(Received for publication, October 7, 1994; and in revised form, November 7, 1994)
From the
A novel protein kinase (named PKD) with an
NH-terminal region containing two cysteine-rich motifs has
been expressed in COS-7 cells and identified as a receptor for phorbol
esters. COS-7 cells transfected with a PKD cDNA construct (pcDNA3-PKD)
exhibit a marked (4.8-fold) increase in
[
H]phorbol 12,13-dibutyrate binding. An antiserum
raised against the COOH-terminal 15 amino acids of PKD specifically
recognized a single 110-kDa band in PKD-transfected cells. PKD prepared
by elution from immunoprecipitates with the immunizing peptide
efficiently phosphorylated the synthetic peptide syntide-2. The enzyme
only poorly phosphorylated a variant syntide-2 where arginine 4 has
been replaced by an alanine. The addition of
[
H]phorbol 12,13-dibutyrate,
1-oleoyl-2-acetylglycerol, or 1,2-dioctanoyl-sn-glycerol in
the presence of dioleoylphosphatidylserine stimulated the syntide-2
kinase activity of PKD in a synergistic fashion (4-6-fold).
Furthermore, the autophosphorylation of PKD was strikingly stimulated
by the same lipid activators (14-24-fold). Similar properties
were found with PKD isolated from mouse lung. The substrate specificity
of PKD is different from that of previously identified members of the
protein kinase C family since it does not efficiently phosphorylate
histone III-S, protamine sulfate, or a synthetic peptide based upon the
conserved pseudosubstrate region of the protein kinase C family. Taken
together, these data unambiguously establish PKD as a phorbol ester
receptor and as a novel phospholipid/diacylglycerol-stimulated protein
kinase.
A rapid increase in the synthesis of lipid-derived second
messengers is an important mechanism for transducing extracellular
signals across the plasma membrane(1, 2) . The second
messenger DAG, ()which is generated through alternative
pathways(3, 4) , activates PKC, a major cellular
target for the potent tumor-promoting phorbol
esters(5, 6) . Molecular cloning has demonstrated the
presence of multiple related PKC isoforms(1, 7) , i.e. classic PKCs (
,
I,
II, and
), novel
PKCs (
,
,
, and
), and atypical PKCs (
,
), all of which possess a highly conserved catalytic domain. The
regulatory domain of both classic and novel PKCs has a tandem repeat of
zinc finger-like cysteine-rich motifs that confers
phospholipid-dependent phorbol ester and DAG binding to these PKC
isoforms(8, 9, 10, 11) . In
contrast, atypical PKCs contain a single cysteine-rich motif, do not
bind phorbol esters, and are not regulated by
DAG(8, 12, 13, 14) . However, other
proteins such as chimaerin(15) , UNC-13(16) , and
Vav(17) , which possess a single cysteine-rich domain, bind DAG
and phorbol esters. These studies emphasize the complexity of the
signaling pathways initiated by DAG but do not exclude the possibility
that other protein kinases, unrelated to the PKC family in their
catalytic domain, could also play a role in mediating the cellular
effects of DAG and phorbol esters.
Recently, we cloned a novel mouse
serine protein kinase, named PKD, that consists of a putative
regulatory and catalytic domain(18) . The
NH-terminal region of PKD contains a putative transmembrane
domain, two cysteine-rich, zinc finger-like motifs, and a pleckstrin
homology domain. Interestingly, the length of the sequence separating
the cysteine-rich motifs in PKD (95 residues) is substantially longer
than that of classic PKCs (28 amino acids) or novel PKCs (35 amino
acids). Furthermore, two amino acids (Ala-146 and Ala-154) in the
consensus of the cysteine-rich motif of PKD differ from those in PKCs.
In contrast to all known PKCs including mammalian, Drosophila,
or yeast isoforms, PKD does not contain sequences with homology to a
typical PKC pseudosubstrate motif upstream of the cysteine-rich region.
