From the Unit of Signal Transduction and Gastrointestinal Cancer, Division of Digestive Diseases, Department of Medicine, The David Geffen School of Medicine at UCLA, UCLA-CURE Digestive Diseases Research Center and Molecular Biology Institute, UCLA, Los Angeles, California 90095-1786
Received for publication, August 7, 2002, and in revised form, October 21, 2002
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ABSTRACT |
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Persistent activation of protein kinase D
(PKD) via protein kinase C (PKC)-mediated signal transduction is
accompanied by phosphorylation at Ser744 and
Ser748 located in the catalytic domain activation loop, but
whether PKC isoforms directly phosphorylate these residues, induce PKD autophosphorylation, or recruit intermediate upstream kinase(s) is
unclear. Here, we explore the mechanism whereby PKC activates PKD in
response to cellular stimuli. We first assessed in vitro PKC-PKD transphosphorylation and PKD activation. A PKD738-753 activation loop peptide was well phosphorylated by immunoprecipitated PKC isoforms, consistent with similarities between the loop and their
known substrate specificities. A similar peptide with glutamic acid
replacing Ser748 was preferentially phosphorylated by
PKC Protein kinase C (PKC)1
is an enzyme family of central importance in signal transduction (1,
2). The ten distinct PKC isoforms thus far identified by molecular
cloning comprise three subfamilies differing in structure and
regulation (1, 3, 4). Classical ( PKCs are implicated as mediators of a diverse array of biological
functions, including both short term alterations in cellular activities
and the long term determination of cell fate (9). PKC isoforms exhibit
distinct expression patterns, and in response to signaling events,
become dynamically targeted to discrete subcellular locations and
anchored in positions adjacent to substrates (10, 11). This
differential dynamic localization, as well as intrinsic substrate
selectivity (12), may confer separate signaling roles to the individual
PKC isoforms. However, despite their recognized importance in signal
transduction, few links have been established between individual PKC
isozymes and the direct targets that specify their individual
biological outcomes.
Protein kinase D (PKD; the murine homologue of human PKCµ) (13, 14)
and two recently identified serine protein kinases termed PKC Physiological activation of intact PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory (20). In response to cellular stimuli, PKD is converted from
a low activity form into a persistently active form that is retained
during isolation from cells. Engagement of specific G protein-coupled
receptors either by multiple peptides (21-24) or lysophosphatidic acid
(23, 25), activation of receptor tyrosine kinases such as the
platelet-derived growth factor receptor (21), signaling via
heterotrimeric and monomeric G proteins (26, 27), and oxidative stress
(28) were demonstrated to induce PKD activation in a wide variety of
cell types including Swiss 3T3 fibroblasts, small cell lung cancer and
pancreatic cancer, normal epithelial and smooth muscle cell lines,
cardiocytes, and lymphocytes (23, 25, 29-34).
Throughout all these studies, multiple lines of evidence have indicated
that PKC activity is indispensable for PKD activation by cellular
stimuli. Cell treatments with phorbol esters or other agents that
bypass surface receptors and directly stimulate PKCs are potent
triggers of cellular PKD activation (20, 35). Cotransfection of PKD,
together with active mutant forms of PKC Our previous studies identified Ser744 and
Ser748 in the PKD activation loop as phosphorylation sites
critical for PKC-mediated PKD activation. Whereas a PKD mutant with
Ser744 and Ser748 mutated to alanines
(PKD-S744A/S748A) could not be activated by cellular stimuli, a mutant
with these residues mutated to glutamic acid residues (PKD-S744E/S748E)
possessed dramatically increased constitutive activity (50). We have
shown that Ser744 and Ser748 are phosphorylated
in vivo during PKD activation in response to phorbol ester
stimulation, in a manner blocked by preincubation with GF 109203X (50,
51). Specifically, in two-dimensional 32P tryptic
phosphopeptide maps, individual spots were selectively eliminated when
PKD forms point-mutated at Ser744 or Ser748
were used in transfections (50). Because kinase-deficient PKD, which
retains Ser744 and Ser748, also became
phosphorylated during stimulation with phorbol ester, we concluded that
transphosphorylation at these sites by an upstream kinase,
e.g. novel PKCs, was a critical factor responsible for in vivo PKD activation (50, 51).
A different group of researchers (52) have questioned the role of
activation loop transphosphorylation in the PKD activation mechanism.
Consequently, we reinvestigated PKD activation loop phosphorylation
using phosphospecific antisera generated against phosphorylated
Ser744 and Ser748 (34). In these studies, we
demonstrated that Ser744 and Ser748
phosphorylation were concomitant with activation, induced by either a
variety of receptor-mediated or -independent stimuli or by transfection
of PKC Cell Culture and Transfections--
Stock cultures of COS-7
cells were maintained in 10-cm tissue culture plates by subculturing
every 3-4 days in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum in a humidified atmosphere containing 10%
CO2 and 90% air at 37 °C. Confluent stock cells were
reseeded at a density of 6 × 104 cells/ml in 6-cm
dishes, 8-18 h prior to transfections. All transfections and
cotransfections were carried out with equivalent amounts of DNA (5 µg/dish for single transfections, 3 µg/dish of each DNA for
cotransfections), using vector pcDNA3 as the control DNA added to
single transfections. Transfections were carried out in Opti-MEM (Invitrogen) using Lipofectin (Invitrogen) at 10 µl/dish.
DNA-Lipofectin complexes were formed according to the protocol provided
by the manufacturer and then cell cultures were layered over with these DNA complexes in a final volume of 2.5 ml/dish in the absence of serum
and incubated at 37 °C in a humidified atmosphere containing 5%
CO2, for 5-6 h or overnight to allow uptake of complexes.
Fetal bovine serum (10% final concentration) in Opti-MEM was then
added to dishes to yield a final volume of 5 ml/dish. Cells were used for experiments after a further 72 h of incubation.
