) was taken at 20× magnification.

For detection of PKD2 expression in adult mice, we isolated RNA from the murine T-lymphocyte cell line EL4. The RNA was reverse-transcribed, and RT-PCR was performed using the sense primer 5'-ctgccctgcacggctgaaga-3' corresponding to bases 643-662 and the reverse primer 5'-cagggaagatctggtagaca-3' corresponding to bases 1641-1660 of human PKD2. The RT-PCR yielded a 1.023-kb fragment encoding the second cysteine-rich domain and the PH domain of murine PKD2. This fragment shared 885 identical bases with human PKD2 (87% identity). In contrast, murine PKD shares only 693 identical bases with human PKD2 (68% identity; data not shown) suggesting that the cloned fragment indeed corresponds to the murine homologue of human PKD2. In situ hybridization using the 1.023-kb probe demonstrated constitutive expression of murine PKD2 in various adult murine tissues. In addition to PKD2 mRNA expression in those tissues identified by Northern blot analysis, murine PKD2 was found to be highly expressed in rapidly proliferating tissues such as colonic crypts and testis (Fig. 2B).

The PKD2 cDNA Directs the Synthesis of a 105-kDa Protein in Vitro and in Vivo-- To confirm the predicted open reading frame, human PKD2 transcripts were synthesized in vitro and translated in a cell free system in the presence of 35S-labeled methionine. The T7 sense transcript produced a polypeptide with an estimated molecular mass of 105 kDa as revealed by SDS-PAGE under reducing conditions (Fig. 3A). These data are similar to the calculated mass of the deduced protein sequence of 97 kDa. The apparent 8-kDa difference in molecular mass might reflect unusual migration of the protein in SDS gels or post-translational modifications. No polypeptides of comparable size were synthesized when only the pcDNA3-FLAG vector was used for in vitro translation (Fig. 3A, lane 1).



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Fig. 3.   The PKD2 cDNA directs the synthesis of a 105-kDa protein. A, PKD2 transcripts were synthesized in vitro and translated in a cell-free system in the presence of 35S-labeled methionine. The T7 sense transcript produced a protein with an estimated molecular mass of 105 kDa as revealed from SDS-PAGE under reducing conditions (PKD2). No protein of comparable size was observed when only the pcDNA3 vector was used for in vitro transcription/translation (-). B, HEK293 cells were transfected with pcDNA3-FLAG (V) or pcDNA3-FLAG-PKD2 (PKD2). 48 h after transfections, cells were lysed. Lysates were immunoprecipitated with anti-FLAG or anti-PKD2 antibody, respectively, and further analyzed by Western blotting with the anti-FLAG or anti-PKD2 antibody as indicated. C, HEK293 cells were transfected with pcDNA3-FLAG-PKD2 (PKD2) or pcDNA3-PKCµ (PKCµ). 48 h after transfections, cells were lysed, immunoprecipitated using the PKD2 antiserum or an antibody directed against the C terminus of PKCµ, and further analyzed by Western blotting with the anti-PKD2 antiserum or the anti-PKCµ antibody, respectively. D, left panel: nontransfected HEK293, HeLa, HL60, and HUVEC cells were immunoprecipitated with the anti-PKD2 antiserum followed by Western blotting with the same antibody. D, right panel: nontransfected HEK293 and HL60 cells were immunoprecipitated with an antibody directed against the N terminus of PKCµ followed by Western blotting with the same antibody. In each case, the experiments shown are representative of at least three independent experiments.