The catalytic domain of PKD contains all 11 distinct subdomains
characteristic of protein kinases(19) . Comparison of the
deduced amino acid sequence of the catalytic domain of PKD with that of
other protein kinases indicates that PKD is a distinct protein kinase
that is distantly related to Ca-regulated kinases (
)but does not belong to any of the protein kinase
subfamilies(18) . (
)In particular, the kinase
subdomains of PKD show little similarity to the highly conserved
regions of the kinase subdomains of the PKC family. For example, the
motif XXDLKXX(N/D) in subdomain VI, which is
important because it guides the peptide substrate into the correct
orientation so that catalysis can occur(20) , is YRDLKLDN in
all PKCs, which differs from that of PKD (HCDLKPEN) in every variable
residue (X). The comparisons of the regulatory and catalytic
domains of PKD with other kinases clearly establish PKD as a novel type
of protein kinase and therefore it is important to elucidate the
regulatory properties of this enzyme.
Johannes et al.(21) recently cloned a human protein kinase called
atypical PKCµ with 92% homology to PKD (extending to 98% homology
in the catalytic domain). In vitro phorbol ester binding
studies and kinase assays with lysates of cells overexpressing PKCµ
showed no increased phorbol ester binding and revealed phorbol
ester-independent kinase activity. In contrast, our bacterial fusion
protein encoding the zinc finger-like domains of PKD bound
[H]PDBu with high affinity(18) . Hence it
was necessary to establish whether PKD can serve as a cellular phorbol
ester receptor in intact eukaryotic cells and whether the kinase
activity of PKD is lipid regulated.
The results presented in this study indicate that PKD is a protein kinase with distinct substrate specificity that is markedly stimulated by DAG analogues and PDBu in a PS-dependent manner and serves as a novel receptor for PDBu in eukaryotic cells.
Mouse lungs were homogenized using a
Polytron homogenizer (10 s at setting 17) in lysis buffer B (lysis
buffer A without Triton X-100). After the homogenization, 1% Triton
X-100 was added. Mouse lung and COS-7 cell lysates were clarified by
centrifugation at 100,000 g for 30 min at 4 °C.
The synthetic peptide EEREMKALSERVSIL corresponding to the COOH-terminal amino acid sequence of PKD was conjugated to keyhole limpet hemocyanin, and antisera were prepared as described previously (22) . Proteins were immunoprecipitated at 4 °C for 2 h with the PA-1 antipeptide antiserum (1:50 dilution) in the absence or the presence of the immunizing peptide (2 µg/µl antiserum). The immune complexes were recovered using protein A coupled to agarose.
Exponentially growing COS-7 cells, 40-60%
confluent, were transfected with either pcDNA-3 or pcDNA3-PKD using
Lipofectin as described in detail by the manufacturer (Life
Technologies, Inc.). Briefly, 2 or 12 µg of DNA were used for 35-
or 90-mm dishes, respectively. The DNA was diluted to 100 µl with
Opti-mem I medium (Life Technologies, Inc.) and then mixed with
Lipofectin (6 or 36 µl) diluted to 100 µl with Opti-mem I
medium. After 15 min, the DNA-Lipofectin complex was diluted to 2 or 10
ml with Opti-mem I medium, mixed gently, and overlaid onto rinsed (1
with Opti-mem I) COS-7 cells. The cultures were then incubated
at 37 °C for 6 h, and the medium was then replaced with fresh DMEM
containing 10% fetal bovine serum. The cells were used for experimental
purposes 72 h later. The level of PKD expression was determined by
[
H]PDBu binding and by Western blot analysis.
Free
[H]PDBu was separated from bound
[
H]PDBu by binding of PKD to
polyethyleneimine-treated GF/F Whatman filters. The filters were washed
rapidly three times with ice-cold 20 mM Tris, pH 7.4, and
bound radioactivity was measured using liquid
scintillation
counting.