Plasmid Constructs and Fusion Proteins--
Plasmid constructs
encoding PKCs and wild-type and mutant PKD forms used in this study
have been described previously (17, 18, 20, 36, 50). The construct
pcDNA3-PKD Immunoprecipitations--
COS-7 cells transfected either with
wild-type or mutant PKD or PKC isoforms were treated as indicated in
the figures and then lysed in 1 ml of lysis buffer (1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 2 mM
dithiothreitol, 1 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride
in 50 mM Tris-HCl, pH 7.4). Small amounts (typically
1/10) of these total lysates were saved and combined with equal
volumes of 2× SDS-PAGE sample buffer (0.2 M Tris-HCl, pH
6.8, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10%
glycerol) for Western blot analysis. To generate PKC or PKD
immunoprecipitates, medium was removed, and lysis buffer was added to
cells on ice. These lysates were cleared by centrifugation at 15,000 rpm for 10 min, and the cleared supernatants were combined with
antibodies and protein A-agarose (30 µl) and placed on a rotator at
4 °C for 3 h. Immune complexes were collected by brief spinning
and washed thoroughly before subsequent kinase assays. Antibodies used
for immunoprecipitations were either the isoform-specific antisera from
Santa Cruz Biotechnology (for PKC Peptide Phosphorylation Assays--
For assays of peptide
phosphorylation by PKD or PKCs, immune complexes were washed twice with
lysis buffer and then twice with kinase buffer consisting of 30 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 2 mM dithiothreitol. Substrate peptides (
PS/PDB phospholipid vesicles used for assays of peptide or
protein phosphorylation by PKD or PKC PKC-PKD Transphosphorylation/PKD Activation
Assays--
For transphosphorylation of full-length wild-type or
kinase-defective PKD proteins by PKC
For measurements of PKC
Autophosphorylation or syntide-2 phosphorylation reactions to assess
activity of the PKD- Western Blot Analysis--
For Western blot analysis, samples or
cell lysates were directly solubilized by boiling in SDS-PAGE sample
buffer. After resolving by SDS-PAGE, proteins were transferred to
Immobilon-P membranes (Millipore) as described previously (21). To
block nonspecific protein binding, membranes were blocked by incubation
with either 5% non-fat dried milk in PBS, pH 7.2 (for PKD C-20 or
the antibody pS748 that recognizes PKD) (34), or 5% bovine
serum albumin/0.1% Tween 20 in PBS (for the antibody pS744 that
recognizes PKD) (34) for 2-3 h at 20-25 °C or overnight at
4 °C. Membranes were then incubated at room temperature for 2-3 h
with antisera, specifically recognizing PKD at a dilution of 1 µg/ml,
the phosphospecific phosphoSer748 recognizing PKD
phosphorylated at Ser748 (1:500) in PBS containing 3%
non-fat dried milk, or the phosphospecific phosphoSer744
raised against a peptide containing phosphoSer744 and
phosphoSer748, chiefly recognizing PKD phosphorylated at
Ser744, at 1:1000 dilution in 5% bovine serum
albumin/0.1% Tween 20 in PBS. Immunoreactive bands were visualized
using horseradish peroxidase-conjugated anti-rabbit IgG and enhanced
chemiluminescence (ECL reagents; Amersham Biosciences) detection.
Materials--
[ Comparison of the PKD Activation Loop Segment with the Known
Substrate Recognition Sequences of PKC Isoforms--
The initial
molecular cloning and expression of PKD and its human homologue PKCµ
was followed by the molecular cloning and expression of another related
protein, termed PKD2, and two other clones thus far identified only at
the cDNA level (human PKC
The amino acid residues in the vicinity of the serine or threonine
residues targeted by different protein kinases comprise characteristic substrate recognition sequences that contribute to
enzyme specificity. The substrate recognition sequences of various PKC
isoforms and PKD/PKCµ were analyzed by Cantley and co-workers (12)
using directed peptide libraries. As shown in Fig. 1B, the
substrate recognition sequences of PKC Phosphorylation of PKD Activation Loop Peptides by Different PKC
Isoforms--
To investigate whether PKC
We first examined immunoprecipitated active PKD in assays of syntide-2,
Next, specific PKC immunoprecipitates (wild-type PKC
As shown in Fig. 2B, PKC
We then used the variant activation loop peptides described in Fig.
1A as substrates to examine whether PKC PKC
Shown in Fig. 3A, the time
courses of PKD autophosphorylation with or without added PKC
In the presence of PS/PDB vesicles that allosterically stimulate PKD
enzyme activity without persistent activation, PKD autophosphorylation increased approximately linearly over a 40-min time course (Fig. 3B). Interestingly, even when PKD activity was directly
stimulated, inclusion of PKC PKC
Experiments using pS748 to monitor Ser748 phosphorylation
over a 2.5-min time course, shown in Fig. 4, revealed the
time-dependent appearance of a phosphorylated band
corresponding to PKD. Thus, these data indicated that PKD could
autophosphorylate at the activation loop after 1 min of incubation in
the presence of activators. We also examined PKD phosphorylation in
these reactions by Western analysis using the commercial antiserum that
recognizes chiefly phosphoSer744 (and to a lesser extent
phosphoSer748 (34)). Results shown in Fig. 4 illustrate
that pS744 immunoreactivity in PKD increased very little over this time
course. Because this antibody binds to a limited extent to
phosphorylated Ser748, this low degree of immunoreactivity
most likely reflects little or no autophosphorylation at
Ser744 but rather corresponds to the increase in
Ser748 phosphorylation observed using pS748.
Significantly, Ser748 autophosphorylation by isolated PKD
occurred slowly, becoming detectable only after a lag of ~1-1.5 min of the reaction. In contrast, PKD incubated with PKC PKC
To substantiate further the role of PKC catalytic activity in the
activation of PKD, we also used an inhibitor, Gö6983, of classic
and novel PKCs including PKC
In control experiments, we activated PKD within cells by stimulation
with PDB and then immunoprecipitated, eluted, and reimmunoprecipitated the enzyme before assaying. Results (Fig. 5B) demonstrated
that PKD activated within cells retained its increased activity
throughout all these procedures. Eluted PKD, incubated with ATP and
PS/PDB and then reimmunoprecipitated and assayed for syntide-2
activity, had a low level of activity that was very similar to the
control enzyme isolated from unstimulated cells (Fig. 5B).
This clearly indicates that allosteric activators do not induce a
persistent increase in PKD activity and that PKD must be phosphorylated
at both serines in the activation loop to stabilize the active form of
the enzyme. In contrast, eluted PKD incubated with PKC
To confirm that an increase in PKD activity, as opposed to that of a
coimmunoprecipitated kinase, was responsible for the increased
catalytic activity of the reimmunoprecipitated PKD, we incubated eluted
kinase-deficient PKD mutant PKD-K618N protein with PKC
We also carried out experiments similar to those in Fig. 5B
but using the insect cell-expressed and purified PKD and PKC Relief of Autoinhibition and Activation Loop Phosphorylation Both
Contribute to PKD Activation--
Autoinhibition is a central feature
of the regulation of protein kinase catalytic activity (64). Previous
results from this laboratory indicated that PKD mutants lacking the
pleckstrin homology domain or the cysteine-rich domain were highly
active in the absence of stimulation (18, 19). These results suggest
that in the intact kinase, the entire N-terminal regulatory domain,
i.e. both the cysteine-rich and PH domains, help to maintain
the enzyme in an inactive, autoinhibited state, and consequently,
removal of either domain facilitates activation. Consistent with this interpretation, a maltose-binding protein-PKD catalytic domain fusion
protein produced in bacteria was demonstrated previously to be
catalytically active (13). Mutation of Ser744 and
Ser748 in the activation loop to glutamic acid residues
introduces negative charges that mimic phosphorylation, which generates
a full-length kinase that is highly active in the absence of
stimulation. Reciprocally, a PKD mutant in which Ser744 and
Ser748 in the activation loop are replaced with
unphosphorylatable alanine residues (PKD-S744A/S748A) is not activated
within cells by stimuli that fully activate wild-type PKD (50). Thus,
we hypothesize that the mechanism of PKD activation is based on a
functional link between activation loop phosphorylation and relief of autoinhibition.
