From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041
Received for publication, July 19, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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The activity of phospholipase D (PLD) is
regulated by a variety of hormonal stimuli and provides a mechanistic
pathway for response of cells to extracellular stimuli. The two
identified mammalian PLD enzymes possess highly homologous C termini,
which are required for catalytic activity. Mutational analysis of PLD1 and PLD2 reveals that modification of as little as the C-terminal threonine or the addition of a single alanine attenuates activity of
the enzyme. Protein folding appears to be intact because mutant enzymes
express to similar levels in Sf9 cells and addition of peptides
representing the C-terminal amino acids, including the simple hexamer
PMEVWT, restores partial activity to several of the mutants. Analysis
of several mutants suggests a requirement for the hydrophobic reside at
the Phosphatidic acid plays a prominent role in phospholipid
metabolism (1) and signal transduction. As a signaling molecule, phosphatidic acid is produced through the action of phospholipase D
(PLD)1 in response to a
variety of cellular stimuli and acts directly as a second messenger or
as a precursor for the formation of another second messenger,
diacylglycerol (see Refs. 2-5 for reviews).
Understanding of mechanisms by which mammalian PLD enzymes are
regulated has been greatly accelerated by the development of a
sensitive in vitro assay for the activity (6, 7) and
identification of two mammalian genes (8-10). The regulation of PLD
activity in vitro allowed the identification of four
distinct mechanisms for stimulation of the enzymes.
Phosphatidylinositol 4,5-bisphosphate (PIP2) was the first
activator and a key element in establishing assays for the enzymes (6,
11-13). This was followed by the elucidation of activation by members
of both the Arf (6, 14) and Rho (15-19) families of small monomeric
GTPases and by classical isoforms of protein kinase C (PKC) via a
phosphorylation independent pathway (20, 21). Subsequent studies with
recombinant proteins in vitro have shown that PLD1 responds
to all of these activators (22), while PLD2 is only stimulated by
PIP2 and Arf (9, 23, 24). Elucidation of these regulatory
mechanisms and investigations to determine which of these pathways are
utilized in the cellular milieu in response to extracellular stimuli
have been reviewed (see Refs. 25-27 for examples).
The availability of the cloned enzymes has also resulted in multiple
attempts to define interaction sites of PLD isozymes with different
activators. Constructs of PLD1 missing the N-terminal third of the
protein yield selective attenuation of regulation by PKC (28-30),
whereas stimulation by other activators remains intact. A site for
interaction with Rho is located in the C-terminal third of the protein
based on interaction of C-terminal regions of PLD1 with RhoA (31, 32)
and selective attenuation of RhoA regulation by inclusion of PLD1
C-terminal constructs in assays (31). In contrast, regions of PLD1
involved in regulation by Arf have not been identified.
The identification of catalytic portions of PLD1 benefited from
homology of the enzyme with other lipid transferases (33). The
predicted participation of both conserved HKD
(HXKXXXXDXXXXXXGXXN) motifs
in catalytic function was verified by expression of mutagenized proteins (32). Extensive mutagenesis in other regions of the protein
has led to a variety of catalytically defective enzymes (30), but the
role of altered regions in catalysis, stability of the enzyme, or
regulation remains to be defined.
Although modification of the N terminus of PLD1 allows apparent normal
activity, modification of the C terminus of the mammalian PLD enzymes
results in proteins that lack catalytic activity (29). In this study,
we demonstrate that PLD1 and PLD2 with alterations in the C terminus
are inactive but functionally intact. Partial restoration of the
activity of these mutant enzymes by addition of various peptides
strongly suggests that the C-terminal threonine of these mammalian
enzymes is a required component of the active site or functionally
helps stabilize the active site for hydrolytic activity.
Materials--
Dipalmitoyl phosphatidylcholine (DPPC) and bovine
brain phosphatidylethanolamine were purchased from Avanti.
PIP2 was purchased from Roche Molecular Biochemicals.
L- Antisera--
Synthetic peptides were cross-linked to
tuberculin-purified protein derivate (Statens Seruminstitut) with
glutaraldehyde (34). The conjugates were used to immunize rabbits (35).
Sera were collected and screened by immunoblot analysis.
