From the Department of Pathology, Beth Israel
Deaconess Medical Center, Harvard Medical School,
Boston, Massachusetts 02215 and § Fraunhofer Institute for
Interfacial Engineering and Biotechnology, 70569 Stuttgart, Germany
Received for publication, December 27, 2002, and in revised form, March 7, 2003
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ABSTRACT |
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Protein kinase D (PKD) is a member of
the AGC family of Ser/Thr kinases and is distantly related to
protein kinase C (PKC). Formerly known as PKCµ, PKD contains protein
domains not found in conventional PKC isoforms. A functional pleckstrin
homology (PH) domain is critical for the regulation of PKD activity.
Here we report that PKD is tyrosine-phosphorylated within the PH
domain, leading to activation. This phosphorylation is mediated by a
pathway that consists of the Src and Abl tyrosine kinases and occurs in response to stimulation with pervanadate and oxidative stress. Mutational analysis revealed three tyrosine phosphorylation sites (Tyr432, Tyr463, and Tyr502),
which are regulated by the Src-Abl pathway, and phosphorylation of only
one of these (Tyr463) leads to PKD activation. By using a
phospho-specific antibody, we show that Abl directly phosphorylates PKD
at Tyr463 in vitro, and in cells
phosphorylation of this site is sufficient to mediate full activation
of PKD. Mutation of the other two sites, Tyr432 and
Tyr502, had no significant influence on PKD activity. These
data reveal a tyrosine phosphorylation-dependent activation
mechanism for PKD and suggest that this event contributes to the
release of the autoinhibitory PKD PH domain leading to kinase
activation and downstream responses.
Protein kinase D
(PKD)1/PKCµ is a
serine/threonine protein kinase with homology to conventional PKC
isoforms in the regulatory domain (1, 2). However, PKD has
additional protein modules not found in other PKCs, including an acidic
domain, a hydrophobic domain, and a pleckstrin homology (PH) domain,
whose functions are yet not fully defined (3-5). Thus, PKD together
with two other homologues, PKD2 (6) and PKD3/PKC PKD plays a major role in Golgi function and organization (15, 20-22).
In addition, nuclear factor- As with other members of the AGC kinase superfamily, PKD
activity and function are tightly regulated by phosphorylation. Two serine residues, Ser203 and Ser916 (in murine
PKD), have been mapped as autophosphorylation sites (11, 25).
Furthermore, two serine residues (Ser744 and
Ser748 in murine PKD), located within the activation loop,
are trans-phosphorylated in cells, and this is necessary for full
catalytic activity (26, 27). Recent studies (26, 28) have shown that
PKCs are upstream kinases for PKD and can directly phosphorylate the
activation loop serine residues. Unlike members of the PKC family, PKD
lacks an autoinhibitory pseudosubstrate sequence (5). However, deletion analysis of the PKD regulatory domain revealed that the amino-terminal pleckstrin homology (PH) domain has an autoinhibitory function, such
that PH domain deletion mutants are constitutively active in cells (3).
Interestingly, PKCs including PKC Here we report that tyrosine phosphorylation of PKD occurs within the
PH domain, leading to activation. This phosphorylation is mediated by
the Src/Abl signaling pathway in response to oxidative stress as well
as pervanadate treatment of cells. We show that three tyrosine
phosphorylation sites within the PKD PH domain are regulated by Src/Abl
signaling, and phosphorylation of one of these (Tyr463) is
responsible for PKD activation. These results provide evidence for a
tyrosine phosphorylationdependent activation mechanism for PKD.
Cell Lines, Antibodies, and Reagents--
The HeLa cell line was
from the American Type Culture Collection and maintained in high
glucose Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum. The anti-Abl, anti-Src (polyclonal), and
anti-PKD/PKCµ antibodies were from Santa Cruz Biotechnology (Santa
Cruz, CA), and anti-Src (monoclonal) and anti-phosphotyrosine (4G10)
were from Upstate Biotechnology, Inc. (Waltham, MA). Anti-HA was
purified in-house from the 12CA5 hybridoma. The anti-pY463 has been
described previously (14). The secondary POX-linked anti-mouse or
anti-rabbit antibodies were from Roche Applied Science. Pervanadate was
prepared by mixing 1 ml of a 20 mM sodium orthovanadate
with 330 µl of 30% H2O2 (Fisher) for 10 min
at room temperature, yielding a 15 mM solution of
pervanadate and H2O2. Residual
H2O2 was inactivated by a 15-min incubation with 10 µl of catalase (8 units/mg, Sigma). Pervanadate solutions were freshly prepared for each experiment. The PKD-specific substrate peptide used was AALVRQMSVAFFFK. Superfect (Qiagen, Valencia, CA) was
used for transient transfections. Purified recombinant catalytic
subunit DNA Constructs--
Full-length PKD expression plasmids
are based on an amino-terminal HA-PKD in pcDNA3, derived from human
PKD/PKCµ, using PCR with the following primer pairs:
5'-GCGGGATCCATGTATCCTTATGATGTTCTTGATTATGCTAGCGCCCCTCCGGTCCTG-3' and 5'-GCGCTCGAGTCAGAGGATGCTGACACGCTC-3'. Amino-terminal
HA-tagged Purification of Recombinant Proteins--
Recombinant
His6- and HA-tagged PKD and PKD.K612W were expressed in
baculovirus-infected Sf9 insect cells. Infection, harvesting, cell lysis, and purification of proteins by
Ni2+-nitrilotriacetate chromatography has been described
(32).
