From the Growth Regulation Laboratory, Imperial Cancer Research Fund, P.O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein kinase D (PKD) is a serine/threonine protein kinase that contains a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC) and a catalytic domain with only a low degree of sequence similarity to PKCs. PKD also contains a pleckstrin homology (PH) domain inserted between the cysteine-rich motifs and the catalytic domain that is not present in any of the PKCs. To investigate the function of the PH domain in the regulation of PKD activity, we determined the kinase activity of several PKD PH domain mutants immunoprecipitated from lysates of transiently transfected COS-7 cells. Deletion of the entire PH domain (amino acids 429-557) markedly increased the basal activity of the enzyme as assessed by autophosphorylation (~16-fold) and exogenous syntide-2 peptide substrate phosphorylation assays (~12-fold). Mutant PKD proteins with partial deletions or single amino acid substitutions within the PH domain (e.g. R447C and W538A) also exhibited increased basal kinase activity. These constitutive active mutants of PKD were only slightly further stimulated by phorbol-12,13-dibutyrate treatment of intact cells. Our results demonstrate, for the first time, that the PKD PH domain plays a negative role in the regulation of enzyme activity.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein kinase C (PKC)1
has been implicated in the signal transduction of a wide range of
biological responses including changes in cell morphology,
differentiation, and proliferation (1-5). Molecular cloning has
demonstrated the presence of multiple related PKC isoforms (3, 6, 7),
i.e. classic PKCs (,
1,
2, and
), novel PKCs
(
,
,
, and
), and atypical PKCs (
and
) all of which
possess a highly conserved catalytic domain.
The newly identified PKD is a mouse serine/threonine protein kinase with distinct structural and enzymological properties (8). The catalytic domain of PKD is distantly related to Ca2+-regulated kinases and shows little similarity to the highly conserved regions of the kinase subdomains of the PKC family (9). Consistent with this, PKD does not phosphorylate a variety of substrates utilized by PKCs indicating that PKD is a protein kinase with distinct substrate specificity (8, 10). However, the amino-terminal region of PKD contains a tandem repeat of cysteine-rich, zinc finger-like motifs that bind phorbol esters with high affinity (8, 10). Immunopurified PKD is markedly stimulated by PDB or diacylglycerol in the presence of phosphatidylserine (10). The human protein kinase PKCµ (11, 12) with 92% homology to PKD (extending to 98% homology in the catalytic domain) is also stimulated by phorbol esters and phospholipids (12). These in vitro results indicate that PKD/PKCµ is a novel phorbol ester/diacylglycerol-stimulated protein kinase. Recently a new mechanism of PKD activation has been identified (13). Specifically, exposure of intact cells to biologically active phorbol esters and membrane-permeant diacylglycerol induces phosphorylation-dependent PKD activation via a PKC-dependent pathway (13). Thus, PKD can function either in parallel to or downstream of PKCs in signal transduction.
The amino-terminal region of PKD also contains a PH domain which is not
found in any of the PKCs. PH domains are molecular structures of
approximately 120 amino acids with limited identity in sequence but
similar three-dimensional structure (14-19). These domains have been
identified in a large number of signaling and cytoskeletal proteins
(for review see Refs. 18-21). It has been suggested that PH domains
mediate intermolecular and/or intramolecular interactions like src
homology domain 2 and 3, but their function and binding partners remain
unclear. In some cases PH domains have been shown to bind
phosphoinositides and their head groups or proteins such as the
subunits of heterotrimeric G proteins (19, 22-25). The integrity of
the PH domain is critical for the activation and subcellular
localization of many PH domain-containing enzymes including Bruton's
tyrosine kinase (14, 26-28),
-adrenergic receptor kinase (25), and
the serine/threonine kinase encoded by the proto-oncogene
c-akt (29). These considerations prompted us to examine the
function of the PH domain in the regulation of PKD activity.
In the present study we demonstrate that a PKD mutant lacking the entire PH domain exhibits high basal kinase activity. This active form of PKD binds phorbol esters as well as the wild type protein but is only slightly further activated by treatment with PDB in vivo. PKD mutants lacking part of the PH domain or with single amino acid substitutions within the PH domain also show high basal kinase activity. Our results indicate that the PH domain of PKD plays a negative role in the regulation of PKD kinase activity.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture and Transfections-- COS-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 10% CO2. Exponentially growing COS-7 cells, 40-60% confluent, were transfected in serum-free medium by using 5 µg of DNA from the various plasmids and 10 µl of Lipofectin reagent (Life Technologies, Inc.) per 60-mm-diameter dish according to the protocol provided by the manufacturer. Briefly, Lipofectin (10 µl) was diluted to 1 ml with Opti-MEM I medium (Life Technologies, Inc.), left for 30 min, and then mixed with the DNA previously diluted in 1 ml of the same medium. After 15 min, the volume of the DNA-Lipofectin mixture was increased to 2.5 ml with Opti-MEM I and overlaid onto rinsed (once with Opti-MEM I) COS-7 cells. The cultures were incubated at 37 °C for 6 h, and the medium was then replaced with fresh DMEM containing 10% fetal bovine serum. The cells were used for experimental purposes 72 h later.