To characterize PKD2 at the protein level, HEK293 cells were transfected with the pcDNA3-FLAG-PKD2 plasmid and cell lysates were analyzed by Western blotting with an anti-FLAG mAb. These experiments also revealed a protein of 105 kDa corresponding to the in vitro translated protein. A protein with an identical molecular mass was identified when lysates of HEK293 cells transfected with FLAG-PKD2 were subjected to immunoprecipitation with a FLAG mAb followed by anti-FLAG-Western blotting. In both cases, a similar protein could not be detected when HEK293 cells were transfected with pcDNA3-FLAG vector alone (Fig. 3B). Next, we raised a polyclonal antiserum against the synthetic peptide GLPTDRDLGGACPPQD corresponding to amino acids 850-865 at the C-terminal region of the predicted amino acid sequence of human PKD2. This peptide was chosen, because it exhibits the least homology to PKCµ, PKD, or PKCnu at the C terminus. A protein band migrating with an identical molecular mass of 105 kDa was obtained when lysates of HEK293 cells transfected with FLAG-PKD2 were immunoprecipitated with the polyclonal anti-PKD2 antibody followed by Western blotting using the same affinity purified antiserum. Identical results were obtained when lysates of HEK293 cells transfected with FLAG-PKD2 were either immunoprecipitated with PKD2 antiserum followed by Western blot analysis with the FLAG antibody or immunoprecipitated with the FLAG antibody followed by Western blot analysis with the PKD2 antiserum (Fig. 3B). In addition, the antibody failed to detect FLAG-PKD2 upon preincubation with the immunizing peptide (data not shown). Thus, the polyclonal antiserum detects transfected PKD2.

To determine whether the PKD2 antiserum cross-reacts with PKD/PKCµ, HEK293 cells were transfected with PKCµ or PKD2. Our results show that the polyclonal PKD2 antiserum preferentially detected PKD2 and only very weakly PKCµ (migrating with a molecular mass of about 115 kDa) upon overexpression of these proteins in HEK293 cells. In contrast, a polyclonal antibody directed against the C terminus of PKCµ detected PKCµ as efficient as PKD2 upon overexpression of the respective plasmids in HEK293 cells (Fig. 3C). Thus, the PKD2 antiserum specifically recognizes PKD2.

To examine endogenous expression of PKD2 in various human cell lines, lysates of HEK293, HeLa, HL60, and HUVEC cells were immunoprecipitated using the PKD2 antibody followed by Western blotting with the same antiserum. For detection of endogenous PKD2 a 10-fold higher concentration of cellular protein was used to immunoprecipitate PKD2 than in the transfection experiments. These experiments revealed an immunoreactive band of 105 kDa corresponding to PKD2 in all human cell lines examined with HUVEC cells exhibiting the lowest level of expression. PKCµ could also be detected in HEK293 cells by a specific antibody directed against the N terminus of PKCµ, which did not cross react with PKD2 (data not shown). PKCµ could clearly be separated from PKD2 running with a higher molecular mass of about 115 kDa (Fig. 3D). Interestingly, in contrast to PKD2, we could not detect expression of PKCµ in HL60 cells in accordance with previous results (2). This suggests that the expression of members of the PKC family can be tissue-specific.

[3H]PDBu Binding to HEK293 Cells Transfected with PKD2-- PKD2 contains a duplex zinc-finger-like motif, which acts as a phorbol ester binding domain in PKCµ/PKD and PKCs. Therefore, we determined whether the expression of PKD2 in HEK293 cells would also confer increased phorbol ester binding to these cells. HEK293 cells were transfected with either pcDNA3-FLAG or pcDNA3-FLAG-PKD2. Cells transfected with FLAG-PKD2 showed a marked 4.8 ± 0.03-fold increase in specific [3H]PDBu binding as compared with HEK293 cells transfected with the vector alone or to untransfected cells (Fig. 4A, top panel). Addition of unlabeled PDBu inhibited [3H]PDBu binding to HEK293 cells transfected with FLAG-PKD2 in a concentration-dependent manner. A half-maximum inhibition of [3H]PDBu binding was achieved at 50 nM PBDu (Fig. 4A, bottom panel). These results indicate that PKD2 can serve as a novel phorbol ester receptor in intact cells.



