From the Department of Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095-1786 and the Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results presented here demonstrate that
protein kinase D (PKD) and PKC Protein kinase C (PKC),1
a major cellular target for the potent tumor-promoting phorbol esters
(1, 2), has been implicated in the mediation of diverse cellular
functions, including short-term regulation of ion fluxes, receptor
ligand binding, and signal transduction, and a wide range of longer
term effects including proliferation, differentiation, transformation,
and regulation of the mammalian cell cycle (3-6). At least 10 PKC
isoforms, i.e. the classic ( The recently identified protein kinase D (PKD) is a mouse serine
protein kinase with distinct structural and enzymological properties
(11-13). In particular, the catalytic domain of PKD, which is
distantly related to Ca2+-regulated protein kinases (11),
possesses only a low degree of sequence similarity to the highly
conserved regions of the kinase subdomains of the PKC family (14).
Accordingly, PKD does not phosphorylate a variety of substrates
utilized by PKCs indicating that PKD is a protein kinase with distinct
substrate specificity (11, 12, 15). The amino-terminal region of PKD
contains a tandem repeat of cysteine-rich regions that bind phorbol
esters and diacylglycerol (11), similar to those found in classical and
novel PKCs (16). However, unlike all PKCs, PKD does not possess a
regulatory pseudosubstrate domain upstream of the first cysteine-rich
motif. An additional structural feature that distinguishes PKD from the
PKC family is the presence of a PH domain, interposed between the
second cysteine-rich motif and the catalytic domain. PH domains are
modular protein domains which mediate protein-protein and lipid-protein
interactions and are found in many cytoskeletal and signal transducing
proteins (17, 18). The PKD PH domain has been found to contribute to
regulation of PKD catalytic activity (15).
Recently we reported that exposure of intact cells to biologically
active phorbol esters, membrane-permeant diacylglycerol or bryostatin-1
induces PKD activation via a PKC-dependent pathway (19,
20). PKD activated within cells via PKC can be immunopurified from cell
extracts in an active state that is independent of exogenously added
cofactors (19). Growth factors and mitogenic neuropeptides that promote
phospholipid turnover and diacylglycerol generation also stimulate this
pathway, indicating that PKD activation is an early event in cell
stimulation (21). We further substantiated the role of PKCs in PKD
activation by coexpression of PKD together with constitutively active
mutant PKC isoforms. These studies revealed that novel PKC Here, we demonstrate, for the first time, that coexpression of PKD with
PKC Cell Culture and Transfections--
COS-7 cells were maintained
by subculture in 10-cm tissue culture plates, every 3-4 days in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum at 37 °C in a humidified atmosphere containing 10%
CO2. For experimental dishes, confluent cells were
subcultured at a density of 6 × 104 cells/ml in 6- or
10-cm dishes on the day prior to transfections. All transfections and
co-transfections were carried out with equivalent amounts of DNA (6 µg/6-cm dish, 12 µg/10-cm dish), using vector pcDNA3 as the
control DNA added to single transfections. Transfections were carried
out in Opti-MEM (Life Technologies, Inc.) using Lipofectin (Life
Technologies, Inc.) at 10 µl/6-cm dish or 20 µl/10-cm dish, added
to cells in a final volume of 2.5 ml/6-cm dish or 5 ml/10-cm dish,
following formation of DNA-Lipofectin complexes according to the
protocol provided by the manufacturer. Cells were allowed to take up
complexes in the absence of fetal bovine serum for 5-6 h or overnight,
then fetal bovine serum (10% final concentration) in Opti-MEM was
added to the dishes to yield a final volume of 5 ml/6-cm dish or 10 ml/10-cm dish. Cells were used for experiments after a further 48-72 h
of incubation.
cDNA Constructs used in Transfections--
The
constructs pcDNA3-PKD encoding PKD (12), pcDNA3-PKD/K618M
encoding kinase-deficient mutant PKD (19), cysteine-rich domain mutant
constructs pcDNA3-PKD Preparation of PKD PH Domain Fusion Protein--
The cDNA
sequence spanning the entire PKD PH domain (aa 418-567) was amplified
by PCR from wild-type PKD using specific oligonucleotide primers
(forward primer, 5'-GATGGATCCGTGAAGCACACGAAGCGGAGG-3'; reverse primer, 5'-GCGGAATTCAGAAATATCTTTGTGTGAGTTGGA-3')
containing restriction sites for BamHI and EcoRI,
respectively (underlined). The resulting PCR product was subcloned as a
BamHI-EcoRI fragment into the vector pGEX4T3
(Pharmacia Biotech Inc.) to generate the bacterial expression construct
pGEX-GST-PKDPH. The 42-kDa GST-PKD PH domain fusion protein (GST-PKDPH)
was expressed in Escherichia coli, purified on
glutathione-agarose beads, eluted with 25 mM reduced
glutathione, dialyzed against phosphate-buffered saline, and stored at
Assays of PKC·GST-PKDPH Fusion Protein Binding in
Vitro--
COS-7 cells transiently transfected with the different PKC
isoforms were lysed 72 h after transfection by removal of growth medium from cells on ice and addition of lysis buffer (50 mM Tris-HCl, pH 7.6, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 10 µg/ml
aprotinin, 100 µg/ml leupeptin, 1 mM AEBSF (Pefabloc),
and 1% Triton X-100), and the resulting extracts were combined with
either GST (control) or GST-PKDPH fusion proteins preadsorbed onto
glutathione-agarose beads. After 2 h at 4 °C, the complexes
were washed 8 times with lysis buffer, and bound proteins were
extracted with SDS-PAGE sample buffer and subjected to SDS-PAGE and
Western analysis using isoform-specific antisera to detect associated
PKC, as described in figure legends.