Immunoblotting and Immunoprecipitation--
Cells were lysed in
lysis buffer (50 mM Tris/HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, pH 7.4) plus protease
inhibitor mixture (Sigma). Lysates were used either for immunoblot
analysis or proteins of interest were immunoprecipitated by a 1-h
incubation with the respective antibody (2 µg) followed by a 30-min
incubation with protein G-Sepharose (Amersham Biosciences). Immune
complexes were washed three times with TBS (50 mM Tris/HCl,
pH 7.4, 150 mM NaCl) and resolved by SDS-PAGE or subjected
to in vitro kinase assays.
Preparation of PS/DOG--
Mixed lipid vesicles were
prepared by drying lipids (diacylglycerol in a PtdSer background;
DOG/PtdSer, 4:140 µM) stored in chloroform/methanol (1:1,
v/v) under a stream of nitrogen. Lipids were reconstituted by
sonication into PKD kinase buffer in an ice bath sonicator at 4 °C
for 10 min at 50% output. Fresh preparations were made for each experiment.
Phosphorylation of PKD by Abl--
Purified PKD (500 ng) and Abl
kinase (25 ng) and lipids (PS/DOG) were mixed in a total volume of 40 µl of kinase buffer (50 mM Tris/HCl, pH 7.4, 10 mM MgCl2, 2 mM dithiothreitol). The
kinase reaction was carried out for 20 min at 37 °C after addition
of 10 µl of ATP solution (75 µM ATP in kinase buffer).
The reaction was stopped with sample buffer, or PKD was
immunoprecipitated, and PKD kinase assays were performed. Samples were
applied to SDS-PAGE and transferred to nitrocellulose.
PKD Kinase Assays--
After immunoprecipitation (anti-PKD for
endogenous and anti-HA for transfected PKD) and washing, 20 µl of
kinase buffer (50 mM Tris/HCl, pH 7.4, 10 mM
MgCl2, 2 mM dithiothreitol) was added to the
precipitates. The kinase reaction was carried out for 20 min at room
temperature after addition of 10 µl of kinase substrate mixture (150 µM PKD-specific substrate peptide, 50 µM
ATP, 10 µCi of [ Pervanadate Treatment of HeLa Cells Activates
PKD--
H2O2 stimulation of cells has been
shown to stimulate PKD activity (13, 14). In order to investigate a
potential role for tyrosine phosphorylation in the activation of PKD,
HeLa cells were treated in a time- and dose-dependent
manner with the phosphotyrosine phosphatase inhibitor pervanadate.
Activation of PKD was monitored by phosphorylation of a PKD-specific
synthetic peptide substrate (33), as well as by autophosphorylation.
Substrate phosphorylation increased by 4-5-fold and
autophosphorylation by 4-fold following a 10-min treatment of cells
with 75 µM pervanadate (Fig.
1A). Pervanadate-induced
activation of PKD peaked by 30 min of stimulation and with a
quantitatively identical response to control PMA treatment of HeLa
cells (Fig. 1B). The production of pervanadate requires the
inactivation of H2O2 with catalase (see
"Experimental Procedures"). In control experiments using catalase,
we demonstrated that there is no residual H2O2
in the pervanadate preparations such that the observed PKD activation
is due to pervanadate treatment alone (Fig. 1C).
Overexpression of either wild-type PKD or a kinase-inactive PKD allele
containing a mutation in the ATP-binding site (K612W) revealed that
phosphorylation of PKD in response to pervanadate is due to PKD
autophosphorylation and not due to co-immunoprecipitating kinase (Fig.
1D).
Pervanadate-induced Tyrosine Phosphorylation of PKD Leads to
Activation--
Since PKD can be activated by pervanadate and
H2O2, agents that elevate intracellular
phosphotyrosine levels, we determined the tyrosine phosphorylation of
PKD in response to these stimuli. Pervanadate treatment increased
tyrosine phosphorylation of either endogenous (Fig.
2A) or overexpressed PKD (Fig.
2B). The control phorbol ester PMA did not induce PKD
tyrosine phosphorylation, despite the fact that it is a potent
activator of PKD. Moreover, there was a good correlation between the
kinetics of PKD activation and tyrosine phosphorylation in
pervanadate-treated HeLa cells (compare Figs. 1B to
2A). Interestingly, PKD kinase activity was not necessary
for pervanadate-induced tyrosine phosphorylation, suggesting that
tyrosine phosphorylation occurs prior to PKD autophosphorylation (Fig.