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Fig. 4.   [3H]PDBu binding to HEK293 cells transfected with PKD2. A, top panel: nontransfected HEK293 cells (-) and HEK293 cells transfected with pcDNA3-FLAG (FLAG) or pcDNA3-FLAG-PKD2 (FLAG-PKD2) were incubated with [3H]PDBu in the presence or absence of 10 µM unlabeled PDBu. [3H]PDBu binding was determined as described in "Experimental Procedures." Data are expressed as a -fold increase above control values, which were obtained in nontransfected HEK293 cells and are the means ± S.E. of three independent experiments each performed in duplicate. A, bottom panel: HEK293 cells transfected with pcDNA3-FLAG-PKD2 were incubated with [3H]PDBu in the presence of increasing concentrations of unlabeled PDBu as indicated and [3H]PDBu binding was determined as described in "Experimental Procedures." B, PDBu stimulates PKD2 autokinase activity in synergy with PS. Lysates of HEK293 cells transfected with pcDNA3-FLAG-PKD2 were immunoprecipitated with the anti-FLAG mAb. PKD2 was eluted from the beads as described under "Experimental Procedures," and PKD2 autokinase activity was determined in the absence or presence of phosphatidylserine (PS; 100 µg/ml), PDBu (250 nM), or PS in the presence of PDBu as described under "Experimental Procedures." Data, further analyzed by scanning densitometry, are expressed as -fold increases above control and unstimulated values, and are the means ± S.E. of three independent experiments.

PDBu Stimulates PKD2 in Synergy with PS-- The preceding findings prompted us to investigate the effects of PDBu on PKD2 kinase activity. To determine the effect of PDBu and PS on PKD2 autokinase activity in vitro, lysates of HEK293 cells were immunoprecipitated with the affinity-purified PKD2 antibody. Autokinase activity of PKD2 eluted from the resultant immunocomplexes was then determined in the presence of PS and/or PDBu. Addition of PS (100 µg/ml) or PDBu (250 nM) alone caused a 3.6 ± 1.2- and 3.4 ± 1.4-fold increase in autophosphorylation of PKD2, respectively. The combination of PS with PDBu caused a synergistic, 11.8 ± 2.3-fold stimulation of PKD2 autophosphorylation (Fig. 4B). Thus, PKD2 is a novel serine threonine kinase that is directly stimulated by PDBu in a phospholipid-dependent manner.

Phorbol Esters and Growth Factors Stimulate PKD2 Kinase Activity in Intact Cells-- To verify kinase activity of the cloned cDNA product in intact cells, FLAG-PKD2 was transfected into HEK293 cells. 48 h after transfection, cells were incubated with PDBu and lysed, and the lysates were immunoprecipitated with the anti-FLAG mAb. As shown in Fig. 5A (left panel), PDBu stimulated autokinase activity of PKD2 in HEK293 cells transiently transfected with PKD2 in a time-dependent manner. A maximum, 3.3-fold activation was achieved after 10 min of incubation with PDBu. The effect of PDBu on PKD2 autokinase activity was also concentration-dependent. A maximum effect was observed at 200 nM PDBu (data not shown).