Further experiments were designed to assess the possible
interdependence(s) between activation loop phosphorylation and relief of autoinhibition. We first examined the effect of activation loop
phosphorylation on the catalytic activity of the isolated PKD catalytic
domain using a wild-type catalytic domain fusion protein, GST-Cat, and
a catalytic domain fusion protein with Ser744 and
Ser748 mutated to unphosphorylatable alanines,
PKD-GST-Cat/S744A/S748A. As shown in the inset in Fig.
6A, control GST had no
catalytic activity. In contrast, and consistent with previous results
using a different PKD catalytic domain fusion protein (MBP-CAT) (13), bacterially expressed GST-Cat vigorously phosphorylated syntide-2. Interestingly, an equivalent amount of GST-Cat/S744A/S748A
phosphorylated syntide-2 to only a slightly lesser extent (~80% of
that achieved by GST-Cat). Thus, even complete prevention of
phosphorylation did not drastically affect the activity of the isolated
catalytic domain, suggesting that activation loop phosphorylation is
not needed for PKD activation in the truncated enzyme.
To assess the effect of preventing activation loop phosphorylation
under circumstances in which PH domain-mediated regulation is removed,
we generated a novel mutant derived from a PH domain deletion mutant
(PKD
Interestingly, like PKD
We next analyzed the activation loop phosphorylation in the cell
lysates from cells transfected with the enzymes assayed in Fig. 6,
A and B, using the phosphospecific antibodies
described above. As shown in Fig. 6C, PKD was
unphosphorylated in unstimulated cells, and stimulation with PDB
induced a dramatic increase in phosphorylation at Ser748
and Ser744, measured using pS748 and pS744, respectively.
PKD-S744A/S748A could not be detected in either unstimulated or
stimulated cells, as the phosphorylation sites responsible for antibody
detection are altered by mutation.
Western analysis of PKD Previous studies from this laboratory demonstrated that
pharmacological inhibition of PKC prior to cell stimulation prevents PKD activation, and cotransfection of PKC Recent additional studies elucidated dynamic movements of PKD in
response to G protein-coupled receptor stimulation (56-59) by
immunocytochemistry and real-time imaging. These studies demonstrated that PKD initially translocates to the plasma membrane, where the
enzyme becomes activated, and then subsequently reverse translocates away from the plasma membrane, to gain access to targets elsewhere in
the cell (56-58), including the nucleus (59). Interestingly, PKC-dependent phosphorylation of activation loop
Ser744 and Ser748 was required for this dynamic
behavior of activated PKD (58, 59). Specifically, preincubation with
selective PKC inhibitors prior to cell stimulation with neuropeptides
including bombesin prevented reverse translocation of PKD. Thus, the
actions of PKC induce multiple facets of PKD function including its
persistent activation and the process by which the activated enzyme is
dispatched within the cell. But as yet, information as to
whether Ser744/Ser748 are directly
phosphorylated by PKC or by an intermediate kinase(s) has been elusive
(20, 36, 51-53).
Here, we investigated the involvement of direct activation loop
phosphorylation in PKC-mediated PKD activation, using in
vitro phosphorylation assays with immunoprecipitated or purified
recombinant PKC isoforms as upstream kinases and PKD activation loop
peptides and intact PKD as substrates. Novel PKC Our results also indicate that both PKC We also examined the mechanism whereby activation loop phosphorylation
triggers PKD activation. Specifically, deletion of the PH domain
renders the enzyme insensitive to activation loop phosphorylation.
Furthermore, mutation of Ser744/Ser748 to
alanines did not abrogate the catalytic activity of PKD forms lacking
either the PH domain or the entire regulatory domain. Collectively,
these results suggest that the major part of PKC-mediated activation
derives from relief of autoinhibition mediated by the regulatory domain
of PKD and that activation loop phosphorylation is a mechanistic
trigger for this process. Our findings complement and extend our
previous studies, establishing PKC, suggesting that PKD containing phosphate at Ser748
is rapidly targeted by this isoform at Ser744. When
incubated in the presence of phosphatidylserine, phorbol 12,13-dibutyrate and ATP, intact PKD slowly autophosphorylated in the
activation loop but only at Ser748. In contrast, addition
of purified PKC
to the incubation mixture induced rapid
Ser744 and Ser748 phosphorylation, concomitant
with persistent 2-3-fold increases in PKD activity, measured using
reimmunoprecipitated PKD to phosphorylate an exogenous peptide,
syntide-2. We also further examined pleckstrin homology domain-mediated
PKD regulation to determine its relationship with activation loop
phosphorylation. The high constitutive activity of the pleckstrin
homology (PH) domain deletion mutant PKD-
PH was not abrogated
by mutation of Ser744 and Ser748 to alanines,
suggesting that one function of activation loop phosphorylation in the
PKD activation mechanism is to relieve autoinhibition by the PH domain.
These studies provide evidence of a direct PKC
-PKD phosphorylation
cascade and provide additional insight into the activation mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUDING REMARKS
REFERENCES
,
I,
II, and
) and novel
(
,
,
, and
) PKCs are allosterically stimulated by
diacylglycerol, produced rapidly in response to increased
activity of phospholipase C/D enzymes (5, 6), and are cellular targets
of the tumor-promoting phorbol esters (7). Classical PKCs also possess
functional Ca2+-binding sites that mediate sensitivity to
Ca2+ signals (1). In contrast, atypical (
/
and
)
PKCs respond to 3'-phosphorylated inositol phospholipids (8) but do not bind diacylglycerol/phorbol esters or Ca2+.
(15)
and PKD2 (16) constitute a new protein kinase subfamily separate from
the previously identified PKCs. Salient features of PKD structure and
function include an N-terminal regulatory domain comprising a putative
transmembrane region, a diacylglycerol/phorbol ester binding
cysteine-rich domain, and a pleckstrin homology (PH) domain and a
C-terminal catalytic domain with a primary sequence and substrate
specificity divergent from those of PKCs. Like the classical and novel
PKCs, PKD catalytic activity is stimulated in vitro by
diacylglycerol or biologically active phorbol esters (17). The
cysteine-rich domain and PH domain both play a role in the negative
regulation of PKD catalytic activity, because deletion of either of
these domains produces a constitutively active enzyme (18, 19).
or PKC
, also dramatically activates PKD in the absence of cell stimulation (20, 36).