2B). To determine whether tyrosine phosphorylation directly contributes to the activation of PKD, we stimulated HeLa cells with
pervanadate and immunoprecipitated PKD. The precipitates were treated
with either purified recombinant catalytic subunit The Src/Abl Signaling Pathway Is Responsible for
Tyrosine Phosphorylation and Activation of PKD--
By using
overexpression of dominant negative alleles of Src and Abl, we recently
showed that these tyrosine kinases contribute to PKD activation in
cells (14). In order to determine whether pervanadate- and
H2O2-stimulated tyrosine phosphorylation of PKD are mediated by Src and/or Abl, HeLa cells were treated with the Src
kinase inhibitors PP1 and PP2 prior to stimulation. Both PP1 and PP2
blunted the activation of PKD in response to pervanadate by 50%,
concomitant with a reduction of tyrosine phosphorylation (Fig.
3A). The control inactive PP3
compound had no effect on PKD activity or tyrosine phosphorylation.
This underscores the requirement of Src in the activation and
phosphorylation of PKD. Moreover, co-expression of constitutively
active Src (Src.Y527F) or Abl (v-Abl p120) together with wild-type PKD
demonstrated that both kinases mediate tyrosine phosphorylation of PKD
in transfected cells (Fig. 3B).
Src/Abl Mediates Tyrosine Phosphorylation of PKD in the
PH Domain--
It is known that a functional PH domain is required
for PKD regulation in cells (3). To determine whether the
tyrosine phosphorylation of PKD occurs within this domain, we first
assessed PKD phosphorylation in pervanadate-treated cells expressing
either wild-type PKD or a PKD. Mapping of Src- and Abl-dependent Tyrosine
Phosphorylation Sites in the PKD PH Domain--
We next used a
mutational approach to determine which tyrosine residues within the PKD
PH-domain are targets of Src and Abl. We mutated six candidate tyrosine
residues (Tyr432, Tyr443, Tyr462,
Tyr463, Tyr501, and Tyr502) into
phenylalanine (Fig. 5A). Each
of these single mutants as well as a triple mutant (PKD*;
Y432F/Y463F/Y502F) were co-expressed with active Src (Src.Y527F) in
HeLa cells, and the level of PKD tyrosine phosphorylation was assessed.
The PKD Y432F, Y463F, and Y502F mutants showed an appreciable loss of
phosphotyrosine signal compared with wild-type PKD or the other three
mutants (Fig. 5B). Thus, Tyr432,
Tyr463, and Tyr502 are likely targets of
Src-dependent tyrosine phosphorylation. Interestingly, upon
co-expression of these PKD mutants with active Abl (v-Abl p120), only
the Y463F mutant showed any appreciable loss of phosphotyrosine signal
(Fig. 5C). These data indicate both overlap as well as
specificity in the Src- and Abl-mediated tyrosine phosphorylation of
PKD.
Src Associates with PKD in Cells--
Because both Src and Abl are
able to induce the tyrosine phosphorylation of PKD in cells, we tested
whether these tyrosine kinases exist in a complex with PKD.
Co-immunoprecipitation experiments were carried out with both
endogenous and transfected PKD. Endogenous Src was found in a complex
with PKD immunoprecipitates following pervanadate stimulation of HeLa
cells (Fig. 6A). Under these
co-immunoprecipitation conditions, we failed to detect
co-immunoprecipitation between endogenous Abl and endogenous PKD (Fig.
6A). Similar results were obtained when PKD and Src were
co-transfected in HeLa cells, where overexpressed Src was
co-immunoprecipitated with overexpressed PKD (Fig. 6B). A
constitutive association between PKD and Src was observed when the two
proteins were overexpressed, and this could not be increased further
with pervanadate treatment. Interestingly, mutation of Y463F had no
effect on this association. Again, overexpressed Abl could not be
detected on PKD immunoprecipitates (Fig. 6B). Moreover, the
kinase activity of Src was not required for this association because
the kinase-inactive Src* allele was still able to co-immunoprecipitate
with transfected PKD (Fig. 6C). Thus, in response to
pervanadate stimulation of cells, Src translocates to a complex that
contains PKD, which likely facilitates the phosphorylation event.
Conversely, the phosphorylation of PKD by Abl is likely mediated by a
transient association between the two kinases that cannot be recovered
under these co- immunoprecipitation conditions.
Abl Phosphorylates and Activates PKD through Tyr463
Phosphorylation--
Tyr463 in human PKD lies in a motif
highly conserved between mammalian PKD isoforms, as well as PKD
orthologues (Fig. 7A). We next
tested whether Abl can directly phosphorylate PKD in vitro using a phospho-specific antibody raised against a phosphopeptide based
on the Tyr463 motif. This antibody only detected PKD when
phosphorylated at Tyr463 in cells but not the Y463F mutant,
demonstrating that it is specific for phospho-Tyr463 (data
not shown and Ref. 14). Incubation of purified PKD with purified Abl
revealed that Tyr463 can be phosphorylated directly by Abl
in vitro (Fig. 7B). Furthermore, PKD
phosphorylated by Abl showed a 2-fold increase in protein kinase
activity compared with PKD incubated alone, suggesting that
Tyr463 phosphorylation partially contributes to PKD
activation (Fig. 7C). This activation was on the same order
of magnitude as the control activators PS/DOG. Finally, we evaluated
whether cellular stimuli, which are known to activate PKD in cells,
also induce PKD tyrosine phosphorylation. Stimulation of cells with
pervanadate or oxidative stress (H2O2) induced
tyrosine phosphorylation of PKD at Tyr463 (Fig.
