From the 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
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ABSTRACT |
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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 PKC 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:
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.
Materials--
[ 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 [ 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 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 [ 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 PKC
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 PKC
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 PKC 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.
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).
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 PKC
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.
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).
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).
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 G 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 PKC 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 PKC 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 PKC 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,
PKC, 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
1,
2, and
), novel PKCs (nPKCs:
,
,
, and
),
and atypical PKCs (aPKCs:
and
), 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 PKC
and PKC
(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 PKC
(9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
-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).
-scintillation
counter. Nonspecific binding was determined in the presence of 1 µM PDBu.
-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 [
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sharing 69 and 64% identical amino acids with PKD/PKCµ and PKC
,
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 PKC
and PKC
/
, 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 PKC
, 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 PKC
(NM 005813) amino acid sequences using the Align
PPC/MacMolly Tetra program. Identical amino acids are denoted in
boldface.
The percentage of amino acid identity between human PKD2 and the other
PKD family members, PKD/PKCµ (mouse/human), and human PKC
(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 PKC
, respectively (Fig. 1B and Table
I).
(91%) but not to any other member of the PKC family
(Table I). Of all other members of the PKC family, PKC
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.
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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.
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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.
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.
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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.
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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.
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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.
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
. This
protein kinase family is only distantly related to PKCs: Although
PKD/PKCµ, PKC
, 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, PKC
, 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 PKC
exhibiting only 34 and 27% homology
in the first cysteine-rich and the kinase domain, respectively. Within
the PKD kinase family, both PKD2 and PKC
exhibit a slightly higher
homology to PKD/PKCµ than to each other.
(25, 26).
and PKC
(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.
, 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.
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.
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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.
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REFERENCES |
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