Received for publication, November 26, 2000, and in revised form, March 16, 2001
The association of phospholipase (PLD)-1 with
protein kinase C-related protein kinases, PKN
and PKN
, was
analyzed. PLD1 interacted with PKN
and PKN
in COS-7 cells
transiently transfected with PLD1 and PKN
or PKN
expression
constructs. The interactions between endogenous PLD1 and PKN
or
PKN
were confirmed by co-immunoprecipitation from mammalian cells.
In vitro binding studies using the deletion mutants of PLD1
indicated that PKN
directly bound to residues 228-598 of PLD1 and
that PKN
interacted with residues 1-228 and 228-598 of PLD1.
PKN
stimulated the activity of PLD1 in the presence of
phosphatidylinositol 4,5-bisphosphate in vitro, whereas
PKN
had a modest effect on the stimulation of PLD1 activity. The
stimulation of PLD1 activity by PKN
was slightly enhanced by the
addition of arachidonic acid. These results suggest that the PKN family functions as a novel intracellular player of PLD1 signaling pathway.
 |
INTRODUCTION |
PKN
is a serine/threonine protein
kinase whose regulatory domain in the N-terminal region contains three
repeats of leucine zipper-like sequences (CZ1-3) and a D region, and
whose catalytic domain in the C-terminal region is highly homologous to
those of PKC1 family members (1). The protein kinase
activity is enhanced by fatty acids such as arachidonic acid (2) and
the small GTP-binding protein Rho through the direct interaction with
the CZ1 region containing the RhoA effector motif class I in a
GTP-dependent manner (3-5). This protein kinase makes a
family that comprises at least three gene products including
PKN
/PRK1, PKN
, and PRK2 (6, 7). The CZ1 regions of PKN
and
PRK2 have the RhoA effector motif class I sequences (8), and they can
interact with RhoA (6, 9-11). We previously reported that the D region
of PKN
contains the autoinhibitory domain (12). It blocks the access of the catalytic domain to the substrate. Arachidonic acid can recover
the catalytic activity from this autoinhibition (12), suggesting that
arachidonic acid causes conformational change of PKN
to release the
autoinhibitory domain. PKN
and PRK2 conserve the domains
corresponding to the autoinhibitory domain of PKN
(6, 12); however,
they are only slightly activated by arachidonic acid (6, 13).
Therefore, PKN
and PRK2 may need some other factors for
conformational changes to release their autoinhibitory domains.
PKN
is ubiquitously expressed but is enriched in the brain (14). In
neurons, PKN
is concentrated in a subset of endoplasmic reticulum as
well as late endosomes, multivesicular bodies, Golgi bodies, secretary
vesicles, and nuclei (15). Thus we have identified several binding
partners of PKN
by employing a two-hybrid screening using the
N-terminal regulatory region of PKN
as a bait (16-20). One of these
partners is the centrosome- and Golgi-localized PKN-associated protein (CG-NAP), localized to centrosomes throughout the cell cycle and the Golgi apparatus at interphase (17). PKN
is localized to the Golgi apparatus and nuclei in cells (6). Thus, there is the
possibility that PKN
and PKN
may play roles in the control of
physiological events in the Golgi apparatus and endosomal compartment such as mitotic Golgi fragmentation and intracellular membrane traffic
through the interaction with some other molecules.
In the present study, we have focused attention on phospholipase D
(PLD)-1 showing overlapping subcellular localization with PKN
and
PKN
: endoplasmic reticulum, Golgi apparatus, late endosomes, and
plasma membrane (21, 22). PLD hydrolyzes phosphatidylcholine to
generate the signaling lipid phosphatidic acid. Phosphatidic acid has
been hypothesized to act as a fusogenic lipid in vesicular transports
such as exocytosis and endocytosis. Many cell types express the known
PLD1 and/or PLD2 isozymes, which become activated in response to a wide
variety of agonists through heterotrimeric G protein-coupled or
tyrosine kinase receptors. It has been proposed that the link between
receptor stimulation and PLD activation is mediated by PKC,
ADP-ribosylation factor, and Rho family members, which have been shown
to stimulate PLD1 directly using in vitro reconstitution
assays (23-29). They can act alone to stimulate PLD1 and in
combination to elicit a synergistic activation (26-28, 30).