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Fig. 5.   A, phorbol esters stimulate PKD2 in intact cells. Left panel: HEK293 cells transfected with pcDNA3-FLAG-PKD2 were incubated with 250 nM PDBu for various times as indicated and lysed. PKD2 was immunoprecipitated using the anti-FLAG mAb, and PKD2 autokinase activity was determined as described under "Experimental Procedures." A typical result was subjected to scanning densitometry. Right panel: activation of PKD2 stimulates phosphorylation of aldolase and histone H1 in vitro. HEK293 cells transfected with pcDNA3-FLAG-PKD2 were incubated with 250 nM PDBu for 10 min and lysed. Lysates were subsequently immunoprecipitated with the anti-FLAG mAb and further analyzed by in vitro kinase assays as described under "Experimental Procedures" using aldolase (top panel, left) or histone H1 (top panel, right) as substrates. Typically, in assays where aldolase was used as substrate, x-ray films had to be exposed for >24 h to observe aldolase phosphorylation. In contrast, when histone H1 was used in the in vitro kinase assays, a marked increase in histone H1 phosphorylation could be detected already after 30-min exposure. Results from several histone H1 phosphorylation assays were subjected to scanning densitometry. Data are expressed as -fold increases above control, unstimulated values and are the means ± S.E. of three independent experiments. B, the pS876 antibody specifically detects phosphorylated PKD2. HEK293 cells 48 h after transfection with pcDNA-3-FLAG-PKD2 were incubated with 250 nM PDBu for 10 min. Cells were subsequently lysed, immunoprecipitated with the anti-FLAG antibody (FLAG) or the pS876 antibody (pS876), and further analyzed by Western blotting with the pS876 or the anti-FLAG antibody, respectively. In some experiments, cell lysates were directly analyzed by Western blotting with the pS876 antibody or pS876 antibody, which had been preincubated with the immunizing peptide (pS876+peptide). C, HEK293 cells 48 h after transfection with pcDNA3-FLAG-PKD2 (PKD2) or pcDNA3-FLAG-PKD2-S876A (PKD2-S876A) were incubated with 250 nM PDBu for 10 min or received an equivalent amount of solvent (-). Cells were subsequently lysed, immunoprecipitated with the anti-FLAG antibody (FLAG), and further analyzed by Western blotting with the pS876 or the anti-FLAG antibody, respectively. In each case, a representative of at least three independent experiments is shown.

The kinase domain of PKD2 is highly homologous to that of PKCµ/PKD. Therefore, we reasoned that PKD2 could phosphorylate similar substrates. PKCµ has been described to phosphorylate aldolase in vitro (17). Aldolase phosphorylation by PKD2 was only detected after prolonged exposure of the blots (>24 h). In contrast, histone phosphorylation by activated PKD2 could already be observed after a very brief exposure of the blots (30 min; Fig. 5A, right panel, top). This suggests that histone is phosphorylated more efficiently than aldolase by activated PKD2. A maximum, 2.6 ± 0.4-fold increase in histone H1 phosphorylation was observed in these cells upon incubation of cells with PDBu (Fig. 5A, right panel).

Serine 876 Is Phosphorylated upon Activation of PKD2-- Recently, Matthews et al. (18) and Vertommen et al. (19) demonstrated that phosphorylation of the serine residue at the C terminus of PKCµ/PKD (Ser916 in PKD) occurs by autophosphorylation and correlates with the activation status of the kinase. Given the high homology of the kinase domains of PKD and PKD2 we were interested whether the corresponding C-terminal serine in PKD2 (Ser876) would also be phosphorylated during activation of the kinase and whether this phosphorylation site could be useful to monitor kinase activity of PKD2. We therefore generated an antibody selectively reactive to a C-terminal phosphopeptide of PKD2 encompassing residues 872-878, where Ser876 was the phosphorylated residue. First, we determined that the antibody specifically detected PKD2. HEK293 cells were transiently transfected with FLAG-PKD2 and incubated with PDBu. Cells were then lysed and immunoprecipitated with the FLAG or the pSer876 antibody and further analyzed by Western blotting with the pSer876 antibody or the FLAG antibody, respectively. As shown in Fig. 5B, the pSer876 antibody specifically detected PKD2 immunoprecipitated with the anti-FLAG antibody. In addition, the FLAG antibody also detected a single 105-kDa band corresponding to PKD2 when transfected PKD2 was immunoprecipitated from the cells using the pSer876 antibody. We now wanted to confirm that the antibody was indeed specific for the C-terminal Ser876 residue of PKD2. The reactivity of the pSer876 antibody for PKD2 isolated from PDBu-treated HEK293 cells transiently transfected with PKD2 was completely blocked by competition with the C-terminal pSer876 immunizing peptide (Fig. 5B). Next we examined whether the pSer876 antibody was indeed activation-specific. HEK293 cells were transiently transfected with FLAG-PKD2, subsequently incubated with either PDBu or solvent, and further analyzed by Western blotting with the anti-FLAG or the pSer876 antibody. As shown in Fig. 5C (top panel, right), equal amounts of transfected protein could be detected in PDBu-treated and nontreated HEK293 cells transiently transfected with FLAG-PKD2. A low level of reactivity of the pSer876 antibody for PKD2 was observed in unstimulated HEK293 cells. This could be due to a low degree of basal phosphorylation of the Ser876 residue upon overexpression of PKD2 in exponentially growing HEK293 cells. However, the pSer876 antibody was very strongly reactive with PKD2 in PDBu-treated HEK293 cells (Fig. 5C, top panel, left). Thus, the pS876 antibody was indeed activation-specific.