Preincubation of cells with the specific PKC inhibitors GF
109203X (37) or Ro 31-8220 (38) that do not directly inhibit PKD
(20) impairs PKD activation by all stimuli, reducing it by 70-80%
when used at maximally effective concentrations (28). Furthermore, PKD
interacts preferentially with PKC
, forming complexes involving the
PH domain (36). These findings imply the existence of PKC-PKD protein
kinase cascade(s), and we have postulated that some
PKC-dependent biological effects involve PKD acting either in parallel or as a downstream intermediate. In particular, PKD has
been reported recently to mediate several important cellular activities
and processes, including function and organization of the Golgi
apparatus (39), metastatic tumor cell invasion (40), epidermal growth
factor receptor signaling (41, 42), Na+/H+
antiporter (43), mitogen-activated protein kinase activation (44),
proliferation (45), and adenomatous transformation (46). Moreover,
critical downstream targets of PKD signaling are beginning to emerge
(47-49). Therefore, the mechanism whereby PKC activates PKD has been
attracting intense interest.
or PKC
in the absence of cell stimulation. These
approaches also produced phosphorylation of kinase-deficient PKD forms
at both Ser744 and Ser748, supporting the view
that PKC, but not PKD, activity induced phosphorylation of these
residues. However, the precise mechanisms involved remain unclear (20,
36, 51-53). In the present study, we use peptides, full-length PKD,
and mutant PKD proteins to examine whether PKC mediates direct PKD
activation loop Ser744 and Ser748
phosphorylation and activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUDING REMARKS
REFERENCES
PH/S744A/S748A was generated by standard
subcloning procedures. Thus, a Bsu36I restriction fragment
within pcDNA-PKD
PH (nucleotides 2305-2827 of PKD) was replaced
with a corresponding Bsu36I fragment excised from
pcDNA3-PKD-S744A/S748A. The GST-Cat and GST-Cat/S744A/S748A fusion
proteins were constructed in pGEX-4T by standard subcloning procedures.
Thus, C-terminal BamHI fragments (corresponding to amino
acids 559-918) of either wild-type PKD or PKD-S744A/S748A were fused
in-frame at the C-terminal of the sequence encoding glutathione
S-transferase in the construct pGEX-4T (Amersham
Biosciences). Fusion proteins were isolated by affinity
chromatography using glutathione-Sepharose 4B (Roche Molecular
Biochemicals) and eluted with 10 mM glutathione.
, PKC
, or PKC
) or the
previously described PA-1 antiserum (17) for PKD (1:100 dilution).
-peptide,
syntide-2, PKD738-754, PKD-738-754/S744E, PKD-738-754/S748E, or the
PKD C-terminal peptide EEREMKALSERVSIL used to generate PA-1 antiserum,
termed PKD CT, each at a final concentration of 2.0 mg/ml), were then 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 µM H3PO4 and
dried, and radioactivity incorporated into peptides was determined by
detection of Cerenkov radiation in a scintillation counter.
were prepared by dehydrating 300 µg of PS under ethanol (200 µl) in a Speedvac dehydrator and then sonicating the dried lipid into kinase buffer (typically 300 µl)
in the presence of added PDB and 0.05% nonionic detergent (Triton
X-100). For assays of syntide-2 phosphorylation by purified PKD or
PKC
, fresh aliquots (typically 2 µg) of enzyme were thawed on ice,
combined with PS/PDB vesicles (final concentrations, 200 µg/ml and
200 nM, respectively, in kinase buffer), and aliquoted to
tubes on ice containing kinase buffer with or without inhibitors as
indicated in the figures. Reactions were initiated by adding a mixture
containing syntide-2 (final concentration, 2 mg/ml), together with 100 µM ATP (including [
-32P]ATP at 2 µCi/assay), and then transferring to a water bath at 30 °C for 7 min. Reactions were terminated by adding H3PO4
as above, spotted to P-81 paper, and washed, and radioactivity was counted in a scintillation counter as above.
, we transfected COS-7 cells
with PKD or PKD-K618N. After 72 h, cells were lysed, and PKD
protein was isolated in immune complexes using PA-1. In each
experiment, parallel dishes were included to examine the persistence of
PKD activation throughout immunoprecipitation, elution from
immunocomplexes, and subsequent reimmunoprecipitation. PKD or PKD-K618N
immunoprecipitates from unstimulated cells were washed twice with lysis
buffer and then twice with kinase buffer. The PKD-PA-1 immune complexes
were then incubated with immunizing peptide (15 µg in ~15 µl of
kinase buffer) overnight on ice to elute PKD proteins. For
transphosphorylation reactions, the supernatant containing eluted PKD
protein was transferred to fresh tubes on ice, adjusted to 20 µl, ATP
(100 µM) was added on ice, and then reactions were
initiated by mixing in purified PKC
(Calbiochem) for a final
concentration of 5.8 µg/ml, combined with PS/PDB vesicles (either
with or without 3 µM Gö6983), and transferred to a
37 °C water bath. Reactions were terminated at 2.5 min by adding 1 ml of ice-cold lysis buffer and were transferred to ice. PKD antibody
(C-20; Santa Cruz Biotechnology) was then added, and PKD was
reimmunoprecipitated for 3 h, washed as before, and then subjected
to syntide-2 assays in the presence of 3 µM Gö6983.
Activation assays using purified PKC
to transphosphorylate purified
PKD (Calbiochem) were conducted similarly beginning with the addition
of ATP to purified PKD in kinase buffer (20 µl).
transphosphorylation/PKD autophosphorylation
by 32P incorporation, purified components (PKD (200 ng),
alone or with PKC
(70 ng) or PKC
(70 ng) in the absence of PS/PDB
vesicles, or PKD (250 ng), alone or with PKC
(150 ng), in the
presence of PS/PDB vesicles and kinase buffer) were combined in
microcentrifuge tubes (final volume, 20 µl) on ice. To initiate
reactions, ATP at 100 µM with or without 1 µCi of
[
32P]ATP/assay was added and incubated at 30 °C
for times indicated in the figures. Reactions were terminated by
addition of 2× SDS-PAGE sample buffer and resolved by SDS-PAGE on 8%
gels. Protein phosphorylation was assessed by autoradiography of dried
gels. For detection of PKC
transphosphorylation/PKD
autophosphorylation at the PKD activation loop by Western blot analysis
using phosphospecific antibodies, components (PKD, 275 ng/lane, with or
without PKC
, 175 ng/lane, were combined in the presence of PS/PDB
vesicles and incubated at 37 °C. At times indicated in the figures,
aliquots were taken and combined with SDS-PAGE gel loading buffer for
subsequent Western analysis.
PH-S744A/S748A mutant were conducted as
described previously for PKD activity measurements (28). Briefly, COS-7
cells were transfected with the different plasmids. After 72 h,
cells were left untreated or treated with PDB (200 nM for
10 min) and then lysed, and PKD or PKD mutant protein was immunoprecipitated from the lysates. Immunoprecipitates were washed twice with lysis buffer and twice with kinase buffer and subjected to
autophosphorylation reactions or syntide-2 phosphorylation reactions as
described (28). Activity of the catalytic domain GST fusion proteins
was measured by immobilization on glutathione-Sepharose beads and
subsequent incubation (30 °C for 10 min) with syntide-2 (2 mg/ml),
together with ATP (100 µM with 1 µCi/assay of
[
-32P]ATP). Reactions were terminated by addition of
H3PO4, spotted to P-81 paper, washed, and
counted as above.
-32P]ATP (6000 Ci/mmol) was
from Amersham Biosciences. Protein A-agarose and
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (Pefabloc) were
from Roche Molecular Biochemicals. The anti-PKD antiserum (C-20)
used in Western blot analysis was from Santa Cruz Biotechnologies, Palo
Alto, CA. pS744 was from Cell Signalling Technologies, Beverly, MA.