7D). However, stimulation of HeLa cells with either
epidermal growth factor, platelet-derived growth factor (PDGF), or
insulin-like growth factor-1 did not induce any appreciable tyrosine
phosphorylation of PKD, indicating that Tyr463
phosphorylation is limited to oxidative stress signaling.
Phosphorylation of Tyr463 Mediates PKD Activation in
Cells--
We next evaluated the contribution of Tyr463
phosphorylation to PKD activation in pervanadate- and
Src/Abl-stimulated cells. First, pervanadate-stimulated PKD activation
was partially blunted in cells expressing a Y463F mutant, when compared
with wild-type PKD. Second, the triple mutant PKD*
(PKD.Y432F/Y463F/Y502F) showed a quantitatively similar activity
compared with Y463F (Fig. 8A). This suggests that phosphorylation of Tyr432 and
Tyr502 does not contribute to PKD activation. In addition,
overexpression of the Y432F or Y502F mutants did not significantly
alter PKD activity in response to pervanadate treatment of HeLa cells
(data not shown).
In order to test whether mutation of these three tyrosine residues to
glutamate would lead to constitutive PKD activity, by effectively
mimicking the negative charge induced by phosphorylation, we
constructed Y432E, Y463E, and Y502E mutants. As a control, glutamine
mutations were also created. Interestingly, only the PKD.Y463E mutant
showed constitutive PKD activity when expressed in cells (Fig.
8B), whereas the other two glutamate mutants were essentially unchanged from wild-type PKD. There was no significant alteration of PKD kinase activity in all of the glutamine mutants (the
slight increase in the Y463Q mutant was not reproducible over the
course of several experiments). Moreover, the constitutive kinase
activity of PKD.Y463E could not be further augmented by pervanadate
stimulation of cells (Fig. 8C). Finally, to reveal a
potential negative regulatory function of Tyr432 or
Tyr502 phosphorylation, we also compared the kinase
activity of two double mutants, PKD.Y463E/Y432F and PKD.Y463E/Y502F. In
response to pervanadate-stimulation of cells, however, neither of these two mutants showed any appreciable decrease in kinase activity compared
with PKD.Y463E (Fig. 8C). Again the implication is that these two tyrosine residues do not significantly contribute to PKD activation.
PKD is a member of a protein kinase family that is distinct from
PKC isoenzymes and that is subject to diverse regulatory mechanisms
that are required for its activation (8). In the present study, we
report an activation mechanism for PKD that is mediated by tyrosine
phosphorylation of residues within the PH domain. Treatment of HeLa
cells with the tyrosine phosphatase inhibitor pervanadate, or with
H2O2, as well as by overexpression of active
alleles of Src and Abl leads to tyrosine phosphorylation and activation
of PKD. By using a mutational approach, we mapped three candidate
tyrosines within the PH domain that serve as targets of Src and/or Abl.
These are Tyr432, Tyr463, and
Tyr502 (Fig. 5). Although mutation of these single sites to
non-phosphorylatable Phe residues reduced PKD tyrosine phosphorylation
(Fig. 5), only mutation of Tyr463 showed an appreciable
loss of protein kinase activity in transfected cells (Fig. 8). Because
mutation of this residue to a Glu induced constitutive protein kinase
activity, we deduced that phosphorylation of this site is both
necessary and sufficient to stimulate PKD activity in cells, both in
response to pervanadate and downstream of the Src/Abl pathway (Fig. 8).
Thus, phosphorylation of PKD at Tyr463 represents an
alternative mechanism for PKD activation and is particularly
interesting in light of the fact that this residue lies within the PH domain.
Unlike PKCs, PKD lacks a classical pseudosubstrate sequence necessary
for autoregulation (5). However, PKD is negatively regulated by an
autoinhibitory PH domain, and deletion of this domain results in a
protein with elevated constitutive protein kinase activity (24).
Similar increases in PKD activity were reported by mutation of critical
residues in this domain such as Trp538 (3). Recently, it
was also shown that serine phosphorylation at Ser738 and
Ser742 in the activation loop mediates the release of the
PH domain (28). Because phosphorylation of the activation loop can be mediated directly by PKC Our data reveal an alternative regulatory mechanism for PKD activation
that may also result in the release of PH domain autoinhibition. Phosphorylation of Tyr463, either by pervanadate
stimulation of cells or downstream of the Src/Abl pathway, leads to PKD
activation, and this can be achieved both in vitro using
purified proteins or in transfected cells. Thus, Tyr463
represents a critical feature of this activation process, because mutation to phenylalanine compromises PKD activity, and mutation to
glutamate results in a constitutively active kinase. It is tempting to
speculate that phosphorylation of Tyr463 leads to release
of the PH domain, thus exposing the activation loop which would now be
accessible to PKC for phosphorylation. This model is supported by the
fact that the Y463E mutant is constitutively phosphorylated at
Ser738/Ser742 when expressed in
cells.2 Future studies will
address the precise interplay between Tyr463 and activation
loop phosphorylation.