Preparation of the Protein Activators of PLD--
Recombinant
Arf proteins were expressed in bacteria and purified through two steps:
batch elution from DEAE-Sephacel and a gel-filtration column as
described (36). A plasmid encoding hexahistidine-tagged protein kinase
C Preparation of Light Membrane Fractions--
Wild-type and
mutant PLD enzymes were expressed in Sf9 cells grown in
monolayer. About 1 × 107 Sf9 cells were
infected with 0.5 ml of high titer recombinant virus (~1 × 108/ml) for 48 h. Cells were washed and harvested in
buffer containing 20 mM NaHepes, pH 7.5, 1 mM
EDTA, 1 mM dithiothreitol, and protease inhibitors (21 µg/ml
N Preparation of Gel-filtered Substrate Vesicles--
Substrate
vesicles made by gel filtration have proven to be more stable and
provide lower background for measurement of PLD activities. The
procedure given here will be presented in more detail
elsewhere.3 A mixture of
phosphatidylethanolamine (600 µM), PIP2 (30 µM), DPPC (60 µM), and
[3H]DPPC (about 500 cpm/pmol) was prepared and dried
under a stream of nitrogen. The lipids were resuspended in PLD reaction
buffer (50 mM NaHepes, pH 7.5, 80 mM KCl, 3 mM EGTA, and 1 mM DTT) containing 1%
n-octyl- Assay of PLD Activity--
Except for the preparation of
substrate vesicles, the assay of PLD activity was measured by the
release of [3H]choline as described previously (6, 7).
Briefly, aliquots of light membrane fractions were mixed in 30 µl of
a buffer containing 50 mM NaHepes, pH 7.5, 80 mM KCl, 3 mM EGTA, 1 mM DTT, 3 mM MgCl2, 3 mM CaCl2,
and 10 µM GTP Construction of Mutants--
The plasmid encoding human
phospholipases D1 and D2 were kindly provided by Michael Frohman and
Andrew Morris (8, 9). DNA encoding PLD1 was subcloned into pFastBacTHb
vector (Life Technologies, Inc.), and the fragment from SphI
to the C terminus of the coding region was deleted and replaced with
mutated products generated by polymerase chain reactions. The 3'
primers used for PCR reactions contained mutations and new stop codons
to give the altered amino acid sequences indicated. All mutations were confirmed by DNA sequencing. Recombinant viruses were obtained by
transformation of DH10Bac cells and selection by blue-white screening
as described by the manufacturer. Viral DNA was isolated and used to
transfect Sf9 cells for creation and amplification of
baculoviruses encoding the mutated PLDs.
PLD1 with a Modified C Terminus Is Catalytically
Compromised--
Recombinant PLD1 that has been modified with 6 histidines at its C terminus was inactive when expressed in Sf9
cells (Fig. 1). Inactive enzyme was also
obtained if the four C-terminal amino acids were deleted from the
enzyme (PLD1-C
The concentrations of peptide that restore activity to mutationally
compromised PLD are substantial, albeit specific. A more potent effect
can be obtained if a myristoylated peptide is utilized. This is shown
in Fig. 2. The acylated peptide shows an
increase in potency of over 1000-fold but not a real increase in
efficacy at the concentrations that can be utilized. The increase in
potency can probably be attributed to localization of the peptide to
the surface of substrate vesicles, thus allowing for more efficient competition of the peptide with the endogenous C terminus of PLD1.
Immunological Characterization of Expressed PLD1
Mutants--
Further mutational analysis of PLD1 and PLD2 was done to
investigate the role of C-terminal residues more fully. The mutant enzymes used in this study are described in Fig.
3A. All of the mutant enzymes
could be expressed in Sf9 cells and were enriched in light
membrane fractions. Antibodies used to detect the PLD1 proteins are
described in Fig. 3B, and the level of expression of each
mutant is shown in Fig. 4 (A
and B). Antisera raised to peptides representing the N
terminus (Q054) or two internal sequences of PLD1 (R654, R653) detected
all of the expressed PLD1 enzymes (Fig. 4A) but not PLD2. In
contrast, antisera to a peptide representing the C terminus of PLD1
recognized both PLD1 and PLD2, but discriminated strongly among the
constructs with mutations near the C terminus. The recognition of both
PLD1 and PLD2 by C-terminal sera is indicative of their highly
homologous C termini, whereas the N-terminal and internal peptides
represent more divergent sequences between the two enzymes.