Figure 1:
Expression of PKD in COS-7 cells and
characterization of the PA-1 antiserum. A, Western blot
analysis of the catalytic domain fusion protein. Various amounts
(0.1-1 µg of protein) of purified catalytic domain fusion
protein (calculated molecular mass is 82 kDa) were analyzed by SDS-PAGE
and transferred to Immobilon membranes. Western blot analysis using the
PA-1 antiserum in the absence or in the presence of immunizing peptide (1.0+) was carried out as described under
``Experimental Procedures.'' B, PKD protein
expression in transfected COS-7 cells. COS-7 cells (lane 1) or
COS-7 cells transfected with pcDNA3 (lane 2) or pcDNA3-PKD (lanes 3 and 4) were washed twice with PBS at 4
°C and solubilized in 2 sample buffer. In each case 2
10
cells were used. Following SDS-PAGE and transfer
to Immobilon membranes, Western blot analysis was carried out using
PA-1 antiserum in the absence (lanes 1-3) or presence (lane 4) of the immunizing peptide. Similar results were
obtained in 14 independent experiments. C, lysates from COS-7
cells (lane 5), COS-7 cells transfected with pcDNA3 (lane
6), or COS-7 cells transfected with pcDNA3-PKD (lanes
7-9) were immunoprecipitated with the PA-1 antiserum in the
absence (lanes 5-7 and 9) or in the presence (lane 8) of the immunizing peptide. The resulting
immunocomplexes were analyzed by Western blotting using the same
antiserum (lanes 5-8). In lane 9, the
immunoprecipitates were washed and incubated with the immunizing
peptide (30 min at 4 °C). The resulting eluate was subjected to
Western blot analysis with the PA-1 antiserum. Similar results were
obtained in four independent experiments.
To examine PKD expression in COS-7 cells, an assembled DNA fragment corresponding to the complete sequence of PKD (spanning bases -125 to 3179) was inserted between the XhoI and XbaI sites of the mammalian expression vector pcDNA3, and the resultant plasmid (pcDNA3-PKD) was transiently transfected in COS-7 cells. Cultures of these cells were lysed, and the extracts were subjected to Western blot analysis using the PA-1 antiserum. As shown in Fig. 1B, PA-1 recognized a single band migrating with an apparent molecular mass of 110 kDa in COS-7 cells transfected with pcDNA3-PKD (lane 3). This band was not detected in lysates from COS-7 cells transfected with pcDNA3 or from untransfected cells or when the immunoblots were incubated with PA-1 antiserum in the presence of the immunizing peptide (lanes 1, 2, and 4). A band migrating with an identical molecular mass (110 kDa) was also obtained when lysates from COS-7 cells transfected with pcDNA3-PKD were immunoprecipitated with the PA-1 antiserum, and the immunoprecipitates were analyzed by Western blotting using the same antiserum (Fig. 1C, lane 7). The detection of the 110-kDa band was blocked by the inclusion of the immunizing peptide during the immunoprecipitation (lane 8). Incubation of PA-1 immunoprecipitates with the immunizing peptide eluted the 110-kDa band from the immunocomplexes (lane 9). Furthermore, no bands were detected in PA-1 immunoblots of PA-1 immunoprecipitates of lysates of either COS-7 cells (lane 5) or COS-7 cells transfected with pcDNA3 (lane 6) or when preimmune serum was used instead of the PA-1 antiserum (data not shown). In the absence of detergents, PKD was recovered in both soluble and particulate fractions of the pcDNA3-PKD-transfected COS-7 cells. These results clearly demonstrate the transient expression of PKD in COS-7 cells and substantiate the specificity of the PA-1 antiserum.
Figure 2:
Expression of PKD in COS-7 cells confers
increased [H]PDBu binding. Left panel,
control COS-7 cells (gray bars) or COS-7 cells transfected
with either pcDNA3 vector alone (open bars) or with the
pcDNA3-PKD (closed bars) all cultured in 90-mm dishes, were
washed twice with DMEM and incubated at 37 °C with 6 ml of binding
medium containing 10 nM [
H]PDBu for 30
min. Cell-associated radioactivity was determined as described under
``Experimental Procedures.'' Nonspecific binding (hatched
bars) was determined in the presence of 10 µM unlabeled PDBu. The values represent the mean ± S.E. of
five independent experiments each performed in triplicate. Right
panel, dose-dependent inhibition of [
H]PDBu
binding by unlabeled PDBu. COS-7 cells cultured in 35-mm dishes were
transfected with either pcDNA3 (open circles) or pcDNA3-PKD (closed circles). After 72 h, the cultures were washed and
incubated at 37 °C with 1 ml of binding medium for 30 min either in
the absence or in the presence of increasing concentrations of
unlabeled PDBu.