Immunoprecipitations--
COS-7 cells transfected with wild-type
or mutant PKD or co-transfected together with different PKC isoforms
were lysed as described above. Small amounts (typically 1/10) of these
total lysates were saved and combined with equal volumes of SDS-PAGE sample buffer (1 M Tris-HCl, pH 6.8, 6% SDS, 0.5 M EDTA, 4% 2-mercaptoethanol, 10% glycerol) for Western
blot analysis. PKD was immunoprecipitated at 4 °C for 3 h with
either the PA-1 antiserum (1:100 dilution) raised against the synthetic
peptide EEREMKALSERVSIL that corresponds to the predicted
COOH-terminal region of PKD, as described previously (12), or a 1:200
dilution of a commercial antiserum (PKD C-20, Santa Cruz
Biotechnologies), which also recognizes the COOH-terminal region of
PKD. PKCs were immunoprecipitated using respective PKC antisera at
1:100 dilution. Immune complexes were recovered using protein A coupled
to agarose.
In Vitro Kinase Assays--
Immune complexes were washed twice
with lysis buffer, then twice with kinase buffer consisting of 30 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol. Autophosphorylation reactions were
initiated by combining 20 µl of immune complexes with 10 µl of a
phosphorylation mixture containing [
For assays of exogenous substrate phosphorylation, immune complexes
were processed as for autophosphorylation reactions, then substrates
(either syntide-2 or Western Blot Analysis--
For Western blot analysis, immune
complexes and proteins associated with glutathione-agarose/GST fusion
protein complexes were washed as for in vitro kinase
reactions, then extracted by boiling in SDS-PAGE sample buffer. Samples
of cell lysates were directly solubilized by boiling in SDS-PAGE sample
buffer. Following SDS-PAGE on 8% gels, proteins were transferred to
Immobilon-P membranes (Millipore), as described previously (21) and
blocked by overnight incubation with 5% non-fat dried milk in
phosphate-buffered saline, pH 7.2. Membranes were incubated at room
temperature for 2 h with antisera specifically recognizing either
PKD or the different PKC isoforms, at a dilution of 1 µg/ml, in
phosphate-buffered saline containing 3% non-fat dried milk.
Immunoreactive bands were visualized using either horseradish
peroxidase-conjugated anti-rabbit IgG and subsequent enhanced
chemiluminescence detection or 125I-labeled protein A
followed by autoradiography.
Materials--
[ An 80-kDa Phosphoprotein Associates with PKD in Cells Coexpressing
PKD and PKC
In agreement with previous results (12, 19-21), PKD isolated from
cells transfected with PKD alone displayed low catalytic activity and
PDBu treatment of these cells resulted in isolation of persistently
activated PKD. Furthermore, PKD isolated from cells co-transfected with
PKD together with active mutant PKCs
Initially, we considered whether this band might be the result of
either nonspecific immunoprecipitation by the PA-1 antiserum or
long-term overexpression of the active mutant PKC
To test whether the 80-kDa band represented a proteolytic fragment of
autophosphorylated PKD, we co-transfected an expression construct,
pcDNA3-PKD/K618M, which encodes a kinase-deficient mutant of PKD,
together with PKC PKC
Since PKC
Although the experiments in Figs. 1 and 2 indicate that PKC Phosphorylation of PKC and PKD Substrates by PKD
Immunoprecipitates--
To further address the specificity of the
PKD-PKC interaction, we measured phosphorylation of exogenous
substrates by PKD immunoprecipitates from cells expressing each PKD-PKC
combination. In initial experiments, we examined phosphorylation of
For assays of PKD immunoprecipitates we used both
Interestingly, in cells co-transfected with PKD and PKC The PKD PH Domain Is Required for Formation of a PKD·PKC
In order to determine the specificity of inhibition of PKD-PKC The PH Domain of PKD Selectively Binds to PKC Our previous studies have shown that PKD is activated in
vivo by treatment with biologically active phorbol esters or
multiple agonists via a PKC-dependent pathway (19-21). The
results presented here demonstrate the formation of a complex between
PKC From our results, it is clear that the PH domain of PKD mediates a
major portion of the binding to PKC Recently, a model for the binding of PH domains to proteins has been
presented (32) in which a candidate sequence
(HIKX8E), identified by sequence homology
analysis, was proposed to act as a target for the PH domains. However,
the putative binding sequence HIKX8E is present
in both PKC In conclusion, our findings demonstrate that coexpression of PKD with
PKC transiently coexpressed in COS-7
cells form complexes that can be immunoprecipitated from cell lysates
using specific antisera to PKD or PKC
. The presence of PKC
in PKD
immune complexes was initially detected by in vitro kinase
assays which reveal the presence of an 80-kDa phosphorylated band in
addition to the 110-kDa band corresponding to autophosphorylated PKD.