Purified PKD (purity
90% by SDS-PAGE), PKC
(purity
95%), and PKC
(purity
95%), produced in cells from
Spodoptera frugiperda, were from Calbiochem. Opti-MEM and Lipofectin were from Invitrogen. The PKC substrate peptide, peptide
, purified PDK-1, and its control substrate peptide, PDKtide, were
obtained from Alexis Biochemicals. Syntide-2 and the PKD activation
loop peptides were synthesized at the peptide synthesis core facility
of CURE-DDRC at UCLA. The C-terminal PKD peptide, PKD CT and pS748,
were synthesized by the central peptide synthesis and antibody
production facility at Imperial Cancer Research Fund, London, United
Kingdom. All other reagents were from standard suppliers or as
described in the text and were the highest grade commercially available.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUDING REMARKS
REFERENCES
/PKD3 and a Caenorhabditis
elegans clone related to PKD2). A canonical region in the
catalytic portion of protein kinases bridges two characteristic amino
acid motifs in subdomains VII (DFG) and VIII (APE). This region
typically forms an elongated loop positioned adjacent to the active
site, referred to as an activation loop/segment because of
targeting of this region for regulatory control of enzyme activity by
upstream protein kinases acting in a cascade fashion (54, 55).
Interestingly, the activation loop segment is 100% conserved among all
members of the novel PKD protein kinase subfamily (Fig.
1A). We recently demonstrated
that Ser744 and Ser748 are not phosphorylated
significantly in resting cells and that phosphorylation of these
residues is rapidly induced by multiple cellular stimuli in a
PKC-dependent manner and is concomitant with persistent
activation (34). Additional studies from this laboratory have shown
that phosphorylation at these sites is also closely linked with dynamic
intracellular redistributions of PKD (56-59). Thus, PKD is regulated
by PKC activity at multiple levels, and direct phosphorylation of the
activation loop may represent a convergence point for these events.
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Fig. 1.
Comparison of PKD family activation loop
sequences, PKC isoform substrate specificities, and peptides used as
substrates to assess PKC-mediated PKD activation loop
phosphorylation. A, activation loop sequences spanning
subdomains VII and VIII in the catalytic region of known PKD family
members. Canonical DFG and APE motifs are in bold, and the
serine residues corresponding to Ser744 and
Ser748 in PKD are in bold and
highlighted by gray boxes. B, the
sequence specificity of PKC , PKC
, and PKC
, emphasizing the
region from
5 to +5 surrounding the targeted serine, and based on
data of Nishikawa et al. (12). Residues are arranged
vertically in decreasing order of preference; slash marks
between residues indicate equivalent values. The residues identified as
matches with the Ser744 site in PKD are circled,
and the residues identified as matches with the Ser748 site
in PKD are in boxes. C, peptides synthesized for this study
based on the sequence of the amino acids 738-754 of PKD.
PKD738-754, the peptide corresponding to the wild-type
sequence in PKD; PKD738-754/S748E, the peptide
corresponding to the sequence with Ser748 replaced by
glutamic acid; PKD738-754/S744E, the peptide corresponding
to the sequence with Ser744 replaced by glutamic acid.
Amino acids corresponding to Ser744 and Ser748
are in bold.
and PKC
possess
similarities to the amino acid sequence surrounding Ser744
and Ser748 in the PKD activation loop, suggesting that
these PKCs might directly phosphorylate these sites in vivo.
In particular, the sequences clustered on both sides of
Ser744, as well as Ser748, in PKD correspond to
favored PKC
recognition
sequences.2 Similarly, the
amino acids in positions adjacent to and C-terminal to
Ser744, or those N-terminal to Ser748, are
strikingly similar to those forming PKC
recognition sequences (Fig.
1B). However, the resemblance between the PKD activation segment and the substrate recognition sequence of atypical PKC
is
relatively limited (Fig. 1B), consistent with our previous findings that this isoform is unable to activate PKD in cotransfection experiments (20, 36, 60).
, PKC
, or PKC
could
recognize the PKD activation segment in vitro, we
synthesized a peptide, RIIGEKSFRRSVVGTPA, comprising amino acids
738-754 of PKD (PKD738-754) (Fig. 1C) as a substrate for
phosphorylation assays. The importance of dual activation loop
phosphorylation in the PKD activation mechanism has been underscored by
previous studies. Specifically, we demonstrated previously that
mutation of both Ser744 and Ser748 to glutamic
acid residues to mimic dual phosphorylation in PKD produced a highly
constitutively active enzyme (50). In contrast, single point mutation
of Ser744 or Ser748 to glutamic acid residues
introduce only minor alterations in PKD
activity.3 Thus,
second site phosphorylation of PKD singly phosphorylated at one of the
activation loop sites is necessary for full activation. Therefore, to
complement assays of PKD738-754 phosphorylation, we also synthesized
peptides with individual glutamic acid changes at the positions
corresponding to either Ser744 or Ser748
(PKD738-754/S744E and PKD738-754/S748E; shown in Fig. 1C)
as substrates that mimic singly phosphorylated PKD activation loop segments in phosphorylation assays.
-peptide, or PKD738-754 phosphorylation, shown in Fig.
2A. Consistent with our
previous studies, activated PKD phosphorylated syntide-2 strongly but
-peptide to only a minimal extent (less than 5% of control),
indicating that this peptide is a poor substrate for PKD (Fig.
2A). Interestingly, PKD738-754 was also poorly
phosphorylated by PKD in these assays (Fig. 2A), suggesting
a lack of intrinsic recognition of the activation loop peptide by
PKD.
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Fig. 2.
Peptide phosphorylation assays of
immunoprecipitated PKD and PKCs. A, PKD was
immunoprecipitated from transiently transfected, PDB-stimulated COS-7
cells, and peptide phosphorylation assays were carried out as described
under "Experimental Procedures" using syntide-2, peptide , and
PKD738-754 as indicated. A representative Western blot analysis to
detect immunoprecipitated PKD using anti-PKD antibody is shown in the
inset. B, PKC
, PKC
, or PKC
, as
indicated, were immunoprecipitated from COS-7 cells transiently
transfected with PKC expression constructs, and peptide phosphorylation
assays were carried out as described under "Experimental
Procedures" using peptide
, syntide-2, PKD738-754, or PKD CT, as
indicated. Results are expressed as the percentage of the
phosphorylation of peptide
and represent the mean ± S.E. of
at least five determinations. Representative Western blot analysis to
detect immunoprecipitated PKC isoforms using specific anti-PKC
,
PKC
, or PKC
antibodies are shown in the inset. C, PKC
isoforms were immunoprecipitated as in B, and peptide
phosphorylation assays were carried out as described under
"Experimental Procedures," using PKD738-754, PKD738-754/S744E
(744E), or PKD738-754/S748E (748E), as
indicated. Results are expressed as the percentage of the
phosphorylation of PKD738-754 and represent the mean ± S.E.
PKC
phosphorylated 744E to a significantly greater extent than did
PKC
, as assessed by Student's t test (p = 0.04, n = 9, shown with an asterisk).