The C-terminal directed antisera recognize the very C terminus of the
molecule. This is demonstrated by competition of this interaction with
a peptide (PLDpepPT) representing the last 6 amino acids of PLD1 but
not with a peptide (PLDpepSA) that represents the N-terminal half of
the peptide used for production of the antisera (Fig. 4B).
Further definition of this recognition is demonstrated by the mutant
proteins. The C-terminal antisera did not detect proteins that had
either deletions at the C terminus or the addition of either a
6-histidine tag (PLD1) or a single alanine residue (PLD2). In addition,
mutation of the C-terminal tryptophan to alanine or the C-terminal
threonine to lysine, glutamic acid, or alanine resulted in failure of
recognition by the antisera. Only two conservative mutations in these
residues, W1073F and T1074S, resulted in retention of detection. In
total, these results suggest that the recognition site for the
C-terminal antigenic region includes a hydrophobic pocket for the
tryptophan and recognition of the threonine's hydroxyl group and
C-terminal carboxyl moiety. The latter is suggested by the failure of
the antisera to recognize the enzymes with residues added to the C terminus.
Effect of C-terminal Mutations on the Activity of PLD1 and
PLD2--
The activities of various recombinant PLD1 enzymes are shown
in Fig. 5A. Preparations
derived from either expression of PLD1 containing the K466A mutation,
which prevents catalytic activity, or expression of G protein
Restoration by peptide of ~20% of wild type activity to the inactive
truncated proteins, PLD1-C
Two mutations in the C terminus of PLD2 also attenuated activity of the
enzyme. In both cases, addition of the C-terminal peptide could restore
up to 30% of wild type activity. In the case of PLD2, activity is
restored to enzyme assayed in the presence of PIP2. Wild
type PLD1 has a lower activity in the presence of PIP2 and
can be strongly activated by the small G protein, Arf, or PKC
Involvement of the C terminus with Rho appears more complex.
Restoration of stimulation by RhoA can also be observed (Fig. 6B), but the lower efficacy of Rho makes these observations
difficult and the extent of restoration relative to Arf and PKC The C-terminal Carboxyl Group Is Required for Catalysis--
A
modest tolerance for change in the C-terminal threonine but the
complete attenuation of activity by the attachment of a hexahistidine
tag or alanine to this residue suggested that the The results with mutations in the C termini of mammalian PLD1 and
PLD2 indicate the fundamental role of this portion of the molecule in
the catalytic action of these enzymes. We hypothesize that C-terminal
residues stabilize a functional conformation of the active site. This
may occur through interaction with residues directly involved in
catalysis or with residues more remotely involved in formation of the site.
The catalytic core of PLD is predicted to form around the conserved HKD
motifs (33, 39). The structure of Nuc, a bacterial endonuclease
belonging to the superfamily of enzymes with these motifs, consists of
a dimer in which the individual HKD motifs of the monomeric
polypeptides interact in the dimeric molecule to form the active site
(40). The recent structure of the PLD from Streptomyces sp.
strain PMF confirms the coordinated interaction of two HKD motifs
within a single polypeptide to form the catalytic site (41). Such
coordination of the two motifs in the mammalian PLD enzymes is
supported by mutagenesis experiments (32) and the evolution of PLD
activity when the N-terminal and C-terminal halves of PLD1 were
coexpressed but not when either half (single HKD domain) was expressed
alone (42). One potential role for the C-terminal residues would be to
stabilize the interaction between these domains. If so, partial
restoration of activity by the free peptide indicates that the
mechanism for such stabilization would not be a simple tethering of one
domain to the other.
The discovery that peptides representing the C terminus can restore
partial activity to inactive enzymes suggests a more localized function
for the C terminus. It is likely that the mechanism for this
restoration is binding of the peptide to a site normally occupied by
the endogenous C terminus of the enzyme. To achieve this, peptides
would have to compete with the endogenous C terminus, which will have a
competitive edge by virtue of its attachment to the rest of the enzyme.