The association between PKD and PKC
was further verified by Western
blot analysis and peptide phosphorylation assays that exploited the
distinct substrate specificity between PKCs and PKD. By the same
criteria, PKD formed complexes only very weakly with PKC
, and did
not bind PKC
. When PKC
was coexpressed with PKD mutants
containing either complete or partial deletions of the PH domain, both
PKC
immunoreactivity and PKC activity in PKD immunoprecipitates were
sharply reduced. In contrast, deletion of an amino-terminal portion of
the molecule, either cysteine-rich region, or the entire cysteine-rich
domain did not interfere with the association of PKD with PKC
.
Furthermore, a glutathione S-transferase-PKDPH fusion
protein bound preferentially to PKC
. These results indicate that the
PKD PH domain can discriminate between closely related structures of a
single enzyme family, e.g. novel PKCs
and
, thereby
revealing a previously undetected degree of specificity among
protein-protein interactions mediated by PH domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
1,
2, and
), novel (
,
,
, and
), and atypical PKCs (
,
) have been identified by molecular cloning techniques (5, 7, 8). The different PKC isoforms exhibit distinct
patterns of expression in different cell types and tissues as well as
distinct subcellular distributions, leading to the view that they play
distinct, rather than redundant roles in signal transduction (5, 9,
10). However, since very few of their specific substrates and binding
partners have been identified, it has not yet been possible to ascribe
to each isoform a unique cellular function.
and
PKC
fully activated PKD within cells, whereas classical PKC
1 or
atypical PKC
did not (19). These results raise the possibility that
PKD functions downstream of novel PKCs in a previously undescribed
signal transduction pathway.
in COS-7 cells leads to the formation of a stable PKD·PKC
complex. Strikingly, we found very little evidence of complex formation
between PKD and the PKC
isoform despite its close similarity to
PKC
, and no evidence for a stable interaction between PKD and
PKC
. Our results also demonstrate that the PH domain is critical for
stable PKD·PKC
complex formation, thus indicating that these
domains can mediate highly selective protein-protein interactions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Cys1, pcDNA3-PKD
Cys2, and pcDNA3-PKD
CRD (22), the PKD mutant
pcDNA3-PKD
NH2 with a deletion of the
NH2-terminal first 66 residues including the putative
transmembrane motif (22) and the PH domain mutants pcDNA3-PKD
PH
lacking the entire PH domain, pcDNA3-PKD
1-4
lacking the
first 4
strands of the 7-stranded
-barrel portion, and
pcDNA3-PKD
lacking the COOH-terminal
-helix have been
described previously (15). An additional PH domain mutant PKD
construct, pcDNA3-PKD
1-7
, encoding PKD lacking all seven
-strands of the
-barrel (deletion of amino acids 429-532) was
constructed using PCR, as follows: primers carrying the AscI
site (forward primer, 5'-GTCAGGCGCGCCGATGTGGCCAGGATGTGGGA-3'; reverse
primer, 5'-GTCAGGCGCGCCAGCCAACAGAGGAGCCCTTG-3') were used to amplify
the COOH-terminal part of the PH domain (from Asp533 to
Gly557). The generated PCR product was
AscI-digested and reinserted into the AscI site
of pcDNA3-PKD
PH, resulting in the plasmid pcDNA3-PKD
1-7
. The cDNAs encoding wild-type and active
mutant PKC isoforms were kind gifts from Dr. Peter Parker, Imperial
Cancer Research Fund, and have been described previously (23).
20 °C in 40% glycerol. Purity and concentration of the
recombinant protein were assessed by SDS-PAGE and Coomassie Brilliant
Blue staining.
-32P]ATP (3 µCi/reaction diluted with cold ATP to give a final concentration of
100 µM) in kinase buffer. Reactions were transferred to a
water bath at 30 °C for 10 min, then terminated by addition of 1 ml of ice-cold kinase buffer and removed to an ice bucket. Immune complexes were recovered by centrifugation, and the proteins were extracted for SDS-PAGE analysis by addition of SDS-PAGE sample buffer.
Dried SDS-PAGE gels were subjected to autoradiography to visualize
radiolabeled protein bands.
-peptide at final concentrations 2.5 or 1.75 mg/ml, respectively) were added in the presence of [
-32P]ATP (2 µCi/reaction diluted with cold ATP to
give a final concentration of 100 µM) in kinase buffer
(final reaction volume, 30 µl), and transferred to a water bath at
30 °C for 10 min. Reactions were terminated by adding 100 µl of 75 mM H3PO4, and 75 µl of the mixed supernatant was spotted to Whatman P-81 phosphocellulose paper. Papers
were washed thoroughly in 75 mM
H3PO4, dried, and radioactivity incorporated
into peptides was determined by detection of Cerenkov radiation in a
scintillation counter.