Now we were interested whether the pSer876 antibody specifically detected only phosphorylated Ser876 or whether the antibody could cross react with other serine residues that can be phosphorylated upon activation of PKD2. Therefore, we generated a FLAG-PKD2 mutant, where the serine at position 876 was replaced by a neutral nonphosphorylatable alanine residue (S876A). HEK293 cells were transiently transfected with FLAG-PKD2 or FLAG-PKD2-S876A. The expression of the construct was confirmed by Western blot analysis using an anti-FLAG antibody reactive with the FLAG epitope tag (Fig. 5C, bottom panel, right). The S876A mutant exhibited a similar level of expression as the wild type construct. However, the pSer876 antibody did not detect PKD2 in PDBu-stimulated HEK293 cells transiently transfected with the PKD2-S876A mutant. Thus, the pSer876 antibody specifically detects phosphorylated Ser876 and no other phosphorylated serine residues in PKD2. When the pSer876 antibody was used to immunoprecipitate PKD2 in kinase assays in vitro, a marked increase in PKD2 autokinase activity as well as histone H1 phosphorylation could be detected upon stimulation of FLAG-PKD2-transfected HEK293 cells with PDBu. These data were comparable to those obtained using the affinity-purified pan-PKD2 antibody for immunoprecipitations (Fig. 6A, left panel).



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Fig. 6.   PDBu and gastrin induce activation of endogenously expressed PKD2. A, left panel: HEK293 cells 48 h after transfection with pcDNA3-FLAG-PKD2 were incubated with 250 nM PDBu for 10 min as indicated (+) or received an equivalent amount of solvent (-). Cells were subsequently lysed, immunoprecipitated with the pS876 antibody (pS876) or the affinity-purified PKD2 antiserum (PKD2), and PKD2 autokinase activity and histone H1 phosphorylation were determined as described under "Experimental Procedures." A, right panel, top: HEK293, HeLa, and HL60 cells were incubated with 250 nM PDBu for 10 min as indicated (+) or received an equivalent amount of solvent (-). Cells were subsequently lysed and further analyzed by Western blotting using the pS876 antibody. The mobility shift of hyperphosphorylated PKD2 (pPKD2) as compared with hypophosphorylated PKD2 (PKD2) is indicated by brackets. A, right panel, middle and bottom: HEK293, HeLa, and HL60 cells were incubated with 250 nM PDBu for 10 min or received an equivalent amount of solvent. Cells were subsequently lysed and immunoprecipitated with the pS876 antibody. PKD2 autokinase activity (middle) and histone H1 phosphorylation (bottom) were subsequently determined as described under "Experimental Procedures." The mobility shift of hyperphosphorylated PKD2 (pPKD2) as compared with hypophosphorylated PKD2 (PKD2) is indicated by brackets. B, top panel: AGS-B cells were incubated with 250 nM PDBu (P) or 100 nM gastrin (G) for 10 min or received an equivalent amount of solvent (-). Cells were subsequently lysed and further analyzed by Western blotting with pS876 antibody. B, middle panels: AGS-B cells were incubated with 250 nM PDBu (P) or 100 nM gastrin (G) for 10 min. Cells were subsequently lysed and immunoprecipitated with the pS876 antibody, and PKD2 autokinase activity (upper middle panel) and histone H1 phosphorylation (lower middle panel) were determined as described under "Experimental Procedures." B, bottom panel: histone H1 phosphorylation in response to PDBu (P) or gastrin (G) was quantified by scanning densitometry. Data are expressed as -fold increases in histone H1 phosphorylation above control, unstimulated values and are the means ± S.E. of three independent experiments. C, top panels: AGS-B cells were incubated with various concentrations of gastrin as indicated and lysed, and the lysates were further analyzed by immunoprecipitation with the pS876 antibody followed by in vitro kinase assays using histone H1 as substrate. Autokinase activity of PKD2 (top) and histone H1 phosphorylation (middle) in response to gastrin are indicated by arrows. C, bottom panel: Histone H1 phosphorylation was quantified by scanning densitometry. Data are expressed as -fold increases above unstimulated, control values. All experiments shown are representative of at least three independent experiments. Typically, x-ray films had to be exposed >= 24 h for the detection of PKD2 autokinase activity and 30 min for the detection of histone H1 phosphorylation.