PKC
phosphorylated S748E to a significantly greater extent than
S744E (p = 0.02, n
9, shown with a
number sign). Phosphorylation of S748E by PKC
was
significantly less than by PKC
(p = 0.04, n
7, shown with a double asterisk).
or PKC
from
transiently transfected COS-7 cells) or PKC
(a mixture of endogenous
wild-type and transfected active mutant enzyme expressed in COS-7
cells) were assayed for phosphorylation of peptide
, syntide-2, or
PKD738-754 (Fig. 2B). Peptide
was identified previously as an excellent model substrate for all PKCs but not PKD (36, 61) and
was therefore used as a normalization control to assess the relative
degree of phosphorylation of PKD738-754 by PKC
, PKC
, or PKC
.
In comparison with control, and consistent with previous studies (62),
syntide-2 was phosphorylated very well by the different PKC isoforms
(Fig. 2B). The extent of syntide-2 phosphorylation ranged
from ~55% (for PKC
and PKC
) to 75% (for PKC
) of peptide
phosphorylation (Fig. 2B). Consistent with intrinsic
recognition of the PKD activation loop by PKC
and PKC
, these
isoforms phosphorylated PKD738-754 to an extent between ~40%
(PKC
) and 50% (PKC
) of those reached in peptide
assays (Fig.
2B). In contrast, a peptide similar to PKD738-754 but with both Ser744 and Ser748 replaced with glutamic
acid residues was not phosphorylated significantly in parallel assays
(data not shown), indicating that the single threonine residue
(corresponding to PKD Thr752) in these peptides was not
phosphorylated by these PKCs.
also phosphorylated PKD738-754
to an extent similar to those reached by PKC
and PKC
(~45% of
the extent of peptide
phosphorylation) suggesting that at least in vitro, this isoform also intrinsically recognizes the PKD
activation loop as a possible substrate. However, previous studies from
this laboratory indicated that PKC
neither interacts with nor
activates PKD significantly (20, 36, 60). Taken together, these results suggest that factors other than intrinsic substrate recognition (e.g. a failure to colocalize with PKD) contribute to the
inability of PKC
to activate PKD in vivo. Purified PDK-1,
an upstream regulator that phosphorylates the activation loop of
different PKC isoforms, as well as protein kinase B, did not
phosphorylate PKD738-754 significantly (data not shown). Also shown in
Fig. 2B, none of these PKCs significantly phosphorylated the
C-terminal PKD peptide that contains a PKD autophosphorylation site
(described under "Experimental Procedures"). These data were
therefore included as a negative control for specific recognition by PKCs.
, PKC
, or
PKC
act as second-site PKD activation loop kinases. Because
these PKCs all phosphorylated PKD738-754 to similar extents, we used this peptide as a normalization control to facilitate comparison of
relative phosphorylation levels. Shown in Fig. 2C, both
PKD738-754/S744E and PKD738-754/S748E were well phosphorylated by
PKC
, indicating that this isoform can act as a second-site kinase to
phosphorylate PKD equally well after an initial phosphorylation at
either Ser744 or Ser748. Interestingly, and in
contrast with PKC
, PKC
phosphorylated the two peptides
differentially, demonstrating a strong preference for
PKD738-754/S748E. These data indicated that this enzyme acts preferentially as a second-site kinase to phosphorylate
Ser744. Interestingly, PKC
phosphorylated these peptides
only modestly, suggesting that this isoform is relatively deficient as
a second-site kinase for the PKD activation loop.
Increases PKD Autophosphorylation--
To further examine
whether PKC-mediated PKD activation was direct, we developed in
vitro activation assays using purified components. We have shown
previously that certain PKCs, notably PKC
, form stable molecular
complexes with the PKD PH domain when cotransfected, together with PKD
in COS-7 cells (36). Therefore, for these studies we selected PKC
,
which potently activates PKD but forms complexes with PKD relatively
poorly. To establish the potential of PKC
to activate intact
purified PKD in vitro by phosphorylation at the activation
loop sites (i.e. corresponding to Ser744 and
Ser748 in PKD), we carried out PKD autophosphorylation
assays under conditions that either supported only autophosphorylation
(i.e. purified PKD, together with
[
-32P]ATP) or a combination of transphosphorylation
and autophosphorylation (i.e. including purified PKC
),
measuring the total extent of PKD phosphorylation over time.
were
dramatically different when assays were carried out in the absence of
PS/PDB vesicles. This was possible, because the preparation of PKC
used here possesses constitutive activity without allosteric effectors
(not shown). Thus, purified PKD incubated alone autophosphorylated to
only a very limited extent, reaching a maximum within the first 2.5 min
of the assay and not increasing significantly thereafter. Similarly,
inclusion of PKC
, a negative control for PKC-mediated PKD
activation, promoted PKD autophosphorylation only very weakly over a
40-min time course, despite having a potent activity demonstrated by
autophosphorylation (Fig. 3A), which was not enhanced in the presence of PS/PDB (not shown). In contrast, inclusion of PKC
promoted a continued strong increase in PKD phosphorylation (Fig. 3A). Collected data from multiple experiments, normalized to
the initial PKD phosphorylation levels and shown in the graph in Fig. 3A, emphasized the selective enhancement of PKD
autophosphorylation by PKC
. From these results, we conclude that
catalytic activation of PKD triggered by PKC
transphosphorylation
permits a dramatic overall increase in PKD autophosphorylation,
consistent with our proposal that PKC
activates PKD by activation
loop phosphorylation.
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Fig. 3.
PKD autophosphorylation in the absence or
presence of PKC . A, PKD alone, PKD + PKC
, or PKD + PKC
were incubated in autophosphorylation assays
with [
-32P]ATP and analyzed by SDS-PAGE and
autoradiography as described under "Experimental Procedures."
Representative autoradiograms are depicted above, and the
corresponding band intensities were quantitated by scanning
densitometry and expressed below as the mean ± S.E.
from three experiments (filled circles, PKD; open
circles, PKD plus PKC
; filled triangles, PKD plus
PKC
). B, PKD or PKD + PKC
were incubated in
autophosphorylation assays in the presence of PS/PDB and
[
-32P]ATP and analyzed by SDS-PAGE and autoradiography
as described under "Experimental Procedures." A representative
autoradiogram is depicted above, and the corresponding band
intensities were quantitated below by scanning densitometry
(filled squares, PKD; open squares, PKD plus
PKC
). For each experiment, similar results were obtained at least
three times.
in the assay increased the initial rate
of PKD phosphorylation (Fig. 3B). These results are
consistent with a modest initial acceleration of PKD
autophosphorylation by PKC
-mediated activation and then continued
autophosphorylation until all the possible sites approached saturation.
Rapidly and Directly Phosphorylates PKD in Both Activation
Loop Sites, Whereas PKD Autophosphorylates at Ser748
Only--
We next verified PKC
-mediated phosphorylation of the
activation loop sites by Western analysis using the specific antibodies we characterized previously, which recognize the phosphorylated state
of Ser744 and Ser748 in PKD (Fig.