-32P]ATP (6000 Ci/mmol) was
from Amersham International (United Kingdom). Protein A-agarose and
AEBSF (Pefabloc) were from Boehringer Mannheim (UK). Antisera (PKD
C-20, PKC
C-20, PKC
C-15, PKC
C-15, and PKC
C-15) used in
Western blot analysis were from Santa Cruz Biotechnologies, Palo Alto,
CA. PKC standard proteins were from Calbiochem. Opti-MEM and Lipofectin
were from Life Technologies, Inc. Glutathione-Sepharose was from
Pharmacia Biotech. Syntide-2 peptide and immunizing peptide
EEREMKALSERVSIL corresponding to the PKD COOH terminus were synthesized
at the Imperial Cancer Research Fund.
-Peptide was from Alexis
Biochemicals. All other reagents were from standard suppliers or as
described in the text and were the highest grade commercially available.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
COS-7 cells transiently transfected with
pcDNA3-PKD either alone or together with PKC
, PKC
, or PKC
expression constructs encoding constitutively active PKC mutants were
incubated with or without 200 nM PDBu for 10 min and lysed.
PKD was immunoprecipitated from the extracts and the resulting
immunoprecipitates were subjected to in vitro kinase assays,
followed by SDS-PAGE analysis and autoradiography to detect the
prominent 110-kDa band corresponding to autophosphorylated PKD.
or
was in persistently
activated form in the absence of PDBu treatments of these cells (Fig.
1A). Strikingly, when PKD
immunoprecipitates from PKD/PKC
co-transfected cells were assayed,
an additional phosphorylated band corresponding to an apparent
molecular mass of approximately 80 kDa was also apparent in the
autoradiograms (Fig. 1A). This band did not appear when PKD
was coexpressed with the active PKC
or
mutant forms despite
their level of overexpression shown by Western blot analysis (Fig.
2), and despite the fact that the PKC
mutant induced PKD activation to essentially the same degree as did the
PKC
mutant (Fig. 1A). This latter result also indicates
that the presence in PKD immunoprecipitates of the phosphoprotein
giving rise to the 80-kDa band resulting from coexpression with PKC
is not uniquely required for the persistent activation of PKD.
Expression of PKC
on its own followed by immunoprecipitation with
the PA-1 antiserum and in vitro kinase assays did not result in the appearance of the 80-kDa band (Fig. 1A). Thus, the
immunoprecipitation and subsequent labeling of the 80-kDa band
required PKD.
View larger version (45K):
[in a new window]
Fig. 1.
Detection of PKD·PKC
complexes within PKD immunoprecipitates by in vitro
kinase assays. A, exponentially growing COS-7
cells (40-60% confluent) were co-transfected with pcDNA3-PKD
(PKD) together with either empty vector pcDNA3 (
) or the vectors
containing the cDNAs encoding the constitutively active PKC
isoforms, as indicated. At 72 h post-transfection, the cultures
were either treated with 200 nM PDBu (PDB) (+)
or left untreated (
) for 10 min. The cultures were then lysed and the
lysates immunoprecipitated with the PA-1 antiserum and further analyzed
by in vitro kinase reactions as described under
"Experimental Procedures." A representative autoradiogram is shown.
Similar results were obtained from each of at least three similar
experiments. B, COS-7 cells were co-transfected with
pCO2-PKC
plasmid encoding the wild-type PKC
(+)
together with either pcDNA3-PKD or empty vector pcDNA3, as
indicated (
). After 72 h of incubation, the cultures were lysed
and the lysates immunoprecipitated with the commercially obtained
anti-PKD antiserum in the absence (
) or presence (+) of the
immunizing peptide (Imm Pep) used to generate the PA-1
antiserum at a concentration of 20 µg/ml lysate, and further analyzed
by autophosphorylation reactions. This experiment was repeated twice
with similar results. A representative autoradiogram is shown. C,
left, COS-7 cells were co-transfected with pcDNA3-PKD (PKD),
pcDNA3-PKDK618M (KM), or empty vector pcDNA3 (
)
together with the plasmid construct encoding constitutive active PKC
(
*), as indicated. At 72 h post-transfection, the cultures were
lysed and the lysates immunoprecipitated with the PA-1 antiserum and
further analyzed by in vitro kinase reactions. This
experiment was repeated three times with similar results. A
representative autoradiogram is shown. C, right, COS-7 cells
were co-transfected with either pcDNA3-PKD (PKD) or
pcDNA3-PKDK618M (KM) together with plasmid constructs
encoding wild-type PKC
(
), active mutant PKC
(
*), or empty
vector pcDNA3 (
), as indicated. At 72 h post-transfection,
the cultures were either treated with 200 nM PDBu (+) or
left untreated (
) for 10 min, as indicated. The cultures were then
lysed and the lysates immunoprecipitated with the PA-1 antiserum and
further analyzed by exogenous substrate phosphorylation assays using
peptide as described under "Experimental Procedures."
View larger version (29K):
[in a new window]
Fig. 2.