Activation of Endogenously Expressed PKD2 by Pharmacological and Physiological Stimuli-- We were now interested whether the pSer876 antibody would also detect active PKD2 in nontransfected cells. Human HEK293, HeLa, and HL60 cells were left unstimulated or were treated with PBDu before whole cell lysates were prepared. Lysates were further analyzed by Western blotting with the pSer876 antibody. A very low level of weak reactivity of the pSer876 antibody for endogenous PKD2 isolated from unstimulated HEK293, HeLa, or HL60 cells was observed in Western blotting experiments (Fig. 6A, right panel). This is likely to be due to a low degree of basal phosphorylation of PKD2 in these nonquiescent cells, because the antibody did not detect the nonphosphorylated PKD2-S876A mutant (Fig. 5C). However, the pSer876 antibody showed a very strong immunoreactivity with lysates prepared from PDBu-treated HEK293, HeLa, or HL60 cells (Fig. 6A, right panel). Interestingly, phorbol ester-activated PKD2 migrated more slowly than inactive PKD2 in SDS-PAGE which is most likely due to hyperphosphorylation of the protein upon activation. Phosphorylation of endogenous PKD2 detected by the pSer876 antibody indeed correlated with PKD2 autokinase activity and histone H1 phosphorylation. Using the pSer876 antibody to immunoprecipitate PKD2 from PDBu-stimulated HEK293, HeLa, or HL60 cells, a marked increase in PKD2 autokinase activity and histone H1 phosphorylation was observed as compared with unstimulated cells (Fig. 6A, right panel). Thus, phosphorylation of PKD2 detected with the pSer876 antibody correlates with endogenous PKD2 kinase activity.