4). In these experiments, we incubated
PKD with PS/PDB and cold ATP, either alone or together with PKC
, and
examined activation loop phosphorylation at 30-s intervals by Western
analysis using the phosphospecific antibodies, pS748 and pS744.
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Fig. 4.
PKD activation loop phosphorylation in the
absence or presence of PKC . Purified PKD, alone or
together with purified PKC
(PKD + PKC
) were incubated
with ATP in the presence of PS/PDB for the times indicated and then
analyzed by SDS-PAGE and Western blot analysis using the
phosphospecific antisera pS744 or pS748 as described under
"Experimental Procedures." To demonstrate that equal amounts of PKD
protein were present, blots were stripped and reprobed with the
anti-PKD antibody C-20 recognizing total protein. Western blots
(upper panels) and corresponding band intensities depicted
in the graph (below), as determined by scanning and
densitometry, are representative of three experiments with similar
results.
exhibited a
rapid increase in pS748 immunoreactivity, detected within 30 s of
the reaction (Fig. 4). Furthermore, when PKC
was included in
reactions with PKD, pS744 immunoreactivity increased rapidly and
produced a robust signal within 1 min, indicating that
transphosphorylation of PKD Ser744 had occurred. These
Western blots were also stripped and reprobed with C-20 to demonstrate
that equal amounts of PKD protein were present in each sample (Fig. 4).
Taken together, data in Fig. 4 showed that PKC
rapidly catalyzed
phosphorylation of both Ser744 and Ser748 in
PKD, whereas autophosphorylation was slower and only occurred at
Ser748. From these data, we conclude that
transphosphorylation is the primary mechanism involved in rapid PKD
activation occurring in cells in response to stimuli but that PKD
autophosphorylation over a longer time could contribute to the overall
increase in enzyme activity.
-mediated Activation Loop Phosphorylation Triggers Persistent
PKD Activation in Vitro--
Results in Fig. 4 verified that PKC
had phosphorylated the PKD activation loop sites in our in
vitro assays using purified components. We next examined whether
PKC
-mediated PKD activation loop phosphorylation was associated with
stable increases in PKD activity by a two-stage assay. In our previous
studies, we have assayed PKD in soluble form by eluting the enzyme from
immunoprecipitates (50). Here, we transfected PKD or the
kinase-deficient PKD mutant PKD-K618N into COS-7 cells,
immunoprecipitated PKD from these cells, and then eluted the enzyme
from immunoprecipitates as described under "Experimental
Procedures." We then incubated the soluble, eluted PKD either alone
or with purified PKC
and unlabeled ATP in the first stage of the
assay. After 2.5 min at 37 °C, the reactions were stopped and
diluted with ice-cold buffer, and PKD was reimmunoprecipitated, and its
catalytic activity was measured by syntide-2 phosphorylation in the
second stage of the assay (Fig.
5B).
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Fig. 5.
PKD activation in vitro by
PKC -mediated PKD activation loop
phosphorylation. A, syntide-2 phosphorylation assays
were carried out with purified PKD (triangles) or PKC
(circles) in the presence of PS/PDB,
[
-32P]ATP, and the indicated concentrations of
Gö6983 as described under "Experimental Procedures." Results
shown are expressed as the percentage of control values in the absence
of inhibitor and represent the mean ± S.E. of six separate
determinations each. B, COS-7 cells were transiently
transfected with constructs encoding either PKD or the kinase-deficient
mutant, PKD-K618N. After 72 h, cells were either left untreated
(open bar) or treated with PDB (striped bar) to
activate the enzyme in vivo, as indicated, and then lysed,
and PKD was immunoprecipitated and eluted as described under
"Experimental Procedures." The eluted enzyme was then either kept
on ice or incubated with ATP and PS/PDB at 37 °C, either with
(filled bars) or without PKC
(dotted bar) and
in the presence (gray bar) or absence (black bar)
of 3 µM Gö6983, as indicated, in PKC-PKD
transphosphorylation/PKD activation reactions and then
reimmunoprecipitated and assayed for syntide-2 phosphorylation activity
as described under "Experimental Procedures." In the presence of
Gö6983, PKD activation by incubation with PKC
was
significantly inhibited, as assessed by Student's t test
(p = 0.02, n = 4, indicated by an
asterisk). A control Western blot is depicted to illustrate
that PKD was reimmunoprecipitated in equal amounts prior to syntide-2
assays (inset). C, purified PKD, either alone
(dotted bar) or together with purified PKC
(black
bar) were incubated in the presence of ATP and in the presence or
absence of 3 µM Gö6983, as indicated, for 2.5 min
at 37 °C and then diluted with cold buffer, and PKD was
immunoprecipitated and assayed for syntide-2 phosphorylation activity
as described under "Experimental Procedures." Results presented are
the mean ± S.E. from at least three experiments, each performed
in quadruplicate.
but not PKD (63). To determine the
concentrations of Gö6983 required to selectively and completely eliminate PKC
activity in peptide phosphorylation assays under our
conditions, we performed syntide-2 phosphorylation assays of PKC and
PKD with increasing concentrations of the inhibitor. Results shown in
Fig. 5A indicated that Gö6983 inhibited PKC
in
peptide phosphorylation assays in a concentration-dependent fashion, with half-maximal inhibition at ~300 nM and
complete inhibition at concentrations above 1 µM. In
contrast, PKD activity was not significantly inhibited by
concentrations of Gö6983 up to 5 µM (Fig.
5A). Thus, in the assays of reimmunoprecipitated PKD, 3 µM Gö6983 was included in the reaction mixture to
eliminate any PKC
activity that might have carried over.
, in the
presence of ATP and PS/PDB, then reimmunoprecipitated and assayed, had
dramatically increased activity, i.e. more than 4-fold that
of the control enzyme (Fig. 5B). Consistent with PKC
activity in the initial incubation having been responsible for the
observed increases in PKD activity, incubating PKD with PKC
in the
presence of 3 µM Gö6983 reduced PKD activation by
~40% (Fig. 5B). Control Western blots, shown in Fig.
5B, demonstrated that equal amounts of PKD protein were
isolated by the reimmunoprecipitation procedures.
in parallel
assays and then processed the protein as before for syntide-2 assays.
Shown in Fig. 5B, these immunoprecipitates contained no
significant activity.
in the
initial transphosphorylations. Results, shown in Fig. 5C, demonstrate that incubation of PKD with PKC
increased the activity of the subsequently immunoprecipitated PKD by more than 2.5-fold in
comparison with the same amount of PKD incubated by itself and then
immunoprecipitated and assayed. Again, in all these syntide-2 assays, 3 µM Gö6983 was included to eliminate any possible
traces of PKC
activity carried over into the PKD immunoprecipitates. Similar to results in Fig. 5B, this increase could be
attenuated significantly by including 3 µM Gö6983
in the incubation mixture to inhibit PKC
activity. Taken together,
the data in Fig. 5 demonstrate that PKC
-mediated direct
phosphorylation of PKD promotes its conversion from an inactive to an
active state.
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Fig. 6.