In this report, we show that PLD1 associates with PKC-related protein
kinases, PKN
and PKN
, in vitro and in vivo.
Furthermore, we demonstrate that PKN
stimulates the PLD1 activity
through the direct interaction in vitro. These results
suggest that the PKN family participates in the PLD1 signaling pathway.
 |
EXPERIMENTAL PROCEDURES |
Antibodies--
Mouse monoclonal anti-FLAG (M2),
anti-(His)6, anti-PKN (PRK1), rat monoclonal anti-HA
(3F10), and rabbit polyclonal anti-PLD were purchased from Sigma,
Covance Research Products, Transduction Laboratories, Roche Molecular
Biochemicals, and BIOSOURCE, respectively.
Plasmid Construction--
The pTB701/HA/hPKN
plasmid used to
express HA-tagged human PKN
in COS-7 cells was constructed by
subcloning the EcoRI fragment of pMhPKN-H4 (1) containing
the full length of hPKN
to the EcoRI site of pTB701/HA
(35). The pTB701/HA/hPKN
N and pTB701/HA/hPKN
CA plasmids were
constructed as follows: the cDNA fragment encoding hPKN
(residues 1-540) was obtained by digesting phPKNH4 with EcoRI and BamHI; the cDNA fragment encoding
hPKN
(residues 561-942) was obtained by polymerase chain reaction;
and each cDNA fragment was inserted into pTB701/HA. The
pTB701/HA/hPKN
plasmid was made by inserting the EcoRI
fragment of pRc/CMV/hPKN
into pTB/701/HA. The pTB701/HA/hPKN
N and
pTB701/HA/hPKN
CA plasmids for the expression in mammalian cells were
made by digesting pRc/CMV/hPKN
with EcoNI, filling the
ends with T4 polymerase, ligating with EcoRI linker, digesting with EcoRI, and inserting the resulting fragments
encoding hPKN
(residues 1-520 and 520-889) into the
EcoRI site of pTB701/HA, respectively. The pTB701/FLAG/hPLD1
used to express FLAG-tagged human PLD1 in mammalian cells was
constructed as described previously (28). The pTB701/FLAG/hPLD1N
plasmid was constructed by deleting the BglII fragment from
pTB701/FLAG/hPLD1. The pTB701/FLAG/hPLD1I plasmid was made by digesting
pTB701/FLAG/hPLD1 with NcoI, filling the ends with T4
polymerase, ligating with BglII linker, digesting with
BglII, and inserting the resulting fragment encoding hPLD1 (residues 228-598) to the BglII site of pTB701/FLAG (35).
The pTB701/FLAG/hPLD1C was constructed by digesting pTB701/FLAG/hPLD1 with NcoI, filling the ends with T4 polymerase, ligating
with BglII linker, digesting with BglII, and
ligating the resulting fragment encoding hPLD1 (residues 598-1070) to
the BglII site of pTB701/FLAG. The pBlueBacHis/GST/hPKN
was made as described (12). The pAcGHLTC/hPKN
plasmid was made by
digesting pTB701/HA/hPKN
with EcoRI and inserting the
resulting fragment into the EcoRI site of pAcGHLTC. The
pRSETB/hPLD1N for (His)6-tagged hPLD1N (residues 1-228)
was constructed as follows: the cDNA fragment encoding the amino
acid residues 1-228 of hPLD1 generated by polymerase chain reaction
was ligated into the BamHI site of pRSETB. The pRSETB/hPLD1I
plasmid used to express (His)6-tagged hPLD1I (residues 228-598) was made by digesting pTB701/FLAG/hPLD1I with
BglII, and the resulting fragment was inserted into the
BglII site of pRSETB.
Expression and Purification of Recombinant PKN
and
PKN
--
The recombinant baculovirus was generated as described
(12). The Sf9 cells expressing recombinant protein were lysed
for 15 min in buffer A (20 mM Tris/HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 5 mM MgCl2, 150 mM
NaCl, and 10 µg/ml leupeptin) containing 1% Nonidet P-40 at 4 °C.