Such a mechanism also indicates that positioning of the C terminus in
the enzyme must be flexible. The inability of the peptides to fully
restore activity may reflect an inability to use peptides at
sufficiently high concentrations and transient interactions of the
peptides with functional residues. Alternately, correct positioning of
the endogenous C terminus may also have more global conformational
effects on the active site that cannot be mimicked by the independent peptides.
One possibility is that the C terminus of one PLD molecule interacts
with the active site of a second PLD molecule. Thus, the active enzyme
would be a dimer and two enzymes with dissimilar mutations might be
complementary. Although this mechanism could exist, complementation by
coexpression of PLD1-K466A with PLD1-CHis6 was not observed
(data not shown).
The importance of the last two residues of the PLD1 C terminus has been
shown by mutagenesis and reconstitution; specific roles for other
upstream residues have not been explored. Elimination of the
hydrophobic side chain of tryptophan 1073 (PLD1-W1073A) results in
complete loss of activity. It is possible that the aromatic group helps
position residues in the catalytic site or that its interaction with a
specific site in the enzyme helps stabilize positioning of the
C-terminal threonine for effective interaction. A role in stable
positioning of the C terminus is attractive in light of the ability of
peptides to restore partial function of the W1073A mutant.
The inability of amidated peptides to restore activity to inactive
enzymes indicates a requirement for an intact C-terminal Two putative PLD enzymes from Drosophila melanogaster and
Caenorhabditis elegans retain C-terminal motifs similar to
the mammalian enzymes. However, the C termini from PLD enzymes found in
the fungi, Saccharomyces cerevisiae (PMEIYN) and
Candida albicans (PMEIYD), depart from this consensus by
incorporating a C-terminal residue that would not function in the
mammalian protein. In these yeast enzymes, the tyrosine residue could
provide the same hydrophobic anchor proposed for the mammalian
tryptophan and a free A requirement for interaction of C-terminal residues with the rest of
the protein for expression of enzymatic activity offers great
opportunity for regulation. Stabilization of this interaction would
yield increased activity of the enzyme, whereas disruption would
inhibit activity. Enhancement of interaction of the C terminus with the
active site would be a means for one or more activators of PLD1 to
exert their stimulatory effects, especially synergism observed by
combinations of activators (16, 19, 20, 22, 43, 44). The apparent
deficient recovery of stimulation with Rho, which is thought to
interact with C-terminal regions of PLD (31, 32), is consistent with
such a mechanism. However, the absence of activity in C-terminal
mutants, the uniform recovery of stimulation by other activators in the
presence of peptide, and similar behavior by PLD2 suggest this is not a
major regulatory paradigm. Alternatively, a potential mechanism for
inhibition or down-regulation of PLD could involve binding of an
inhibitory molecule to or modification of the C terminus such that
interaction of the C-terminal residues with the active site was
disrupted. The similarity of C-terminal regulation in both PLD1 and
PLD2 makes this a potential common site to attenuate both PLD pathways in a cell.
2-position but not an absolute requirement for the hydroxyl side
chain of threonine at the C terminus. The inability of peptides
amidated at their C termini to effect restoration of activity indicates
the involvement of the C-terminal
carboxyl group in functional
activity of these enzymes. The ability of peptides to restore activity
to PLD enzymes mutated at the C terminus suggests a flexible
interaction of this portion of the molecule with a catalytic core
constructed on conserved HKD motifs. Participation of these C termini
residues in either stabilization of the catalytic site or the enzymatic
reaction itself remains to be determined. This requirement for the C
terminus provides an excellent potential site for interaction with
regulatory proteins that may either enhance or down-regulate the
activity of these enzymes in vitro.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-[choline-methyl-3H]Dipalmitoyl
PC was purchased from DuPont. Peptides and modified peptides were
synthesized by Genosys Biotechnologies or the biosynthesis facility of
the Howard Hughes Medical Institute (University of Texas Southwestern
Medical Center, Dallas, TX).
(6H-PKC
) was constructed using the polymerase chain reaction.