Determination of PKD binding specificity
toward different PKC isoforms by Western blotting. Exponentially
growing COS-7 cells were either transfected with empty vector
pcDNA3 ( ) or pcDNA3-PKD (A) or co-transfected with
vectors containing the cDNAs encoding either wild-type (
and
; C, D, and G) or constitutively active PKC
isoforms (
*,
*, and
*; B, E, and F)
together with either pcDNA3-PKD (+) or empty vector pcDNA3
(
), as indicated. At 72 h post-transfection, the cultures were
lysed and the lysates were immunoprecipitated with either PA-1
antiserum (A-F) or the PKC
antiserum (G).
After washing, the immune complexes were either extracted with gel
loading buffer and analyzed by SDS-PAGE followed by Western blot
analysis using the isoform-specific polyclonal antisera against either
the different PKCs (A-F) or PKD (G, PKD W.Blot)
or analyzed by in vitro kinase assays (G, IVK),
as indicated. Arrows indicate the position of PKD in
anti-PKC
immunoprecipitates (G) and PKC
in PKD
immunoprecipitates (D and F). Similar results
were obtained in at least three experiments.
. To test this, we
used a commercially available antiserum (rather than PA-1) to perform
PKD immunoprecipitations from cells coexpressing PKD together with the
wild-type PKC
, followed by in vitro kinase assays. As
shown in Fig. 1B, this assay also led to the isolation of
immune complexes containing 110- and 80-kDa phosphoproteins. Thus, it
was possible to substitute either the primary antibody used in the
immunoprecipitation or the wild-type for the active mutant PKC
and
still retain both bands. In addition, both autophosphorylated PKD and
the coprecipitated 80-kDa phosphoprotein band were eliminated when the
immunoprecipitation reactions were carried out in the presence of the
immunizing peptide used to generate the PA-1 antiserum (Fig.
1B).
, and performed immunoprecipitations with the PA-1
antiserum followed by in vitro kinase assays. As shown in
Fig. 1C, this assay again resulted in the appearance of the
80-kDa band. Importantly, whereas the intensity of this band was
similar to that seen when the wild-type PKD was used for
co-transfection, the intensity of the 110-kDa band was drastically reduced in comparison with that generated by the co-transfection with
wild-type PKD2 (Fig.
1C, left). These data also indicate that the kinase activity of PKD is not required for the association with the 80-kDa phosphoprotein.
Specifically Associates with PKD--
Previous studies
indicated that
-peptide, a peptide based on the pseudosubstrate
domain of PKC
, is a substrate for all PKCs (24), but is a poor
substrate for PKD (11, 12, 15). In agreement with these studies,
anti-PKD immunoprecipitates from lysates of cells transfected with PKD
(or the kinase-deficient mutant, PKD/K618M) did not phosphorylate
-peptide, even when PKD was activated within cells by PDBu
treatments (Fig. 1C, right). Surprisingly, PKD
immunoprecipitates from lysates of cells co-transfected with PKD
(either wild-type or PKD/K618M) and PKC
(either constitutive active
or wild-type) contained activity which strongly phosphorylated
-peptide. Since this activity was associated with both catalytically active and inactive forms of PKD, and PKD does not phosphorylate the
peptide, we conclude that the activity was due to an associated protein
kinase (e.g. contributed by the 80-kDa phosphoprotein) whose
activity did not require the catalytic activity of PKD.
has an apparent molecular mass of approximately 80 kDa in
SDS-PAGE, and since the 80-kDa phosphoprotein had appeared only when
PKD was coexpressed together with mutant or wild-type PKC
, it seemed
plausible that coexpression of PKD and PKC
resulted in the formation
of a PKD·PKC
complex that persists during immunoprecipitation of
PKD. To test this hypothesis directly, we used Western blot analysis to
examine whether PKC
was present in PKD immunoprecipitates. In view
of the results indicating that the 80-kDa band is not detected in PKD
immunoprecipitates from lysates of cells co-transfected with PKD
together with either PKC
or PKC
(Fig. 1A), we also performed Western blot analysis to assess whether these isoforms were
present in PKD immunoprecipitates. Since PKC
is abundantly expressed
in COS-7 cells, we also tested for the presence of this isoform in PKD
immunoprecipitates. Lysates of cells either transfected with PKD or
co-transfected with PKD and PKCs
,
, or
were either examined
directly by Western blot analysis using polyclonal antibodies that
specifically recognize PKCs
,
,
, or
or subjected to immunoprecipitation reactions with PA-1 antiserum followed by Western
analysis. As shown in Fig. 2, D and F,
anti-PKC
immunoblotting of PKD immunoprecipitates indicated that
both wild-type and mutant PKC
associated with PKD. Similar analysis
did not detect the presence of PKCs
,
, or
(either wild-type
or constitutively active) in PKD immunoprecipitates from the
corresponding cell lysates (Fig. 2, A-C and E).