The lysate was then centrifuged at 30,000 × g for 20 min at 4 °C, and the resulting supernatant was collected. An
appropriate volume of glutathione-Sepharose 4B was added to the
supernatant, and the mixture was rotated for 1 h at 4 °C. After
washing with buffer A, the bound proteins were eluted with elution
buffer (50 mM Tris/HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 1 µg/ml leupeptin, and 10 mM reduced glutathione).
Cell Culture--
COS-7 and HeLa cells were maintained in
Dulbecco's modified Eagle's medium with 10% fetal calf serum. U937
cells were grown in RPMI 1640 medium supplemented with 10% fetal calf
serum. The cells were incubated at 37 °C in a humidified atmosphere
of 5% CO2. Transfection to COS-7 cells by electroporation
was performed as described (6).
Immunoprecipitation--
Cells were lysed with buffer A
containing 1% Nonidet P-40 at 4 °C, and the resulting lysate was
centrifuged at 10,000 × g for 10 min. The supernatants
were incubated with the indicated antibodies at 4 °C for 2 h
and with protein A-Sepharose for 1 h. The resulting
immunoprecipitates were subjected to PLD assay, SDS-polyacrylamide gel
electrophoresis, and immunoblot analysis with the indicated antibodies.
In Vitro Binding Assay--
Two µg of the indicated protein
was incubated at 4 °C for 2 h with 2 µg of either
(His)6-tagged hPLD1N (residues 1-228) or hPLD1I (residues
228-598) and then precipitated with glutathione-Sepharose 4B beads.
The (His)6-tagged hPLD1 fragment that was co-precipitated with the indicated GST-fused proteins was detected by immunoblotting with an anti-(His)6 antibody.
In Vitro PLD Assay--
The immunoprecipitate with
anti-FLAG from COS-7 cells expressing FLAG-tagged hPLD1 was
reconstituted with the purified proteins in the presence of
phosphatidylethanolamine/phosphatidylinositol 4,5-bisphosphate/[choline-methyl-[3H]dipalmitoylphosphatidylcholine
vesicles in a molar ratio of 16:1.4:1 (36). The mixture was incubated
in 20 mM Hepes, pH 7.5, 3 mM EGTA, 80 mM KCl, 2.5 mM MgCl2, 2 mM CaCl2, and 1 mM dithiothreitol
with or without 40 µM arachidonic acid, and then the
production of [3H]choline was determined (37). The
incubation time was 30 min at 37 °C.
 |
RESULTS |
Association of PKN
and PKN
with PLD1--
To analyze
the association of PKN family members with PLD1, we performed the
immunoprecipitation with the anti-FLAG antibody using the lysate of
COS-7 cells expressing FLAG-tagged PLD1 and either HA-tagged PKN
or
PKN
. The interaction was analyzed by Western blotting with an
anti-HA antibody. As shown in Fig. 1, both PKN
and PKN
were clearly co-immunoprecipitated with
FLAG-tagged PLD1.

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Fig. 1.
PKN and
PKN interact with PLD1 in COS-7 cells.
Either HA-tagged PKN or PKN and FLAG-tagged PLD1 were
co-expressed in COS-7 cells. The cell lysate was immunoprecipitated
with the anti-FLAG antibody as described under "Experimental
Procedures." The cell lysate and the immunoprecipitate were analyzed
by SDS-polyacrylamide gel electrophoresis followed by Western blotting.
The anti-FLAG antibody was used to detect the expression (lanes
l and 2) and immunoprecipitation level (lanes
3 and 4) of FLAG-tagged PLD1 (lower panel).
The amounts of HA-tagged PKN (lane 1) and PKN
(lane 2) expressed and co-immunoprecipitated (lanes
3 and 4) with PLD1 were visualized using the anti-HA
antibody (upper panel). The immunoprecipitation with normal
mouse IgG (lanes 5 and 6) was done as the control
experiment.
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|
To define the association between endogenous PLD1 and PKN
or PKN
in mammalian cells, we next carried out the immunoprecipitation experiments using the specific antibodies. As shown in Fig.
2B, endogenous PKN
was
clearly co-immunoprecipitated with endogenous PLD1 from HeLa cells.