Constitutive activity and activation loop
phosphorylation status of PKD PH domain deletion mutants. COS-7
cells were transiently transfected with constructs encoding PKD,
PKD-S744A/S748A, PKD PH, or PKD
PH-S744A/S748A or with vector
pcDNA3 as control. After 72 h, cells were either left
unstimulated (open bars) or stimulated with PDB
(filled bars) as indicated and then lysed. PKD proteins were
then either immunoprecipitated from lysates and assayed for
autophosphorylation or syntide-2 phosphorylation activity or analyzed
directly by SDS-PAGE and Western analysis, as described under
"Experimental Procedures." A, PKD activity was analyzed
by syntide-2 assays as described under "Experimental Procedures."
Syntide-2 phosphorylation activity of GST-PKD catalytic domain fusion
proteins (WT, GST-Cat; S744A S748A,
GST-Cat-S744A/S748A) is presented in the inset. Data
represent the means ± S.E. from three separate experiments.
B, PKD proteins (S744A/S748A, PKD-S744A/S748A;
PH, PKD
PH;
PH-S744A/S748A,
PKD
PH-S744A/S748A) were subjected to in vitro kinase
(IVK) autophosphorylation assays. A representative
autoradiogram (above), together with compiled data,
quantitated by scanning densitometry (below), are shown. Means ± S.E. from three experiments are depicted in the graph. C,
cell lysates from COS-7 cells transfected with PKD proteins and treated
as described in A were subjected to Western analysis using
the phosphospecific antibodies pS748 or pS744 to detect activation loop
phosphorylation or the PKD antibody C-20 to detect total PKD protein,
respectively, as described under "Experimental Procedures." Similar
results were obtained in three separate experiments.
PH) we described previously (18). Whereas PKD
PH retains a
wild-type activation loop sequence, the newly generated mutant has
Ser744 and Ser748 mutated to alanine residues
(PKD
PH/S744A/S748A). We first compared the activity of this mutant,
by both syntide-2 phosphorylation (Fig. 6A) and
autophosphorylation assays (Fig. 6B), to that of wild-type
PKD, PKD-S744A/S748A, and PKD-
PH, from stimulated or unstimulated
cells. PKD from unstimulated cells had very low activity, and PDB
stimulation of cells dramatically activated the enzyme by both types of
assay. PKD-S744A/S748A was not active either constitutively or after
cell stimulation with PDB. In contrast, PKD
PH from unstimulated
cells was dramatically active in comparison with the wild-type PKD, and
PDB stimulation of cells did not further stimulate the activity of this
mutant (Fig. 6, A and B). These results with PKD,
PKD-S744A/S748A, and PKD
PH were consistent with those we described
previously (18, 50).
PH, PKD
PH-S744A/S748A from unstimulated
cells was also very active, indicating that activation loop mutation to
alanines did not abrogate the function of the enzyme. Thus, this mutant
displayed constitutive syntide-2 phosphorylation activity that was
~5-fold higher than wild-type PKD and about 60% of PKD
PH (Fig.
6A). Results of analysis by autophosphorylation assays was
similar (Fig. 6B). These results are consistent with the
notion that removal of the PH domain releases PKD from autoinhibition. Although in these experiments, expression levels of
PKD
PH-S744A/S748A were, on average, slightly less than (0.75-fold)
those of PKD-
PH (Fig. 6C), it was not clear whether this
difference could fully account for the measured differences in activity
between these two enzymes. Thus, it remains possible that activation
loop phosphorylation could influence the overall activity in the
absence of the PH domain.
PH using pS748 and pS744 indicated that
whereas the basal phosphorylation of these residues in unstimulated cells was slightly increased in comparison with wild-type enzyme, significant increases (in the case of pS748) or even very dramatic increases (in the case of pS744) in immunoreactivity were produced in
the protein upon PDB stimulation of cells. These results were interesting taken in the light of those in Fig. 6, A and
B, as they clearly indicate that the high constitutive
activity of PKD
PH can be dissociated from quantitative
phosphorylation at the activation loop, as this event was dramatically
induced during cell stimulation but was not associated with a
corresponding increase in enzyme activity. Taken together, these
results strongly suggest that the function of activation loop
phosphorylation is to reverse an autoinhibitory effect of the PH domain.
CONCLUDING REMARKS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUDING REMARKS
REFERENCES
or PKC
, together with wild-type PKD, activates PKD in cells via dual phosphorylation of
Ser744/Ser748. As these sites also become
phosphorylated in kinase-deficient forms of PKD, we have proposed that
PKD activation depends on PKC-mediated transphosphorylation rather than
on PKD autophosphorylation (34, 50, 51).
and PKC
directly
phosphorylated a peptide derived from the PKD activation loop in
vitro to levels approaching those of model peptides including
peptide
and syntide-2. To assess phosphorylation of the individual
serine residues, we generated peptides in which either site was
selectively replaced with a negatively charged residue and examined
second-site phosphorylation of these variant peptides. Results of these
assays suggest that PKC
could act as a second-site kinase to
phosphorylate either Ser744 and Ser748 whereas
PKC
could preferentially act as a second-site kinase for PKD
Ser744.
and PKC
transphosphorylate Ser744 and Ser748 in intact
proteins. We therefore conclude that PKC
and PKC
mediate PKD
activation by direct phosphorylation of Ser744 and/or
Ser748 in pathways of importance in vivo.
Although PKD did not significantly phosphorylate an activation loop
peptide, Western analysis using the phosphospecific antibodies revealed
that the full-length enzyme autophosphorylated, albeit slowly, at
Ser748. Given the known substrate specificity of PKC
,
and our results suggesting that PKC
acts as a second-site kinase for
PKD Ser744, it appears possible that initial PKD
autophosphorylation at Ser748 can, in some circumstances,
positively influence or prime Ser744 for
transphosphorylation by PKC
.
as a direct upstream kinase for
PKD in a PKC-PKD signaling cascade in which PKD mediates some of the
critical signaling events controlled by the PKC family.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jim Sinnett-Smith, Osvaldo Rey, and Cliff Hurd for helpful discussions and Peter Parker, Cancer Research UK, London, for kindly providing PKC expression constructs.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK 55003, DK 56930, and P50 CA90388.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.
Recipient of National Institutes of Health Mentored Research
Scientist Career Development Award K01 DK 02834.
§ Ronald S. Hirshberg Professor of Cancer Research. To whom correspondence should be addressed: 900 Veteran Ave., Warren Hall Rm. 11-124, Dept. of Medicine, School of Medicine, UCLA, Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399; E-mail: erozengurt@mednet.ucla.edu.
Published, JBC Papers in Press, October 28, 2002, DOI 10.1074/jbc.M208075200
2
PKC favors negatively charged residue
at position +4 to the targeted serine, which might be mimicked by
negatively charged phosphoserine.
3 T. Iglesias, R. T. Waldron, and E. Rozengurt, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C, PKD, protein kinase D; PDB, phorbol 12,13-dibutyrate; PS, phosphatidylserine; GST, glutathione S-transferase; PH, pleckstrin homology; PBS, phosphate-buffered saline.
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REFERENCES |
---|
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