However, longer exposure of the autoradiograms did reveal a faint band
of anti-PKC
immunoreactivity (not shown), indicating that PKC
may
also associate with PKD but to a much lesser degree.
was
present in PKD immunoprecipitates from cells co-transfected with these
two proteins, these results were obtained using antibodies directed
against PKD. To substantiate further the existence of a PKD-PKC
interaction, we also performed immunoprecipitations using an antibody
directed against the opposite partner in the association,
i.e. PKC
, followed by Western analysis to detect the
interacting PKD protein. As illustrated in Fig. 2G, in
vitro kinase assays revealed the presence of an autophosphorylated
band in the position corresponding to PKD (110 kDa) that depended on the presence of co-transfected PKD. Furthermore, Western blot analysis
revealed the presence of immunoreactive PKD when both proteins were
co-transfected. Thus, the results shown in Fig. 2G
demonstrate the presence of PKD in PKC
immunoprecipitates from
lysates of cells co-transfected with PKD and PKC
.
-peptide by endogenously expressed and transfected PKC isoforms in
anti-PKC immunoprecipitates. Results shown in Fig.
3A demonstrate that PKC
or
PKC
immunoprecipitates from mock-transfected COS-7 cells contained
low levels of
-peptide phosphorylation activity that was
dramatically increased by transfection of these isoforms. PKC
immunoprecipitates from mock-transfected cells contained similar
activity to the immunoprecipitates from PKC
- or PKC
-transfected cells. These results demonstrate the ability of each PKC tested to
phosphorylate
-peptide.
View larger version (18K):
[in a new window]
Fig. 3.
Determination of PKD binding specificity
toward different PKC isoforms by phosphorylation of exogenous
substrates. Exponentially growing COS-7 cells were
transfected with either empty vector pcDNA3 ( ) or plasmid
constructs encoding the constitutively active PKC isoforms, or
co-transfected with pcDNA3-PKD together with either empty
vector or the active PKC isoforms, as indicated. At 72 h
post-transfection, the cultures were lysed, the lysates were
immunoprecipitated with the PA-1 antiserum, and subjected to exogenous
substrate phosphorylation assays as described under "Experimental
Procedures." A and B, lower
panel,
-peptide phosphorylation assay. B,
upper panel, syntide-2 phosphorylation assay. The results are from
at least three experiments, each performed in duplicate (B),
or from a single experiment performed in triplicate
(A).
-peptide, a poor
substrate for PKD (Fig. 1), and syntide-2 (25, 26), a synthetic peptide
previously demonstrated to be an excellent substrate for PKD (11),
thereby exploiting the distinct substrate specificity of PKCs and PKD.
In this way, we measured both PKD activation and the retention of PKC
activity bound to PKD in the same immune complexes. In agreement with
previous results, when the PA-1 antiserum was used to immunoprecipitate
PKD from cells overexpressing only PKD, syntide-2 assays revealed a low
basal activity that was dramatically increased upon PDBu stimulation of
cells (Fig. 3B, upper graph). Again, these
immunoprecipitates did not phosphorylate
-peptide to any significant
extent, even after the cells had been stimulated with PDBu (Fig.
3B, lower graph). In contrast with these results, when PKD
was immunoprecipitated from cells co-transfected with PKD and either
mutant or wild-type PKC
, both syntide-2 and
-peptide
were strongly phosphorylated, indicating the presence of both PKD and
PKC
enzyme activities. These results extend those of Fig. 1 by
providing evidence that PKC
activity, and not that of PKC
or
PKC
, is preferentially associated with PKD. Thus, phosphorylation of
the PKC
peptide, an excellent PKC substrate, correlates with the
appearance of the 80-kDa band in the in vitro kinase assays
(Fig. 1) and with the detection of immunoreactive PKC
in PKD
immunoprecipitates (Fig. 2).
,
phosphorylation of syntide-2 was also nearly maximal even in the absence of PDBu stimulation even though phosphorylation of
-peptide was only slightly above control levels. This result demonstrated that,
in agreement with results in Figs. 1 and 2, PKC
had activated PKD
during coexpression but is retained in the PKD immune complexes only
slightly, implying that the involvement of PKCs in PKD activation is
mediated by a transient event rather than, or in addition to, the
PKD-PKC association itself. As expected, immunoprecipitates from cells
co-transfected with PKD and PKC
phosphorylated syntide-2 in a manner
indistinguishable from that from cells expressing only PKD, and did not
phosphorylate
-peptide. These results confirm that PKD
preferentially forms complexes with PKC
rather than with PKC
, and
does not form complexes with PKC
.
Complex--
Recent reports have shown that PH domains within the
Bruton's tyrosine kinase and the serine-threonine kinase PKB/Akt can mediate association of these proteins with multiple isoforms of PKC
(27, 28). In contrast, our results demonstrated that PKD preferentially
associates with PKC
. To examine whether the PH domain of PKD could
mediate this specific association of PKD with PKC
, we co-transfected
COS-7 cells with wild-type PKC
together with expression constructs
encoding either intact PKD, PKD lacking the entire PH domain, or PKD
with the PH domain truncated by partial deletions (15). PKD
immunoprecipitates from these cells were subjected to Western blot
analysis and phosphorylation of
-peptide to assess the presence of
PKC
in the immune complexes (Fig. 4, A and B). In agreement with data shown in Fig. 2,
wild-type PKC
was co-immunoprecipitated with intact PKD (Fig.
4B). Similarly, these immunoprecipitates contained PKC
activity, as revealed by
peptide assays (Fig. 4A). In
contrast, when PKC
was coexpressed with any of the PKD mutants
containing either complete or partial deletions of the PH domain, both
PKC activity (Fig. 4A) and PKC
immunoreactivity (Fig.