Fig. 2A shows that PLD1 also interacted with PKN
in U937
cells, although the interaction with PKN
was weaker than that with
PKN
. These data suggest that the association of PKN
and
PKN
with PLD1 is physiological.

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Fig. 2.
Endogenous PKN
and PKN interact with endogenous PLD1 in
cells. A, the association of PKN with PLD1 in U937
cells. B, the association of PKN with PLD1 in HeLa cells.
The cell lysates of HeLa and U937 cells were immunoprecipitated
with the anti-PLD1 antibody ( PLD1) followed by immunoblots with
PRK1 (A) and C131 (B) to detect PKN
and PKN , respectively. The immunoprecipitation with normal rabbit
IgG was done as the control experiment (normal).
sup, supernatant of cell lysate; IP,
immunoprecipitate.
|
|
To identify the interaction site(s) of PKN
or PKN
with PLD1, we
made the deletion mutants of PKN
and PKN
as shown in Fig. 3A. FLAG-tagged PLD1 was
co-expressed with HA-tagged PKN
N (residues 1-540), HA-tagged
PKN
CA (residues 561-942), HA-tagged PKN
N (residues 1-520), or
HA-tagged PKN
CA (residues 520-889) in COS-7 cells. The cell lysate
was subjected to immunoprecipitation with the anti-FLAG antibody, and
the interaction was analyzed by Western blotting with the anti-HA
antibody. As shown in Fig. 3B, right, PKN
clearly bound to PLD1 at the N- and C-terminal regions. PKN
also
associated with PLD1 at both N- and C-terminal regions (Fig. 3B, left).

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Fig. 3.
The N terminus and C terminus of
PKN and PKN interact
with PLD1 in COS-7 cells. A, the constructs of PKN
and PKN . Schematic representations of the structures of PKN and
PKN are shown on the top. CZ, charged amino
acid-rich region with leucine zipper-like sequences; D,
~130 amino acid stretch between the CZ region and catalytic domain,
which has weak homology to the E4/V0/C2 domain of PKC, and the
C-terminal part of it functions as a fatty acid-sensitive
autoinhibitory domain; P1 and P2, two distinct
proline-rich regions. B, HA-tagged N-terminal or C-terminal
fragments of PKN (left) and PKN (right)
were co-expressed with FLAG-tagged PLD1 in COS-7 cells. After
immunoprecipitation of FLAG-tagged PLD1 with the anti-FLAG antibody,
the amounts of co-immunoprecipitated HA-tagged N-terminal (lane
3) and C-terminal (lane 4) fragments were detected by
immunoblotting using the anti-HA antibody (upper panel). The
expression (lanes 1 and 2) and
immunoprecipitation (lanes 3 and 4) levels of
PLD1 were visualized by immunoblotting with the anti-FLAG antibody
(lower panel). As a control, immunoprecipitation with normal
mouse IgG was done (lanes 5 and 6).
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Next, we analyzed the interaction site(s) of PLD1 with PKN
or
PKN
. The lysate from COS-7 cells co-expressing either HA-tagged PKN
or PKN
and three different deletion mutants of FLAG-tagged PLD1 (Fig. 4A) was
immunoprecipitated with the anti-FLAG antibody and analyzed by Western
blotting with the anti-HA antibody. Fig. 4B shows that both
PKN
and PKN
associated with the PLD1 (residues 1-228) containing
phox domain and PLD1 (residues 228-598) containing pleckstrin
homology-like domain, conserved regions I and II, and a
part of the loop domain of PLD1 (31-34).

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Fig. 4.
Dissection of the PLD1 interaction with
PKN and PKN .
A, schematic representation of the structure of PLD1 is
shown on the top. The possible functions of each box have
been proposed or demonstrated in Ref. 31. PX, phox domain;
PH, pleckstrin homology-like domain; CRI-IV,
highly conserved regions in the PLD family (31); LOOP, loop
region; CT, C terminus. B, the N-terminal
(lane 1), internal (lane 2), and C-terminal
(lane 3) fragments of PLD1 and either PKN
(left) or PKN (right) were co-transfected into
COS-7 cells. After immunoprecipitation with the anti-FLAG antibody, the
amounts of co-immunoprecipitated HA-tagged PKN and PKN were
detected by immunoblotting with the anti-HA antibody (upper
panel). The immunoprecipitate of FLAG-tagged PLD1 with the
anti-FLAG antibody is indicated in the middle panel. The
expressions of PKN and PKN were visualized using immunoblotting
with the anti-HA antibody (lower panel).