Both in vitro and in cells, Abl appears to be a direct
upstream kinase for Tyr463. Src, on the other hand, may
also have a function in phosphorylating two additional sites in the PKD
PH domain, Tyr432 and Tyr502. In this regard,
it is interesting to note that Src translocates to a complex containing
PKD in response to pervanadate stimulation of cells, whereas we have
failed to detect any appreciable association of Abl with PKD under the
same conditions (Fig. 6). This suggests that the phosphorylation of PKD
at Tyr463 by Abl occurs transiently, and any complex
between the two proteins cannot be recovered under detergent lysis
conditions. Mutation of Tyr432 and Tyr502 to
non-phosphorylatable residues results in a decrease in overall tyrosine
phosphorylation (Fig. 5). It is worth noting that we have not directly
demonstrated tyrosine phosphorylation of these sites. However, because
mutation of these residues to phenylalanine does not significantly
alter PKD activity, and also mutation to glutamate does not result in
increased kinase activity, it appears that phosphorylation of
Tyr432 or Tyr502 would not contribute to PKD
activity. Similarly, mutation of Tyr432 or
Tyr502 does not compromise Tyr463
phosphorylation. It is also worth noting that the loss of
phosphotyrosine signal observed in the phenylalanine mutations is not
due to a loss of Tyr463 phosphorylation (data not shown). A
role for phosphorylation of Tyr432 and Tyr502
may be to serve as docking sites for SH2 domain-containing proteins, adding another level of complexity to the regulation of PKD in cells.
Finally, it is important to note that although the Y463F PKD mutant is
significantly compromised in protein kinase activity in stimulated
cells, some residual activity remains (Fig. 8A). Coupled
with the finding that PKD tyrosine phosphorylation is also not
completely eliminated in either PKD. Phosphorylation of Tyr463 may also provide a docking site
for SH2-containing molecules. However, we speculate that this is
unlikely because the PKD.Y463E mutant is active per se in
immune complex kinase assays. Instead, we postulate that the negative
charge at this site induces conformational changes within the PH
domain, leading to release of autoinhibition, as discussed above. This hypothesis does not exclude other models of PKD activation, such as
release mediated by activation loop phosphorylation. Indeed, the
precise mechanism by which the PH domain is released may depend on the
upstream pathways that converge to regulate PKD. For example, in
response to mitogens as well as bradykinin stimulation of cells, we
have failed to detect Tyr463 phosphorylation (Fig.
7D) (14), despite the fact that these stimuli fully activate
PKD by inducing activation loop phosphorylation. Conversely, both
pervanadate and H2O2 stimulation of a variety of cell types leads to Tyr463 phosphorylation as well as
activation loop phosphorylation (14). Thus, it is likely that in
response to growth factor signaling, diacylglycerol binding and
activation loop phosphorylation primarily control PKD activation,
whereas in response to pervanadate and oxidative stress signaling,
Tyr463 and activation loop phosphorylation are the primary determinants.
Taken together, our results point to an important function of
Tyr463 in the PKD PH domain controlling its activity. By
using both loss of function and gain of function approaches, we show
that tyrosine phosphorylation at this residue results in increased PKD
activity. We cannot fully rule out that the other two tyrosine residues
Tyr432 and/or Tyr502 are also important for
some aspect of PKD activity or function. The fact that both Src/Abl and
PKCs may converge on PKD to tightly regulate its activation presents an
attractive model that we are currently testing. The outcome of these
and future studies will likely impact the diverse array of
physiological responses that have been attributed to PKD.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
(7), represent a
family of protein kinases whose regulation and function are distinct
from conventional PKC isoenzymes (8). PKD is activated in response
to numerous stimuli including platelet-derived growth factor (PDGF)
(9), triggering of the B-cell receptor (10) or T-cell receptor complex
(11, 12), oxidative stress (13, 14), and through G-protein-coupled
receptors (15-17). Genotoxic agents have also been shown to activate
PKD via a caspase 3-dependent mechanism (18, 19).
B (NF-
B) as well as the
serum-response element have been shown to be targets of
PKD-dependent signaling (14, 23). PKD-mediated activation
of serum-response element-driven genes was shown to occur via a Raf-1
kinase-dependent mitogen-activated protein kinase pathway
(23), and the activation of PKD by mitogens suggests a role for PKD in
cellular proliferation (24). Moreover, activation of NF-
B by PKD
indicates an essential role for this kinase in promoting cellular
survival in response to oxidative stress (14).
, have been shown to bind to PKD,
more specifically to the PH domain (29, 30). In addition, direct
phosphorylation of the PKD activation loop serines by PKC
has been
shown to release PH domain autoinhibition (28). However, the precise
mechanism by which the PH domain exerts negative regulation on PKD
activity, and how this is relieved, has not been elucidated.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-isoform of human protein phosphatase 1 (PP-1
) was from
Calbiochem, and human recombinant CD45 protein-tyrosine phosphatase was
from Biomol (Plymouth Meeting, PA). The active, purified Abl kinase
fragment was from Calbiochem. The Src family kinase inhibitor PP1 was
from Biomol, and PP2 and the control PP3 were from Calbiochem.