To measure the
affinity of [H]PDBu binding to the full-length
PKD, lysates of COS-7 cells transfected with pcDNA3-PKD were
immunoprecipitated with the PA-1 antiserum, and PKD was eluted from the
immunocomplexes by incubation with the immunizing peptide (Fig. 1C, lane 9). As shown in Fig. 3,
the specific binding of [
H]PDBu to the eluted PKD
preparation was saturable; Scatchard analysis of the data revealed a K
of 2.2 nM. This value is comparable
with the K
determined for PDBu binding to
PKC
(11) .
Figure 3:
Analysis of [H]PDBu
binding to immunopurified PKD. PKD eluted from lysates of COS-7 cells
transfected with pcDNA3-PKD was incubated with various concentrations
of [
H]PDBu. Specific binding of
[
H]PDBu to PKD was measured as indicated under
``Experimental Procedures.'' Results are of a representative
experiment, with each point determined in duplicate. The values are
expressed as picomoles bound per assay. Inset, Scatchard
analysis of [
H]PDBu binding to PKD. B/F,
bound/free.
To determine
the effect of PS and PDBu on the kinase activity of PKD, lysates of
COS-7 cells transfected with either pcDNA3 or pcDNA3-PKD were
immunoprecipitated with the PA-1 antiserum. The syntide-2 kinase
activity eluted from the resultant immunocomplexes was measured in the
absence or presence of various effectors. As shown in Fig. 4, a
marked increase in syntide-2 kinase activity was detected in the
eluates of PA-1 immunoprecipitates of COS-7 cells transfected with
pcDNA3-PKD as compared with those obtained from COS-7 cells transfected
with pcDNA3 (closed barsversusopen bars,
respectively). The immunoprecipitation of this activity with PA-1 was
virtually abolished by addition of the immunizing synthetic peptide.
Furthermore, preimmune serum failed to immunoprecipitate syntide-2
kinase activity from COS-7 cells transfected with pcDNA3 PKD (results
not shown). Addition of PS (100 µg/ml) or PDBu (250 nM)
singly caused only a small increase (1.7 ± 0.1- and 1.3 ±
0.1-fold, respectively) in the syntide-2 kinase activity obtained from
pcDNA3-PKD-transfected cells. In contrast, the combination of PS (100
µg/ml) with PDBu (250 nM) caused synergistic stimulation
of syntide-2 kinase activity (4.3 ± 0.2-fold, n = 3). The stimulation of syntide-2 kinase activity by PDBu
in the presence of PS was dose-dependent; half-maximum stimulation was
obtained at approximately 25 nM PDBu in the presence of PS (Fig. 4). Addition of Ca did not have any
effect on PKD activity either in the absence or in the presence of PDBu
and PS (Fig. 4, inset). Interestingly, the syntide-2
kinase activity of the bacterially expressed catalytic domain of PKD
was not affected by PS, PDBu, or both, providing evidence that these
effectors act through the regulatory domain of PKD (results not shown).
Thus, PKD is a novel serine/threonine kinase that is directly
stimulated by PDBu in a phospholipid-dependent manner.