Briefly, synthetic oligonucleotides encoding the amino acid sequence,
NASMAGHHHHHHGALDR were inserted into the hinge region of the lepine
PKC
gene in place of amino acids
307-327.2 The 6H-PKC
was
expressed in Sf9 cells following infection with recombinant
baculovirus and purified through consecutive steps of
nickel-nitrilotriacetic acid affinity and Hi-Trap heparin
chromatography. The final purity of proteins was evaluated by
separation on SDS-polyacrylamide gel electrophoresis (37) and detection
by staining with silver. Protein concentration was determined by
staining with Amido Black (38).
-p-tosyl-L-lysine
chloromethyl ketone, 21 µg/ml tosylphenylalanyl chloromethyl ketone,
21 µg/ml phenylmethylsulfonyl fluoride, 12.5 µg/ml pepstatin A, and
21 µg/ml
N
-p-tosyl-L-arginine
methyl ester). The particulate materials of cells were sedimented by
centrifugation at 400 × g for 5 min. The pellets were
resuspended in the same buffer, homogenized, and loaded into the bottom
of centrifuge tubes. Two volumes of 2.5 M sucrose in the
same solution were added to give a final concentration of 47% (w/v).
This was overlaid with equal volumes of solutions containing 40% and
20% sucrose and the gradient was subjected to centrifugation at
80,000 × g for 60 min at 4 °C. Membranes that
migrated to the interface between 20% and 40% sucrose were collected
as the light membrane fraction enriched in PLD activity.
-D-glucopyranoside and sonicated for
2 min at room temperature to form mixed micelles. Vesicles were formed
by gel-filtration of the micelles through a column (0.7 cm × 30 cm) of AcA34 (Biosepra), which had been equilibrated with PLD reaction
buffer before loading. Fractions (~300 µl each) were collected and
the peak of radioactive vesicles pooled and used as substrate for the
assay of PLD activity. The recovery of [3H]DPPC and
unlabeled lipids was uniform and ranged from 30% to 40%.
S. When indicated, Arf and PKC
were added to final concentrations of 5 µM and 50 nM, respectively. Reactions were started by the addition of
substrate vesicles and incubated at 30 °C for 60-90 min. Reactions
were stopped by the addition of 200 µl of 10% trichloroacetic acid
and 100 µl of 10 mg/ml bovine serum albumin. Samples were centrifuged
to remove precipitated lipids and proteins and the supernatants
analyzed for released [3H]choline by liquid scintillation
spectroscopy. Unless indicated otherwise, all assays represent the
average of duplicate samples.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4). This inactivity is due to an apparent catalytic
deficiency, as activity could be partially restored to the expressed
enzymes by addition of peptides representing the C terminus of PLD1
(Fig. 1). Although the addition of peptides at millimolar
concentrations had little effect on activity of the wild-type enzyme,
peptide ST, which represents the last 15 amino acids of PLD1, restored
about 15% of wild-type activity to both enzymes that had been modified
at the C terminus. At higher concentrations, all of the peptides had
inhibitory effects on the assay of PLD activity. A shorter peptide (PT)
containing only the last 6 amino acids of the C terminus could also
restore activity to the inactive enzymes but was less potent than the
longer ST peptide. In contrast, a peptide (SA) representing amino acids
that were N-terminal to peptide PT was ineffective. This indicates that
the C-terminal residues of PLD1 play a crucial role in PLD
activity.
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Fig. 1.
Comparison of different peptides affecting
wild type PLD1 and two PLD1 mutants. Assays were carried out with
3 µg of membrane protein, increasing concentrations of the peptides,
PLDpepST, PLDpepSA, or PLDpepPT, as indicated, and in the presence of
50 nM PKC , 5 µM Arf, and 10 µM GTP
S as described under "Experimental
Procedures."
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Fig. 2.
Effects of peptide myr-IPLDT on the
activities of recombinant PLD1 and the expressed mutants
PLD1-CHis6 and PLD1 4. Assays
containing 3 µg of membrane protein and the indicated amount of
myr-IPLDT (myrGCGIVPMEVWT) were conducted at 30 °C for 60 min with
50 nM PKC
, 5 µM Arf, and 10 µM GTP
S.
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Fig. 3.
A, mutations of PLD1 and PLD2 used in
this study. Deletions of amino acids (indicated by ) are all from
the C terminus. B, illustration of peptides used to make
antisera. Sequences of the actual peptides used for production of
antisera are: PLDpep-SI, SLKNEPRVNTSALQKI; PLDpep-RS,
RRHTFRRQNVREEPREMPS; PLDpep-KD, KDKNEPVQNLPIQKSIDDVD; PLDpep-ST,
SVGTKEAIVPMEVWT.