12-Phorbol 13-myristate acetate (PMA) was from Sigma. Bovine brain
PtdSer and sn-1,2-dioleoylglycerol were from Avanti Polar
Lipids (Alabaster, AL).
PH-PKD (HA-
PH-PKD) and HA-tagged PKD.K612W
(HA-PKD.K612W) were created by PCR using the above pairs and using
non-tagged
PH-PKD or PKD.K612W (both described previously (23, 31))
as templates and cloned into pcDNA3 via BamHI and
XhoI. Mutagenesis was carried out by PCR using QuickChange
(Stratagene, La Jolla, CA) with the following primer pairs:
PKD.Y432F, 5'-GGATGGATGGTCCACTTCACCAGCAAGGACACG-3' and
5'-CGTGTCCTTGCTGGTGAAGTGGACCATCCATCC-3'; PKD.Y432E,
5'-GGATGGATGGTCCACGAAACCAGCAAGGACACG-3' and
5'-CGTGTCCTTGCTGGTTTCGTGGACCATCCATCC-3'; PKD.Y432Q,
5'-GGATGGATGGTCCACCAAACCAGCAAGGACACG-3' and
5'-CGTGTCCTTGCTGGTTTGGTGGACCATCCATCC-3'; PKD.Y443F,
5'-CTGCGGAAACGGCACTTTTGGAGATTGGATAGC-3' and
5'-GCTATCCAATCTCCAAAAGTGCCGTTTCCGCAG-3'; PKD.Y462F,
5'-GACACAGGAAGCAGGTTCTACAAGGAAATTCTT-3' and
5'-AGGAATTTCCTTGTAGAACCTGCTTCCTGTGTC-3'; PKD.Y463F,
5'-GACACAGGAAGCAGGTACTTCAAGGAAATTCCTTTATCT-3' and
5'-AGATAAAGGAATTTCCTTGAAGTACCTGCTTCCTGTGTC-3'; PKD.Y463E, 5'-GACACAGGAAGCAGGTACGAAAAGGAAATTCTTTTATCT-3' and
5'-AGATAAAGGAATTTCCTTTTCGTACCTGCTTCCTGTGTC-3'; PKD.Y463Q,
5'-GACCACGGAAGCAGGTACCAAAAGGAAATTCTTTTATCT-3' and
5'-AGATAAAGGAATTTCCTTTTGGTACCTGCTTCCTGTGTC-3'; PKD.Y501F,
5'-ATCACTACGGCAAATGTAGTGTTTTATGTGGGAGAAAATGTGGTC-3' and
5'-GACCACATTTTCTCCCACATAAAACACTACATTTGCCGTAGTGAT-3'; PKD.Y502F, 5'-ACGGCAAATGTAGTGTATTTTGTGGGAGAAAATGTGGTC-3' and
5'-GACCACATTTTCTCCCACAAAATACACTACATTTGCCGT-3'; PKD.Y502E,
5'-GCAAATGTAGTGTATGAAGTGGGAGAAAATGTG-3' and
5'-CACATTTTCTCCCACTTCATACACTACATTTGC-3'; and PKD.Y502Q,
5'-GCAAATGTAGTGTATCAAGTGGGAGAAAATGTG-3' and
5'-CACATTTTCTTCCACTTGATACACTACATTTGC-3'. All constructs were
verified by DNA sequencing. v-Abl p120, Src wild-type, Src*, and
Src.Y527F constructs have been described (14).
-32P]ATP in kinase buffer). To
terminate, the samples were centrifuged, and the supernatants were
spotted onto P81 phosphocellulose paper (Whatman). The papers were
washed three times with 0.75% phosphoric acid and once with acetone
and dried, and activity was determined by liquid scintillation
counting. For autophosphorylation assays, the reaction was performed in
the absence of substrate peptide and was stopped by the addition of
sample buffer. Samples were resolved by SDS-PAGE and transferred to nitrocellulose.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
PKD activation by pervanadate. 2 Mio
HeLa cells were treated for 10 min with pervanadate in a
dose-dependent manner (A) or treated with 75 µM pervanadate in a time-dependent manner
(B). PKD was immunoprecipitated (IP) and
autophosphorylation or substrate phosphorylation kinase assays were
performed. C, 2 Mio HeLa cells were treated for 10 min with
H2O2 (10 µM), catalase (5 µl of
a 1 unit/ml stock), or pervanadate (75 µM) as indicated.
PKD was immunoprecipitated, and an autophosphorylation kinase assay was
performed. D, wild-type PKD or PKD.K612W was overexpressed,
and cells were treated with pervanadate (PV) (10 min, 75 µM). PKD was immunoprecipitated, and an
autophosphorylation kinase assay was performed. Immunoblots of
immunoprecipitated PKD revealed equivalent expression of
PKD in each experiment. Numbers represent relative PKD
autophosphorylation activity compared with respective controls. All
results are typical of three independent experiments.