Figure 4:
Synergistic stimulation of kinase activity
of PKD by PDBu and PS. COS-7 cells transfected with pcDNA3 (open
bars) or with pcDNA3-PKD (closed bars) were lysed, and
the lysates were incubated with the PA-1 antiserum. PKD was then eluted
from the resultant immunoprecipitates and analyzed in a syntide-2
phosphorylation assay as described under ``Experimental
Procedures.'' The specific activity of
[-
P]ATP was 450 cpm/pmol. Kinase activity
was measured either in the absence(-) or presence (+) of 100
µg/ml PS and without or with 25, 250, or 500 nM PDBu as
indicated. The values are the means ± S.E. (n =
4). Similar results were obtained in two independent experiments. Inset, effect of Ca
on PKD activity. PKD
syntide-2 kinase activity was measured in the absence (-) or in
the presence (PS + PDBu) of 250 nM PDBu and 100 µg/ml
PS either without (closed bars) or with (hatched
bars) 0.5 mM CaCl
. Similar results were
obtained with 0.2 mM CaCl
, but an inhibitory
effect was noticed at 1 mM CaCl
.
Figure 5:
Synergistic stimulation of kinase activity
of PKD by OAG or diC8 in the presence of PS. COS-7 cells transfected
with pcDNA3 (open bars) or with pcDNA3-PKD (closed
bars) were lysed, and the lysates were immunoprecipitated with the
PA-1 antiserum. PKD was then eluted from the immunoprecipitates and
analyzed in a syntide-2 phosphorylation assay as described under
``Experimental Procedures.'' The specific activity of
[-
P]ATP was 490 cpm/pmol. Kinase activity
was measured either in the absence(-) or presence (+) of PS
at 100 µg/ml and without or with OAG or diC8, as indicated. The
values are the means ± S.E. (n = 4). Similar
results were obtained in two independent
experiments.
Figure 6: Synergistic stimulation of PKD autophosphorylation by PDBu, OAG, and diC8 in the presence of PS. COS-7 cells transfected with pcDNA3-PKD were lysed, and the lysates were incubated with the PA-1 antiserum. PKD was then eluted from immunoprecipitates and analyzed in an autophosphorylation assay as described under ``Experimental Procedures.'' Autophosphorylation was determined in the absence(-) or in the presence (+) of PS at 100 µg/ml. PDBu (20 and 200 nM), OAG (2 and 20 µM), and diC8 (2 and 20 µM) were added as indicated. Similar results were obtained in two independent experiments.
Figure 7:
Kinase activity and autophosphorylation of
mouse lung PKD. Mouse lungs were homogenized, and the lysates were
incubated with the PA-1 antiserum. PKD was then eluted from
immunoprecipitates by incubation with the immunizing peptide and
analyzed in a syntide-2 kinase assay (upper panel) and in an
autophosphorylation assay (lower panel) as described under
``Experimental Procedures.'' The specific activity of the
[-
P]ATP was 540 cpm/pmol. The kinase assay
and the autophosphorylation assay were performed in the
absence(-) or presence of 100 µg/ml PS (+) and without
or with 200 nM PDBu, 20 µM OAG, and 20 µM diC8, as indicated.
The results presented here indicate that PKD is a protein
kinase with distinct substrate specificity that is stimulated by
phorbol ester or diacylglycerols in a phospholipid-dependent manner and
can serve as a receptor for PDBu in intact cells. The following lines
of evidence support our conclusion. 1) COS-7 cells transfected with
pcDNA3-PKD displayed a marked increase in high affinity PDBu binding.
2) The kinase activity of PKD, as measured with the substrate
syntide-2, was regulated by lipids. The addition of PDBu, OAG, or diC8
in combination with PS markedly stimulated the kinase activity of PKD
in a synergistic fashion. 3) The same lipid effector combinations
strikingly increased the autophosphorylation of PKD in a synergistic
fashion. In agreement with these findings, we have previously
demonstrated that the bacterially expressed NH-terminal
region of PKD, which contains two cysteine-rich zinc finger-like
domains bound [
H]PDBu in a specific
fashion(18) . To further substantiate the validity of our
conclusions we also isolated PKD from mouse lung tissue. We demonstrate
for the first time the existence of PKD activity in an animal tissue
and show that its regulatory characteristics are similar to those of
recombinant PKD expressed in COS-7 cells.