4B) in PKD immunoprecipitates were sharply reduced. Control
Western blots demonstrated that PKC
was expressed in each
transfection in similar amounts, as was each of the wild-type or mutant
PKD proteins (Fig. 4B).
View larger version (28K):
[in a new window]
Fig. 4.
Role of the PKD PH domain in mediating
PKD·PKC complex formation.
A, exponentially growing COS-7 cells were transfected with
either pcDNA3-PKD (open bars) or co-transfected with
PKD
PH lacking the entire PH domain (
PH), PKD
1-4
lacking the first amino-terminal four
-sheets (
4
),
PKD
1-7
lacking all seven
sheets (
7
), or PKD
1
lacking the carboxyl-terminal
-helix (
1), as indicated,
together with wild type PKC
(filled bars). At 72 h
post-transfection, the cultures were lysed, the lysates were
immunoprecipitated with the PA-1 antiserum, and subjected to
-peptide phosphorylation assays as described under "Experimental
Procedures." B, Western blot analysis of PKC
in PKD immunoprecipitates (upper panel), PKC
in cell
lysates (center panel), and PKD mutants in cell lysates
(lower panel) from the transfected cells. C,
exponentially growing COS-7 cells were co-transfected with wild-type
PKC
together with PKD deletion mutants PKD
NH2 lacking
an amino-terminal portion containing the transmembrane region
(
NH2), PKD
PH (
PH), PKD-
Cys1 lacking the first
cysteine-rich region (
Cys1), PKD-
Cys2 lacking the second
cysteine-rich region (
Cys2), or PKD
CRD lacking the entire
cysteine-rich domain (
CRD). (In the experiment shown, a control
Western blot of cell lysates indicated that the PKD
CRD protein was
expressed to somewhat higher levels than the other PKD truncation
mutants, thereby accounting for the somewhat higher intensity of this
band in the blot of PKC
immunoprecipitates.) At 72 h
post-transfection, these cells were lysed and the lysates subjected to
immunoprecipitations with PKC
or PKD antibodies, as indicated.
Immunoprecipitates were washed and analyzed by SDS-PAGE prior to
Western analysis. PKD immunoprecipitates were probed using the
anti-PKC
antiserum, and PKC
immunoprecipitates were probed using
the anti-PKD antiserum, as indicated. The arrow indicates
the expected position for PKD
PH protein.
interaction by deletion of the PKD PH domain, we also examined the
effect of other deletion mutants of PKD on the PKD-PKC
interaction. COS-7 cells co-transfected with PKC
together with PKD mutants lacking either a portion of the amino terminus containing the hydrophobic sequence of PKD, the first or second cysteine-rich regions,
or the entire tandem cysteine-rich domain (22) were lysed and
immunoprecipitated either with the PA-1 antiserum or with the PKC
antiserum. As shown in Fig. 4C, Western analysis of each of
these reciprocal immunoprecipitates revealed the presence of the
opposite binding partner in the association. Similar to the results
shown in Fig. 4, A and B, deletion of the PH
domain prevented the detection of both binding partners in the
respective immunoprecipitates (Fig. 4C). Taken together,
these data indicate that the PH domain of PKD is necessary for
efficient complex formation with PKC
. However, it appears that the
PH domain is not the only determinant of binding to PKD, as some PKC
immunoreactivity and activity was associated with immunoprecipitates of
the truncated PKD mutants (Fig. 4, A and B).
--
To further
examine the possibility that the PH domain of PKD interacts
preferentially with PKC
, we incubated lysates of cells transfected
with activated PKC
or
or wild-type PKC
or
with a fusion
protein, GST-PKDPH, which contains the full-length PH domain (residues
429-557) of PKD. The recovered fusion protein was extracted and
subjected to Western blot analysis to detect associated PKCs. As shown
in Fig. 5A, PKC
(activated
and wild-type) proteins were retained by their ability to associate
with the GST-PKDPH fusion protein. In contrast, much less PKC
was
recovered from parallel cell lysates with this fusion protein. Control
Western blot analysis of cell lysates confirmed that PKC
was indeed
overexpressed in these cells, allowing us again to infer that there was
a preferential binding of PKD to PKC
over PKC
. To address this
issue more quantitatively, we performed Western blot analysis using
different amounts of transfected cell lysates and purified protein
standards (PKC
and PKC
) to determine the absolute amounts of each
PKC isoform produced in cell lysates and recovered by the fusion
protein. As determined from data shown in Fig. 5B, each PKC
isoform was present in the cell lysates in similar amounts
(approximately 1.5 µg/ml for each PKC in the initial lysates). We
then examined the binding of GST-PKDPH to PKC
and PKC
in cell
lysates as a function of fusion protein concentration. These
experiments demonstrated that the fusion protein bound to PKC
at a
concentration as low as 0.1 µg/ml. In contrast, very little PKC
was bound to the fusion protein even at a 100-fold higher concentration
(Fig. 5B).
View larger version (42K):
[in a new window]
Fig. 5.