The preceding results demonstrated that transfected as well as endogenously expressed PKD2 are potently activated by pharmacological stimuli in various human cell lines. Next, we wanted to determine whether PKD2 could also be stimulated by physiological stimuli. Binding of gastrin to the seven-transmembrane domain CCKB/gastrin receptor activates heterotrimeric G proteins of the Galpha q subfamily, which stimulate phospholipase C to induce the rapid formation of the intracellular messengers DAG and inositol 1,4,5-trisphosphate (20, 21), which in turn activate classical and novel PKCs and mobilize Ca2+, respectively (22, 23). Therefore, we examined whether gastrin could activate PKD2 in a human gastric cancer cell line, AGS, which has been stably transfected with the CCKB/gastrin receptor and hence termed AGS-B (10). Because the pSer876 antibody recognized active endogenous PKD2 from pharmacologically stimulated cells, we reasoned that this antibody might also be useful to detect PKD2 that had been activated by physiological stimuli. As shown in Fig. 6B (left panel, top), gastrin stimulated phosphorylation of PKD2 at Ser876 to the same degree as a maximum efficient concentration of PDBu in Western blots. These observations were confirmed in immune complex kinase assays: Gastrin-stimulated PKD2 autokinase activity was comparable to that in response to PDBu. The degree of histone phosphorylation in response to gastrin was even slightly higher than in response to PDBu (12.6 ± 3- and 11 ± 2.2-fold, respectively; Fig. 6B, left panel, bottom). The effect of gastrin on both PKD2 autokinase activity and histone H1 phosphorylation was concentration-dependent: Half-maximum and maximum effects were achieved at 5 and 30 nM gastrin, respectively (Fig. 6B, right panel, and data not shown). Thus, gastrin potently activates endogenously expressed PKD2, and phosphorylation at Ser876 can be used to monitor both activation of transfected and endogenous PKD2 by pharmacological and physiological stimuli.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report we describe the cloning and functional characterization of a novel serine threonine kinase. This kinase has been termed PKD2, because it was found to be highly homologous to the family of protein kinases comprising PKD/PKCµ and PKCnu . This protein kinase family is only distantly related to PKCs: Although PKD/PKCµ, PKCnu , and PKD2 possess two cysteine-rich zinc finger domains at their N termini, they lack a pseudosubstrate domain but possess a pleckstrin homology domain that is not found in any other member of the PKC family. Furthermore, the kinase domains of PKD/PKCµ, PKCnu , and PKD2 are more closely related to calcium calmodulin-dependent kinases than to other members of the PKC family of serine threonine kinases. The closest homologue of the PKC family was found to be PKCbeta exhibiting only 34 and 27% homology in the first cysteine-rich and the kinase domain, respectively. Within the PKD kinase family, both PKD2 and PKCnu exhibit a slightly higher homology to PKD/PKCµ than to each other.

PKD2 was found to be widely expressed in various human and murine tissues and cell lines, particularly in highly proliferative tissues such as colonic mucosa and testis. Compared with PKCµ, mRNA expression of PKD2 was higher in the pancreas but lower in kidney and liver. In addition, using a specific polyclonal antibody against PKD2 we could detect expression of PKD2 protein in human HL60 cells, which do not express PKCµ (2). This suggests that the various isoforms of the PKD family could, at least in part, be expressed and function in a tissue-specific manner. This might have been overlooked so far, because an anti-PKD/PKCµ antibody directed against the C terminus of PKD/PKCµ also detected PKD2. In addition, the different members of the PKD kinase family could also have distinct functions: Recently, it has been reported that PKCµ can be proteolytically cleaved by caspase 3 at a 374CQNDS378 site creating a constitutively active fragment of the PKCµ catalytic domain, which sensitizes cells to the cytotoxic effects of various genotoxic agents (24). PKD2 does not contain such a motif at the corresponding position and hence might have distinct functions in apoptosis.

Our data further demonstrate that PKD2 is a novel phorbol ester receptor. Transient expression of PKD2 in HEK293 cells gave rise to a marked increase in [3H]phorbol ester binding. Analysis of PKD2 kinase activity demonstrated that autophosphorylation of PKD2 is synergistically enhanced by phorbol esters in the presence of PS. Phorbol ester also markedly activate PKD2 autokinase activity in intact cells. In contrast to PKCµ (17), PKD2 was found to phosphorylate histone H1 more efficiently than aldolase in vitro. Given that the kinase domain of PKD2 and PKD/PKCµ are highly homologous, the difference in substrate specificity could result from differences in the PKD2 regulatory domains, particularly in the linker regions between the two zinc fingers and the PH domain, which exhibit the highest dissimilarities between PKD2 and PKD/PKCµ. Modification of substrate specificity by the regulatory domain has also been demonstrated for PKCeta (25, 26).