Johannes et al.(21) recently cloned a human protein kinase termed PKCµ with 92% homology to PKD. While it is highly likely that the two kinases are functional homologs, Johannes et al.(21) failed to demonstrate any phorbol ester binding and regulation and consequently concluded that this enzyme is an atypical PKC. These authors suggested that two amino acid substitutions in the cysteine-rich region could be responsible for these results. In addition, they suggest that the spacing of the cysteine-rich motifs by 87 amino acids (as opposed to 28-35 amino acids in phorbol ester-binding PKCs) could result in an inappropriate conformation for efficient PDBu binding. In contrast, our results provide compelling evidence indicating that PKD is a novel target for phorbol esters as well as for DAG.
The structural differences between the catalytic
domains of PKD and PKCs (see the Introduction) suggested that these
proteins could have important functional differences. We examined the
ability of full-length PKD to phosphorylate a variety of potential
substrates. These studies revealed that PKD preferentially
phosphorylated the synthetic peptide syntide-2. The poor
phosphorylation of a variant syntide-2 with arginine-4 replaced by an
alanine indicates that the presence of a basic amino acid upstream of
the phosphorylatable serine seems to be essential for efficient
phosphorylation. In contrast, PKD did not efficiently catalyze
phosphorylation of a number of substrates utilized by PKCs such as
protamine sulfate, histone III-S, and a peptide substrate based on the
sequence of the pseudosubstrate region of PKC. Importantly, the
PKC
peptide is very efficiently phosphorylated by classic, novel,
and atypical PKCs(25, 26) . Although PKCs
,
, and
utilize histone as a substrate more effectively than
the novel PKCs(29, 30) , it has become apparent that
this difference is not an intrinsic property of the catalytic domains
but is conferred by the influence of the regulatory domain. Indeed,
novel PKCs such as
and
that have been rendered
constitutively active by proteolysis or mutation phosphorylate histone
as efficiently as the classic
PKCs(31, 32, 33) . In this context, the
inability of both full-length PKD and of the bacterially expressed
catalytic domain of PKD (18) to efficiently phosphorylate
histone clearly emphasizes the functional differences between PKD and
PKCs.
Taken together, our results indicate that PKD is not an
atypical PKC since we clearly demonstrate that PKD binds phorbol esters
and that the activity of this enzyme is markedly stimulated by PDBu,
OAG, or diC8 in the presence of PS. Moreover, we found fundamental
differences in both substrate specificity and catalytic domain protein
structure between PKD and PKCs. In addition, PKD can be distinguished
from PKCs by other important structural features. 1) PKD does not
contain a typical PKC pseudosubstrate motif, which is present upstream
of the cysteine-rich region in all PKCs. 2) The NH terminus
of PKD possesses a highly hydrophobic stretch of amino acids,
suggesting a transmembrane domain, which is not found in any of the
PKCs. 3) PKD contains a pleckstrin homology domain inserted between the
cysteine-rich motifs and the catalytic domain. Pleckstrin homology
domains have recently been identified in a variety of intracellular
signaling and cytoskeletal proteins but are not present in
PKCs(34) . We conclude that PKD cannot be classified in any of
the PKC subfamilies.
Phorbol esters act as potent tumor promoters
and induce a variety of responses in many cultured cell types including
effects on ionic channels, second messenger production, cell-cell
communication, membrane transport, protein phosphorylation, and
cellular growth, morphology, differentiation, and
transformation(1, 5, 6, 35, 36) .
The identification of another target for phorbol esters, PKD, which is
expressed in many organs and tissues as well as in cultured cells (18) raises the possibility that some of the
actions of phorbol esters could be mediated partially or exclusively by
PKD.
One of the earliest responses of many cell types to extracellular stimuli is an increase in the synthesis of DAG, the physiological second messenger generated through multiple pathways(3, 4) . The results presented here demonstrate, for the first time, that the protein kinase activity of PKD is synergistically stimulated by diacylglycerol analogs in combination with PS. Thus, PKD could be a novel component in the signal transduction of many growth factors, regulatory peptides, and cytokines that elevate DAG in their target cells.