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Direct Interaction PKN
and PKN
with PLD1--
To investigate
whether PKN
and PKN
directly bind to PLD1, we performed an
in vitro binding analysis using the purified proteins. (His)6-PLD1N (residues 1-228) and (His)6-PLD1I
(residues 228-598) were bacterially expressed and purified as
described (17). The PKN
or PKN
fused to GST was expressed and
affinity-purified from Sf9 cells. The proteins were mixed, and
each GST-fusion protein was pulled down by glutathione-Sepharose beads.
The bound proteins were detected by Western blotting with the
anti-(His)6 antibody. Fig. 5
shows that GST-PKN
pulled down both (His)6-PLD1N and
(His)6-PLD1I. On the other hand, GST-PKN
associated with
(His)6-PLD1I, but not with (His)6-PLD1N. GST
itself did not interact with (His)6-PLD1N or
(His)6-PLD1I. In the co-immunoprecipitation study, PKN
associated with both PLD1N and PLD1I in COS-7 cells, suggesting that
the interaction of PKN
with PLD1 (residues 1-228) in COS-7 cells is
mediated by some other molecules.

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Fig. 5.
In vitro interactions of the N-terminal and
internal fragments of PLD1 with PKN and
PKN . GST-fused PKN , PKN , and GST
were expressed in Sf9 cells and affinity-purified with
glutathione-Sepharose beads. (His)6-PLD1N and PLD1I
expressed and purified from Escherichia coli were incubated
with GST-PKN ( ) or GST-PKN ( ). Each GST-fused protein was
precipitated as described under "Experimental Procedures." As a
control, GST was used instead of these protein kinases
(GST). The amounts of (His)6-PLD1N
(left) or PLD1I (right) co-precipitated with
GST-fused proteins were detected by immunoblotting with the
anti-(His)6 antibody.
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Stimulation of PLD1 by PKN
in Vitro--
Because the
activators for PLD1 such as PKC and RhoA stimulate the activity of PLD1
through direct interaction, we investigated whether PKN
and PKN
can stimulate PLD1. FLAG-tagged PLD1 expressed in COS7 cells was
immunoprecipitated with the anti-FLAG antibody and was used for this
assay. Fig. 6 shows that PKN
stimulated the activity of PLD1 in the presence of phosphatidylinositol
4,5-bisphosphate, whereas PKN
had a modest effect on the stimulation
of PLD1 activity. The PLD1 stimulation by PKN
was slightly enhanced
by the addition of arachidonic acid, which is a potential activator of
PKN
. This stimulation was independent of ATP (data not
shown).

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Fig. 6.
In vitro activation of PLD1 by
PKN and PKN .
Immobilized FLAG-tagged PLD1 was incubated at 37 °C for 30 min with
the indicated concentrations of GST-PKN ( ) or PKN ( ) in 20 mM Hepes, pH 7.5, 3 mM EGTA, 80 mM
KCl, 2.5 mM MgCl2, 2 mM
CaCl2, and 1 mM dithiothreitol with
(right) or without (left) 40 µM
arachidonic acid (AA). As a control, the indicated
concentrations of GST were incubated instead of these protein kinases
(mock). The PLD1 activity was determined as described under
"Experimental Procedures." The error bars represent the
differences of triplicate determinations. The results shown are
representative of three separate experiments.
|
|
 |
DISCUSSION |
In the present study, we demonstrated that the members of the PKN
family, PKN
and PKN
, associated with PLD1 and that PKN
stimulated PLD1 activity in vitro. This stimulation was
ATP-independent, suggesting that it is mediated by direct binding
rather than phosphorylation of PLD1. The interaction of PKN
with
PLD1 was weaker than PKN
(Figs. 2 and 3). Nevertheless, PKN
stimulated PLD1 activity to a greater extent than PKN
(Fig. 6).