Deletion Mutants and Site-directed Mutagenesis--
A PH domain
deletion mutant was generated by direct-rapid mutagenesis of large
plasmids using PCR (30) with rTth DNA polymerase XL with proofreading
capability (GeneAmp XL PCR Kit, Perkin-Elmer). The PH domain deletion
mutant of PKD (PKDPH), lacking amino acids Val-429 to Gly-557, was
made directly in PKD-cDNA cloned in pBluescript-SK (+) using
oligonucleotide primers starting upstream (reverse primer) and
downstream (forward primer) from the desired deletion and also
containing the unique restriction site AscI which is not present in wild type PKD (Table I,
deletion mutant and Fig. 1, PKD
PH).
PCR was performed by using a DNA Thermal Cycler (Perkin-Elmer) applying
a short number of cycles (9 cycles) starting with a large amount of
template DNA (1 µg). The PCR parameters were optimized for the
oligonucleotides used. After PCR, template DNA was eliminated by
DpnI digestion. Amplified DNA was cut with AscI
and ligated. The resulting deletion construct was then subcloned into
the mammalian expression vector pcDNA3 (pcDNA3-PKD
PH) for
transient expression in COS-7 cells.
|
|
Immunoprecipitation-- Transfected COS-7 cells were washed three times in ice-cold PBS and lysed in buffer A (50 mM Tris-HCl, pH 7.6, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 10 µg/ml aprotinin, 100 µg/ml leupeptin, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, and 1% Triton X-100). PKD was immunoprecipitated at 4 °C for 3 h with the PA-1 antiserum (1:50 dilution) raised against the synthetic peptide EEREMKALSERVSIL that corresponds to the carboxyl-terminal region of PKD, as described previously (10). The immune complexes were recovered using protein A coupled to agarose.
Autophosphorylation Assay--
PKD autophosphorylation was
determined in an in vitro kinase assay as described
previously (13). Briefly, the immunoprecipitates were washed once with
buffer A, twice with buffer B (buffer A minus Triton X-100), twice with
kinase buffer (30 mM Tris-HCl, pH 7.6, 10 mM
MgCl2), and 20 µl of PKD immune complexes were mixed with
20 µl of kinase buffer containing 100 µM final
concentration of [-32P]ATP (specific activity of
400-600 cpm/pmol) for 10 min at 30 °C. The reaction was then
stopped by adding an equal volume of 2 × SDS-PAGE sample buffer
(1 M Tris-HCl, pH 6.8, 0.1 mM
Na3VO4, 6% SDS, 0.5 M EDTA, 4%
2-mercaptoethanol, 10% glycerol) and analyzed by SDS-PAGE. The gels
were dried and autoradiographs were scanned in a LKB Ultrascan XL
densitometer, and the labeled band corresponding to autophosphorylated
PKD or the different mutants was quantified using an Ultrascan XL
internal integrator.
Exogenous Substrate Phosphorylation--
The phosphorylation of
various peptides by immunoprecipitated wild type PKD or mutants was
carried out under the same conditions as the in vitro kinase
assay adding a final concentration of 2.5 mg/ml syntide-2,
epsilon-peptide, myelin basic protein, or histones. After 10 min at
30 °C, the reaction was terminated by adding 100 µl of 75 mM H3PO4 and spotting 75 µl of
the supernatant on P-81 phosphocellulose paper, and free
[-32P]ATP was separated from the labeled substrate by
washing four times (5 min each) in 75 mM
H3PO4. The P-81 papers were dried, and
radioactivity incorporated into the different peptides was determined
by Cerenkov counting.