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Fig. 4.
Immunological detection of PLD1, PLD1
mutants, PLD2, and PLD2 mutants. About 3 µg of membrane protein
were resolved by SDS-polyacrylamide gel electrophoresis and transferred
onto nitrocellulose. A, transferred blots were immunoblotted
with Q054 (anti-N terminus), R654 and R653 (anti-internal sequences),
and Q056 and Q057 (anti-C terminus) antisera. B, blots were
immunoblotted with antisera or "blocked" antisera. For blocking, 20 µl of antiserum were preincubated with 10 µl of 3 mM
indicated peptide (ST referring to PLDpepST, SA
referring to PLDpepSA, and PT referring to PLDpepPT; see
Fig. 1 for details) at 4 °C overnight. All sera were used for
immunoblotting at final dilutions of 1:500.
subunits, provide controls for measurement of endogenous Sf9 PLD
activity in these preparations of membranes. Truncation of the last two
or four amino acids yielded inactive enzyme. Of six point mutations in
the two C-terminal amino acids, three resulted in inactive proteins
while three retained partial activity. Modification of tryptophan to
phenylalanine was tolerated very well, but substitution of alanine in
this position resulted in total loss of activity. This indicates that
the aromatic residue is important for C-terminal function. Replacement
of Thr-1074 with either serine or alanine allowed expression of about
20% of wild type activity. In contrast, substitution of this threonine with the charged residues, lysine or glutamic acid, completely attenuated activity of the enzyme.
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Fig. 5.
A, comparison of responses of PLD1
proteins to the peptide Myr-IPLDT (myrGCGIVPMEVWT). The assays were
conducted with (open bar) or without
(solid bar) 1 µM peptide Myr-IPLDT
and in the presence of activators as described in Fig. 1. B,
effects of peptide myr-IPLDT on the basal activities of wild type PLD2
and PLD2 mutants. Assays contained 4 µg of membrane protein, the
indicated amount of myr-IPLDT, and no other protein activators and were
carried out at 30 °C for 60 min.
2 and PLD1-C
4, PLD1-W1073A, and
PLD1-CHis6 indicates the these enzymes are expressed in a functionally intact form but are unable to maintain an active catalytic
site. The inability of the peptide to restore activity to the
PLD1-T1074K and PLD1-T1074E mutants may reflect the production of
incorrectly folded protein but more likely reflects an inability of the
peptide to compete with a more stable association of the endogenous C
terminus (see "Discussion"). The addition of a C-terminal peptide
to expressed enzymes with endogenous activity resulted in inhibitions
of about 15-30%; this is due to use of the peptide at concentrations
optimal for measuring restoration of activity to inactive proteins but
at which inhibitory effects are beginning to be observed with wild type
enzyme. Use of even higher concentrations of peptide will cause
nonspecific inhibition of activities observed with both wild-type and
restored enzymes.
. Fig.
6A shows that peptide could
effectively restore stimulation by either regulatory molecule to
catalytically compromised enzymes. These results indicate that the role
of the C terminus in catalysis by the PLD isozymes is fundamental to
activity and not selectively employed by these differential regulators
of the enzymes.
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Fig. 6.
Effects of peptide myr-IPLDT (myrGCGIVPMEVWT)
on the activities of wild type PLD1 and PLD1-CHis6 in the
presence or absence of activators. Assays contained either 3 (A and B) or 4 (C) µg of membrane
protein and were incubated with substrate at 30 °C for either 60 (A and B) or 90 (C) min. Samples were
assayed with (open bars) or without
(solid bars) 1 µM MyrIPLDT peptide.
A, assays contained 50 nM PKC and 5 µM Arf as indicated. B, assays contained 1 µM RhoA as indicated and results are the average of
quadruplicate samples. C, assays contained 1 µM RhoA and 30 nM PKCa as indicated. Results
with PLD1-CHis6 are the average of quadruplicate samples.