There may be at least two possible explanations for the lower
stimulatory effect of PKN
. First, the binding nature of PKN
with
PLD1 was somewhat different from that of PKN
. PKN
directly
interacted with the N-terminal (residues 1-228) as well as the
internal (residues 228-598) region of PLD1 (Fig. 5), whereas PKN
interacted directly with the internal region (Fig. 5) and indirectly
with the N-terminal region (Fig. 4). Although PKN
and PKN
are
closely related, this difference in the binding may reflect the
distinct effects of these proteins on PLD1 activity. Second, insect
cell-expressed PKN
used in the PLD1 assay (Fig. 6) was relatively
unstable and susceptible to proteolysis (data not shown). Because
endogenous PKN
in mammalian cells is distributed in membrane and
nuclear fractions (6), PKN
may need some lipids or binding proteins
to form a stable structure. This instability of the enzyme may lower
the effect on the PLD1 activity measured in vitro. Further,
in the case of RhoA, lipid modification such as geranylgeranylation
greatly enhance the stimulatory effect on PLD1 but does not affect the
binding (38), suggesting that RhoA-lipid interaction may help to induce catalytically active conformation of PLD1. Similarly, some
conformational change of PKN
by association with lipids (or other
proteins) might be required to achieve effective stimulation of PLD1.
We observed that endogenous PLD activity in COS-7 cells was
significantly enhanced by the expression of PKN
(data not shown),
suggesting that PKN
may work as an effective stimulator in
vivo.
PKC
is well known to stimulate PLD1 activity by direct interaction
in a phosphorylation-independent manner. The interaction and the
stimulation are enhanced by the presence of phorbol ester (25, 27-29),
indicating the importance of the conformational change of PKC
induced by phorbol ester binding as well as intracellular translocation
of PKC
to the membrane. Proteolysis studies indicate that the
N-terminal regulatory region of PKC
mediates this stimulation (25).
The mechanism of PLD1 stimulation by the PKN family presented here
seems to be similar to that by PKC
. However, we observed stimulatory
effects with the C-terminal catalytic region of PKN
but not with the
N-terminal regulatory region (data not shown). Further analyses will be
needed to clarify the difference in the mechanism of PLD stimulation
between the PKN and PKC families.
PKN
and PKN
associated with PLD1 through the N- and
C-terminal regions (Fig. 3). We have reported that the N-terminal
region of PKN
directly interacts with the C-terminal catalytic
region and that this interaction inhibits its catalytic activity (39). Thus, the PLD1 stimulation by the C-terminal region of PKN
may be
affected by the interaction between its N-terminal region and PLD1.
PLD1 stimulation by PKN
was enhanced by the presence of arachidonic
acid, which is a potential activator of PKN
(Fig. 6). Arachidonic
acid activates PKN
probably by inducing conformational change to
release the N-terminal region and the autoinhibitory region from the
catalytic region (12). This conformational change may help the
associated PLD1 to form active conformation. Although PKN
has the
conserved domain corresponding to the autoinhibitory domain of PKN
,
arachidonic acid has little effect on the protein kinase activity (6)
and PLD1 stimulation by this enzyme (Fig. 6, right),
suggesting that conformation of PKN
is not sufficiently changed by
arachidonic acid.
PKN family proteins interact with the Rho family small GTPase (3-5).
PKN
is activated by the binding of RhoA to its leucine zipper-like
domain in a GTP-dependent manner, suggesting that the interaction with GTP-bound RhoA also causes the conformational change of PKN
to expose the catalytic domain to substrate. Thus, RhoA might stimulate PLD1 activity by activating the PKN family in
addition to the direct interaction with PLD1.
In summary, the PKN family protein kinases modulate the activity
of PLD1 by direct protein-protein interaction. Further analysis including the relationship between other cofactors activating PLD1 and
the PKN family will provide important insights into the physiological
role of PLD1 and PKN family.
We thank Dr. Y. Nishizuka for encouragement.
The abbreviations used are:
PKC, protein kinase
C;
PRK, protein kinase C-related protein kinase;
PLD, phospholipase D;
HA, hemagglutinin;
h, human;
GST, glutathione
S-transferase.
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