Western Blot Analysis-- For Western blot analysis of either PKD or PKD mutants expressed in transfected COS-7 cells, 50 µg of total protein from cell lysates were mixed with the same volume of 4 × SDS-PAGE sample buffer, boiled for 10 min, and analyzed by SDS-PAGE followed by transfer to Immobilon membranes at 100 V, 0.4 A at 4 °C for 4 h using a Bio-Rad transfer apparatus. The transfer buffer composition was 200 mM glycine, 25 mM Tris, 0.01% SDS, and 20% CH3OH. Membranes were blocked for 1 h at room temperature in 5% non-fat dried milk in PBS, pH 7.2, and incubated for 3 h with PA-1 antiserum (1:500 dilution) in PBS containing 3% non-fat dried milk. Immunoreactive bands were visualized using either 125I-labeled protein A (0.1 µCi/ml) and autoradiography or horseradish peroxidase-conjugated anti-rabbit IgG and subsequent enhanced chemiluminescence detection.
PDB Binding to COS-7 Cells-- [3H]PDB binding to intact COS-7 cells was performed as described previously (31). Briefly, cultures were washed twice with DMEM and incubated with binding medium (DMEM containing 1 mg/ml bovine serum albumin and 10 nM [3H]PDB) at 37 °C for 30 min. The cultures were then rapidly washed at 4 °C with PBS containing 1 mg/ml bovine serum albumin and lysed with NaOH/SDS, and bound radioactivity was determined by scintillation counting. Nonspecific binding was determined in the presence of 10 µM unlabeled PDB.
Materials--
[-32P]ATP (370 MBq/ml),
125I-labeled protein A (15 mCi/ml), and enhanced
chemiluminescence reagents were from Amersham Int. (United Kingdom).
PDB was obtained from Sigma. The inhibitor GF I was from LC
Laboratories. Protein A-agarose was from Boehringer Mannheim. Other
items were from standard suppliers or as indicated in the text.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of PKDPH in COS-7 Cells--
The region comprising
nucleotides 1408-1796 in the cDNA encoding PKD (corresponding to
amino acids 429-557 coding for the PH domain) was deleted and the
mutated PKD, lacking the entire PH domain, was subcloned into the
mammalian expression vector pcDNA3. The resulting construct
(PKD
PH) was transiently transfected into COS-7 cells. To examine the
expression of PKD
PH, lysates from these cells were analyzed by
Western blotting with the PA-1 antibody directed against the carboxyl
terminus of PKD. As can be seen in Fig. 2
(left), PKD
PH migrated in SDS-PAGE gels with an apparent
molecular mass of ~96 kDa instead of 110 kDa as expected after
deletion of the 128 amino acids corresponding to the PH domain. The
level of immunoreactive PKD
PH was comparable to that of PKD (Fig. 2,
left). In addition, cells transfected with either pcDNA3-PKD or pcDNA3-PKD
PH showed a similar increase
(5-6-fold) in specific [3H]PDB binding as compared with
that obtained in COS-7 cells transfected with the vector alone (Fig. 2,
right). Thus, the level of expression of PKD
PH, judged
either by immunoblotting or [3H]PDB binding, is similar
to that of PKD in transiently transfected COS-7 cells.
|
Deletion of the PH Domain Causes PKD Activation--
To examine
the function of the PH domain in the regulation of PKD activity, COS-7
cells transiently transfected with pcDNA3-PKD or
pcDNA3-PKDPH were treated with or without 200 nM PDB
for 10 min and then lysed and immunoprecipitated with the PA-1
antibody. The immune complexes were incubated with
[
-32P]ATP and analyzed by SDS-PAGE, autoradiography,
and scanning densitometry to determine the level of PKD
autophosphorylation. In agreement with previous results (10, 13), PKD
isolated from unstimulated cells had low catalytic activity that was
markedly activated by PDB stimulation of intact cells. In striking
contrast, PKD
PH exhibited a high level of basal catalytic activity
(16-fold increase compared with unstimulated PKD) which was not
affected by treatment with PDB (Fig.
3A).
|
|
|
Partial Deletions and Single Amino Acid Substitutions within the PH
Domain Also Lead to PKD Activation--
The preceding results suggest
that deletion of the PKD PH domain leads to an active form of PKD. To
substantiate this conclusion, we analyzed the activity of a set of PKD
mutants containing partial deletions or single amino acid substitutions
within the PH domain. The partially deleted PKD mutants were generated
by reinserting the PH domain sequence of PKD containing partial
deletions into PKDPH to produce PKD
and PKD
1-4
. As a
control, the entire sequence of the PH domain was also reinserted into
PKD
PH (PKD
PH+PH). Fig. 1 shows a schematic representation of the
different mutants.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the tertiary structure of several PH domains, some bound
to ligands, has been determined, the function of this domain remains
incompletely understood (18-21). The PH domains of a variety of
enzymes have been mutated or deleted in an effort to elucidate their
function. Recent studies with Akt (23, 29, 37), G protein-coupled
receptor kinase (38), Bruton's tyrosine kinase (14, 26-28),
-adrenergic receptor kinase (25), the Ras exchange factor Sos
(39-42), and phospholipase C-
(43) have demonstrated that the PH
domain plays an important role in the regulation of the enzyme
activity. Most studies have shown that the integrity of the PH domain
is necessary for enzyme activation.