Binding of different PKCs to the GST-PKDPH
fusion protein. COS-7 cells transiently transfected with active
mutant PKC (
*), wild type PKC
(
), wild-type PKC
(
),
or active mutant PKC
(
*) were lysed after 72 h of
incubation. Equivalent amounts of cell lysates were combined with
immobilized GST or GST-PKDPH, as indicated and then processed by
incubations, washing procedures, and Western blot analysis as described
under "Experimental Procedures." A, GST or GST-PKD PH (1 µg each) was incubated with lysates from cells transfected with
active mutant PKC
(
*), wild type PKC
(
), wild-type PKC
(
), or active mutant PKC
(
*) prior to further processing.
B, upper panels, different amounts of standard PKC
and
proteins, as indicated, were subjected to Western blot analysis
using the isoform-specific antisera. Middle panels, lysates
were prepared from cells transfected with either PKC
or PKC
and
subjected, in parallel, to Western blot analysis using the indicated
amounts of cell lysates (diluted 1:1 with 2 × SDS-PAGE gel
loading buffer), as indicated. Lower panels, GST (10 µg)
or different amounts of GST-PKDPH (100 ng, 300 ng, 1 µg, 3 µg, 10 µg and 15 µg) were preadsorbed to glutathione-agarose beads,
combined with lysates from cells transfected with either wild-type
PKC
or PKC
, as indicated, and then incubated and further
processed as in A. Results shown are representative of at
least three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and PKD and thus, identify a new aspect of the relationship
between these two enzymes. Although further studies will be required to
address the exact physiological role of this complex, the association of PKD and PKC
could play a part in the regulation of the activity and/or subcellular localization of these enzymes. Indeed, recent findings from Cantley and co-workers (29) indicated that transfected PKD/PKCµ formed complexes with endogenous phosphatidylinositol 4-kinase and phosphatidylinositol 4-phosphate 5-kinase enzymes present
in COS-7 cells. Importantly, truncation mutants of PKD/PKCµ lacking a
portion of the molecule between the NH2-terminal
hydrophobic region and the PH domain failed to retain the binding to
the lipid kinases (29). In view of the results presented here, we
conclude that different domains of the regulatory region of the PKD
molecule are involved in mediating interactions with different
proteins. Together, these results suggest the attractive possibility
that PKD/PKCµ may act as a scaffold protein, through its different domains, promoting the assembly of signaling enzymes.
, other (even larger) deletions
of the PKD molecule being without effect. Recently, two important
signaling proteins, PKB/Akt and BTK, have been shown to interact via
their PH domains with multiple isoforms of PKC (27, 28, 30).
Interestingly, there are important differences between these studies
with BTK and PKB/Akt and the findings presented here with PKD. 1) The
studies with BTK and PKB/Akt indicated that the PH domain of these
protein kinases interacts with multiple isoforms of PKC. In contrast,
our results show that PKD associates preferentially with PKC
. In
fact, the fusion protein containing only the PKD PH domain was
sufficient to isolate the wild-type PKC
from cell lysates, and in
this "pull-out" experiment, the PH domain mirrored the results seen
with the intact PKD, i.e. it exhibited a striking preference
for binding PKC
over the closely related PKC
, and did not
interact with PKC
. 2) The binding of BTK PH domain to PKC
requires a region within the C1 regulatory domain in the vicinity of
the pseudosubstrate domain of this enzyme (31). We find that PKD
associates with an active PKC
mutant with a deletion from amino
acids 155 to 171 within its pseudosubstrate domain (as well as with
wild-type PKC
). Therefore, the interaction of the PKD PH domain with
PKC
does not require the pseudosubstrate portion of the molecule.
and PKC
, and consequently is unlikely to play a
critical role in determining the differential binding of the PH domain
of PKD to different PKCs found in the present study.
leads to the formation of a stable PKD·PKC
complex. Strikingly, we found very little evidence of complex formation between
PKD and the PKC
isoform despite its close similarity to PKC
, and
no evidence for a stable interaction between PKD and PKC
. Our
results also demonstrate that the PH domain is critical for stable
PKD·PKC
complex formation. We conclude that the PKD PH domain can
discriminate between closely related structures of a single enzyme
family, e.g. novel PKCs
and
, thereby revealing a
previously undetected degree of specificity among protein-protein interactions mediated by PH domains.
![]() |
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.
Contributed equally to the results of this study.
§ Supported by a postdoctoral research fellowship from the Imperial Cancer Research Fund. Present address: Dept. of Medicine, UCLA School of Medicine, Warren Hall, Room 11-124, Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399.
¶ To whom all correspondence should be addressed. Present address: Dept. of Medicine, UCLA School of Medicine, Warren Hall, Room 11-124, Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399.
2
In results from a large number of experiments,
no increases in band intensity associated with in vitro
kinase analysis of the kinase-deficient mutant PKD/K618M have been
seen. As we do see slight increases in the intensity of this band only
when PKC is coexpressed, we consider likely the possibility that a
small degree of transphosphorylation of the mutant by PKC
has
occurred in vitro.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; PKD, protein kinase D; PH, pleckstrin homology; PDBu, phorbol 12,13-dibutyrate; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|