-isoform of human
protein phosphatase 1 (PP-1
) or the phosphotyrosine phosphatase
(CD45). Dephosphorylation of PKD recovered from pervanadate (or
H2O2, data not shown)-treated cells resulted in
a significant decrease in kinase activity (Fig. 2C). PKD
activity could not be completely reduced by CD45 treatment, despite its
ability to reduce PKD tyrosine phosphorylation by ~80%. This
indicates that either tyrosine phosphorylation of PKD is involved in
the initial activation step, which is then followed by
autophosphorylation, or that the upstream tyrosine kinase is
co-immunoprecipitated with PKD, leading to a steady state between
phosphorylation and dephosphorylation in the in vitro
reaction.
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Fig. 2.
PKD tyrosine phosphorylation contributes to
its activation. A, 2 Mio HeLa cells were treated with
PMA (10 min, 100 nM) or with 75 µM
pervanadate for the indicate time. PKD was immunoprecipitated
(IP), and tyrosine phosphorylation (anti-pY; 4G10) was
analyzed. B, wild-type PKD or PKD.K612W were overexpressed,
and cells were treated with pervanadate (PV) (10 min, 75 µM). PKD was immunoprecipitated, and tyrosine
phosphorylation (anti-pY; 4G10) was analyzed. C, 2 Mio HeLa
cells were treated with 75 µM pervanadate as indicated.
PKD was immunoprecipitated, and precipitates were treated for 15 min
(room temperature) with phosphatases (PP-1 or CD45), and substrate
phosphorylation kinase assays were performed or tyrosine
phosphorylation (anti-pY; 4G10) was determined. The immunoblots were
stripped and re-probed against PKD protein in all experiments. All
results are typical of three independent experiments.
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Fig. 3.
Src/Abl mediates pervanadate-induced tyrosine
phosphorylation and activation of PKD. A, 2 Mio HeLa
cells were pre-treated for 1 h with respective Src kinase family
inhibitors (PP1 and PP2), control
(PP3), or left untreated and then stimulated for 10 min with
75 µM pervanadate (PV). PKD was
immunoprecipitated (IP) and tyrosine phosphorylation
(anti-pY; 4G10) as well as activity (auto- and substrate
phosphorylation) were determined. B and C,
wild-type PKD and active Src (Src.Y527F) or Abl (v-Abl.p120) were
overexpressed in 2 Mio HeLa cells, and PKD was immunoprecipitated and
analyzed for tyrosine phosphorylation by immunoblot analysis. The
nitrocellulose was the stripped and re-probed for PKD. Src and Abl
expression was controlled by immunoblotting with anti-Src and anti-Abl.
All results are typical of three independent experiments.
PH mutant. Deletion of the PH domain
resulted in a near complete loss of PKD tyrosine phosphorylation when
compared with the control wild-type protein (Fig.
4A). Similar results were obtained in cells expressing either active Abl (v-Abl p120) or active Src (Src.Y527F), whereby tyrosine phosphorylation of the
PKD.
PH mutant was significantly blunted compared with wild-type PKD
(Fig. 4B). Thus, the PH domain of PKD is the major target of
pervanadate/Src/Abl-dependent tyrosine phosphorylation.
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Fig. 4.
Tyrosine phosphorylation mediated by
pervanadate and Src/Abl occurs in the PKD PH domain. A,
HA-tagged wild-type PKD or PKD. PH was overexpressed, and cells
were treated with pervanadate (10 min, 75 µM).
PKD was immunoprecipitated (IP) and immunoblotted with
anti-pY (4G10). The nitrocellulose membrane was stripped and re-probed
with anti-PKD (PKD). B, HA-tagged wild-type
(WT) PKD or PKD.
PH were co-expressed with active
Src (Src.Y527F) or Abl (v-Abl.p120), and PKD was immunoprecipitated and
analyzed for tyrosine phosphorylation. The nitrocellulose was the
stripped and re-probed for PKD expression (anti-PKD). Src and Abl
expression was controlled by immunoblotting with anti-Src and anti-Abl.
All results are typical of three independent experiments.
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Fig. 5.
Mapping the Src- and Abl-mediated tyrosine
phosphorylation sites in the PKD PH domain. A,
schematic overview of PKD domains (HR, hydrophobic region;
C1a and C1b, C1 domains; PH, pleckstrin homology;
KD, kinase domain) and the pleckstrin homology domain
primary and predicted secondary structure ( ,
-helix;
,
-sheet; VL, variable region). Tyrosine
residues within the PH domain are highlighted. B
and C, wild-type PKD (PKD), PKD* (PKD.Y432F/Y463F/Y502F) or
other indicated mutants were overexpressed in combination with active
Src (Src.Y527F, B) or active Abl (v-Abl p120, C).
PKD was immunoprecipitated, and tyrosine phosphorylation (anti-pY;
4G10) was determined. The immunoblots were stripped and re-probed
against PKD protein (anti-PKD). Src or Abl expression was controlled by
immunoblotting with anti-Src or anti-Abl. All results are typical of
three independent experiments.
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Fig. 6.
Co-immunoprecipitation of Src and PKD.