The newly identified PKD contains a PH domain inserted between the second cysteine-rich motif and the kinase catalytic domain (9). The presence of the PH domain distinguishes PKD from all known members of the PKC family. In the present study, we have deleted or mutated the PH domain of PKD and analyzed the regulatory properties of the resulting PKD mutants.
In striking contrast to most previous studies showing that functional
PH domains are necessary for enzyme activation, our results demonstrate
that partial or complete deletions of the PH domain of PKD (see Fig. 5)
resulted in PKD mutants that exhibit a high level of basal kinase
activity. Single amino acid substitutions (e.g. R447C and
W538A) within the PH domain also promoted an activated state of PKD.
The R447C mutation was tested because a comparable mutation (R28C) in
the PH domain of Bruton's tyrosine kinase is responsible for the
induction of X-linked agammaglobulinemia in humans and
X-linked immunodeficiency in CBA/N mice (14, 26, 27), and a
similar mutation in the serine/threonine kinase Akt abolished
activation of this enzyme by platelet-derived growth factor (29, 37).
The W538A mutation alters the only invariant amino acid in the
carboxyl-terminal -helix and is conserved in all known PH domains
(18, 20). Interestingly, the W538A mutation was as effective as partial
or complete deletions of the PH domain in promoting an active state of
PKD.
Recent results demonstrated that treatment of intact cells with PDB,
cell-permeant diacylglycerols, or bryostatin induces rapid PKD
activation that was maintained during cell disruption and
immunoprecipitation (13, 44). Several lines of evidence including the
use of PKC inhibitors and cotransfection of PKD with constitutively
activated mutants of PKC and -
(13) indicate that PKD can be
activated by phosphorylation in intact cells through a
PKC-dependent signal transduction pathway. Our results
demonstrate that PKD carrying deletions of the PH domain or the single
amino acid substitution W538A within the
-helix of the PH domain
were only slightly stimulated further by treatment of the cells with PDB, implying that PKD rendered active by PH domain mutation is already
fully activated. We conclude that the integrity of the PH domain is
critical for maintaining unstimulated PKD in a state of low catalytic
kinase activity.
The low basal activity of many protein kinases is maintained by the
interaction between an autoinhibitory domain located within the enzyme
with its catalytic site, thereby preventing the binding of substrates
(45). For example, all members of the PKC family from yeast to human
PKCs possess an autoinhibitory motif that is located upstream of the
first cysteine-rich domain (46). In contrast, PKD does not contain a
pseudosubstrate region in a comparable position (9). Although it is
conceivable that PKD is also regulated by autoinhibition, as many other
regulated protein kinases, the putative autoinhibitory region has not
been identified yet. In view of our results, it is tempting to
speculate that the PH domain of PKD has an inactivating function by
functioning as an autoinhibitory domain. This suggests a novel role of
PH domains in enzyme regulation. In the context of this model, partial or complete deletions of the PH domain of PKD or single amino acid
substitutions within this domain should stabilize an active conformation of PKD as, in fact, it is shown by the mutational analysis
presented here. Alternatively, our results cannot exclude the
possibility that the PH domain of PKD could bind an inhibitory ligand(s) that is released by allosteric stimulation or by
phosphorylation-dependent activation induced by treatment
of intact cells with PDB. However, PH domain ligands such as
phosphoinositides and subunits of G proteins promote correct
subcellular localization and enzyme activation rather than inhibition
of enzyme activity (18-21). Future studies should attempt to
distinguish between these alternative models. Regardless of the precise
mechanism(s), our results demonstrate, for the first time, that the PH
domain of PKD plays a negative role in the regulation of PKD kinase
activity.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a postdoctoral fellowship of the European Molecular
Biology Organization.
§ To whom correspondence should be addressed. Present address: 900 Veteran Ave., Warren Hall Rm. 11-124, Dept. of Medicine, UCLA School of Medicine, Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399.
1 The abbreviations used are: PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; GF, I GF 1092030X; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PDB, phorbol-12,13-dibutyrate; PH, pleckstrin homology; PKD, protein kinase D; PAGE, polyacrylamide gel electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|