Phosphorylation of kinases on the C terminus is thought to provide an electrostatic anchor that structures the kinase and/or alters its surface to regulate protein-protein interactions (27-29). Using a site-specific antibody approach we could identify Ser876 as a phosphorylation site within the C-terminal region of PKD2. The pSer876 antibody preferentially recognized active PKD2 and mutational analysis confirmed that this phospho-antibody was specific for the C-terminal Ser876 site of the kinase. The degree of Ser876 phosphorylation was very similar to the degree of PKD2 autokinase and catalytic activity. Phosphorylation of Ser876 might therefore be useful to monitor kinase activity of PKD2.

We further demonstrate that PKD2 is not only regulated by pharmacological but also by physiological stimuli such as neuropeptides. Gastrin has been implicated in a wide range of fundamental biological responses such as secretion, growth, and transformation (30). Upon binding to the CCKB/gastrin receptor, gastrin stimulates multiple signaling pathways, including phosphoinositide breakdown, Ca2+ mobilization, and activation of PKCs (20, 21). Here we demonstrate that stimulation of AGS-B cells with gastrin induces a striking activation of PKD2. PKD2 recovered by immunoprecipitation from gastrin-stimulated cells is fully active in the absence of lipid effectors such as PS or PDBu as shown by autophosphorylation of the kinase as well as by phosphorylation of the exogenous substrate histone H1. Thus, PKD2 is a novel component in the signal transduction pathways mediated by the CCKB/gastrin receptor. PKD has been characterized as a downstream effector of PKCs, in particular novel PKCeta and PKCepsilon (5, 6). Preliminary data using selective inhibitors of PKCs suggest that PKD2 might be regulated in a similar way. However, the precise mechanism of PKD2 activation and the PKC isoforms involved require further investigation.

Phorbol esters and gastrin induce a variety of responses in many cultured cell types, including proliferation and transformation (30, 31). The identification of PKD2 as a novel target for phorbol esters and gastrin raises the possibility that some of the biological actions of these factors could, at least in part, be mediated by this kinase. PKD2 is expressed in many tissues along with other isoforms of the PKD family, but exhibits also some tissue-specific expression. Further studies are needed to elucidate overlapping and distinct cellular functions of the different members of the PKD family, PKD/PKCµ, PKCnu , and PKD2.


    ACKNOWLEDGEMENTS

We thank Michael J. Seckl for critical reading of the manuscript, Nancy Meyer and Johannes Waltenberger for the HUVEC cells, and Franz Oswald and Ulrike Kostezka for the FLAG-pcDNA3 expression vector.


    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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF309082.

Sponsored by Fonds voor Wetenschappelijk Onderzoek-Vlaanderen and Interuniversity Network for Fundamental Research P4/26.

Dagger Dagger Supported by a grant from the Deutsche Forschungsgemeinschaft/SFB 518/B3. To whom correspondence should be addressed: Abt. Innere Medizin I, Medizinische Universitätsklinik Ulm, Robert Koch Strasse 8, Ulm D-89081, Germany. Tel.: 49-731-50201; Fax: 49-731-5023402; E-mail: thomas.seufferlein@medizin.uni-ulm.de.

Published, JBC Papers in Press, November 2, 2000, DOI 10.1074/jbc.M008719200

2 The PKD2 nucleotide sequence has been submitted to GenBankTM under accession number AF309082.


    ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; cPKC, classical PKC; nPCK, novel PKC; aPKC, atypical PKC; CCK, cholecystokinin; DAG, diacylglycerol; DMEM, Dulbecco's modified Eagle's medium; ECL, enhanced chemiluminescence; EST, expressed sequence tag; PDBu, phorbol 12,13-dibutyrate; PH, pleckstrin homology; PKD, protein kinase D; PS, phosphatidyl-L-serine; PAGE, polyacrylamide gel electrophoresis; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription-polymerase chain reaction; HUVEC, human umbilical vein endothelial cells; kb, kilobase(s); bp, base pair(s); PBS, phosphate-buffered saline; DTT, dithiothreitol; mAb, monoclonal antibodies.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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