A, HeLa cells were treated with pervanadate (PV)
(10 min, 75 µM). Endogenous PKD was immunoprecipitated
( -PKD), and co-immunoprecipitation of Abl or Src was
evaluated by immunoblotting with anti-Abl (
-Abl) or
anti-Src (
-Src). Blots were stripped and re-probed with
anti-PKD. B, wild-type (wt) PKD or PKD.Y463F were
co-expressed with either wild-type Abl or wild-type Src. Cells were
treated with pervanadate (10 min, 75 µM), and PKD was
immunoprecipitated, and co-immunoprecipitation of Abl or Src was
evaluated by immunoblotting with anti-Abl (
-Abl) or
anti-Src (
-Src) or as control anti-PKD
(
-PKD). C, wild-type Src or kinase-inactive
Src (Src*) were co-expressed with wild-type PKD, PKD
immunoprecipitated with anti-HA (
-HA), and immunoblotted
with anti-Src (
-Src), or a control, anti-PKD
(
-PKD). All results are typical of three independent
experiments.
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Fig. 7.
Abl directly phosphorylates PKD at residue
Tyr463 resulting in increased PKD activity.
A, alignment of the amino acid sequences surrounding
Tyr463 in human (Homo (H.)
sapiens) PKD/PKCµ and in other PKD isoforms from mouse
(Mus (M) musculus) and the nematode
worm (Caenorhabditis (C) elegans). The
potential phosphorylation site is highlighted, and identical
amino-acids are underlined. B, recombinant,
purified PKD was incubated either alone or together with purified Abl
in vitro with cold ATP for 20 min at 37 °C, resolved by
SDS-PAGE, and immunoblotted with anti-pY463. The nitrocellulose
membrane was stripped and re-probed with anti-PKD. C,
in vitro activation of PKD by Abl. Recombinant, purified PKD
was incubated either alone or together with purified Abl or PS/DOG
micelles and cold ATP in vitro for 20 min at 37 °C. PKD
was immunoprecipitated, and a substrate kinase assay was performed.
Precipitates were resolved by SDS-PAGE and immunoblotted for
precipitation of equal amounts of PKD. D, HeLa cells were
treated with pervanadate (10 min, 75 µM, positive
control), H2O2 (10 min, 10 µM),
epidermal growth factor (EGF) (5 min, 50 ng/ml), PDGF (5 min, 50 ng/ml), or insulin-like growth factor-1 (IGF-1) (5 min, 50 ng/ml). PKD was immunoprecipitated (IP), and
tyrosine phosphorylation ( -pY; 4G10) was analyzed by immunoblotting
(left panel). HeLa cells were transfected with PKD and
treated with H2O2 (10 min, 10 µM). PKD was immunoprecipitated, and tyrosine
phosphorylation of Tyr463 (anti-pY463) was analyzed on
immunoblots (right panel).
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Fig. 8.
PKD activity is dependent on
Tyr463 phosphorylation. A-C, wild-type PKD
(PKD) or the indicated PKD mutants were expressed, and cells
were treated with pervanadate (PV) (10 min, 75 µM) as indicated. PKD was immunoprecipitated
(IP) (anti-HA) and a substrate kinase assay performed. PKD
expression and tyrosine phosphorylation (A,
-pY) was determined by immunoblotting. All results are typical
of three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, as demonstrated by in vitro
experiments (34), this suggests a hierarchical PKC-PKD pathway that
leads to PKD activation and release of the PH domain. In addition,
because PKCs are also able to bind to the PKD PH domain, this may also contribute to a conformational change leading to release of the PH
domain, thus exposing the activation loop (29, 30). Thus far,
structural evidence for such a model has not been presented.
PH (Fig. 4B) or PKD.Y463F (Fig. 8A), it is reasonable to speculate that
pervanadate stimulation of cells may lead to the phosphorylation of
additional tyrosine residues outside of the PH domain, and which may
contribute to PKD kinase activity. Moreover, the constitutive
protein kinase activity of the PKD.
PH mutant is only modestly
increased in cells exposed to either oxidative stress, pervanadate, or
upon co-transfection with active Src or Abl (data not shown). Again
this modest increase may be due to additional phosphorylations outside
of the PH domain or by increased activation loop phosphorylation
mediated by unknown mechanism(s). Future studies will address these possibilities.
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ACKNOWLEDGEMENTS |
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We thank B. Schaffhausen and A. Hausser for generously providing expression plasmids. We also thank members of the Toker laboratory for insightful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA 75134 (to A. T.) and by Deutsche Forschungsgemeinschaft Grant STO 439/1-1 (to P. S.).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.
We dedicate this paper to the memory of Franz-Josef Johannes, a friend, colleague, teacher, and mentor, whose contributions to the PKCµ/PKD field will be greatly missed.
¶ To whom correspondence should be addressed: Dept. of Pathology, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-8535; Fax: 617-667-3616; E-mail: atoker@caregroup.harvard.edu.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M213224200
2 P. Storz and A. Toker, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PKD, protein kinase D; BCR, B-cell receptor complex; DOG, dioleoylglycerol; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PKC, protein kinase C; PMA, 12-phorbol 13-myristate acetate; PtdSer, phosphatidylserine; TCR, T-cell receptor complex; HA, hemagglutinin; PS, phosphatidylserine; SH2, Src homology 2.
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