Error bars record the average error.
cannot be adequately assessed. However, some deficiency in the ability of RhoA to activate PLDs with C-terminal disruptions is indicated by
the failure of RhoA to synergize with PKC
when the deficient enzymes
are restored with peptide (Fig. 6C). Although this may indicate that Rho directly uses the intact C terminus to partially effect stimulation of activity, it is also consistent with an indirect
role in which anchoring of the C-terminal residues is important to help
stabilize other proximal elements of the protein which mediate
regulation by Rho.
-carboxyl group of
the threonine was functionally important. Therefore, amidated peptides
were tested for their ability to restore activity to inactive enzymes
(Fig. 7). Amidation of the C terminus of
either the shorter or longer C-terminal peptides inhibited their
ability to restore activity to inactive mutants of PLD1.
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Fig. 7.
Effects of amidated peptide on the activities
of wild type or mutant PLD1 enzymes. Assays contained 3 µg of
membrane protein, all activators as in Fig. 1, and the indicated amount
of regular peptide (solid circles and
solid squares) or amidated peptide
(open circles and open
squares).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carboxyl
group. It is possible that this carboxyl group participates directly in
catalytic action of the enzyme. However, conservation of the HKD motifs
(HXKXXXXDXXXXXXGXXN) and
structural data from Nuc (40) suggest that all of the amino acid
residues directly involved in catalysis are in place. It is more likely
that the C-terminal carboxyl group may be stabilizing or activating
catalytic residues through salt bridges or hydrogen bonds. In the
structure of Nuc (40), both serines of the HKD motifs (GSXN)
contribute symmetrically to stabilization of the active site. In the
mammalian PLD isozymes, the serine position in the first HKD motif is a glycine, suggesting a significant departure in structure from this
homologous core domain. It is tempting to speculate that the C-terminal
threonine could supply this role in PLD1. However, the partial activity
of the enzyme when alanine occupies this position rules out this role
for the hydroxyl group and such a functional role would depend on
interactions through the
-carboxyl moiety. A second invariant
residue of interest in the HKD motif is the aspartyl residue. In Nuc,
this residue is located more than 25 Å from the catalytic action but
appears through several hydrogen bonds to help stabilize structural
elements containing residues in the active site (40). Interaction of
the C terminus of PLD with one of the conserved aspartyl residues may
then influence the active site from a distance.
-carboxyl group from the asparagine or
aspartic acid could fulfill the requirement of the last amino acid.
However, compensatory changes in the surfaces of the enzymes with which
these C termini interact would presumably be required to accommodate
the different side chains of these C-terminal residues. The C termini
of identified PLDs in bacteria and plants are much more variable. This
and overall differences in structural components and regulatory
properties of the latter enzymes probably indicate that the functional
requirement of the C terminus defined for the mammalian enzymes will
not be a conserved feature in the broader family of PLD enzymes.
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ACKNOWLEDGEMENT |
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We thank Dr. William Singer for help with the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM31954 and a grant from the Robert A. Welch Foundation.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.
To whom correspondence should be sent: Dept. of Pharmacology,
University of Texas Southwestern Medical Center, 5323 Harry Hines
Blvd., Dallas, TX 75235-9041. Tel.: 214-648-2835; Fax: 214-648-2971; E-mail: paul.sternweis@email.swmed.edu.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M006404200
2 W. D. Singer and P. C. Sternweis, manuscript in preparation.
3 X. Jiang, S. Gutowski, W. D. Singer and P. C. Sternweis, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are:
PLD, phospholipase D;
PIP2, phosphatidylinositol
4,5-bisphosphate;
PKC, protein kinase C;
DPPC, dipalmitoyl
phosphatidylcholine;
6H-PKC, hexahistidine-tagged protein kinase
C
;
myr, myristoyl;
DTT, dithiothreitol;
GTP
S, guanosine
5'-3-O-(thio)triphosphate.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Athenstaedt, K.,
and Daum, G.
(1999)
Eur. J. Biochem.
266,
1-16 |
2. | English, D. (1996) Cell Signal. 8, 341-347[CrossRef][Medline] [Order article via Infotrieve] |
3. | Hodgkin, M. N., Pettitt, T. R., Martin, A., Michell, R. H., Pemberton, A. J., and Wakelam, M. J (1998) Trends Biochem. Sci. 23, 200-204[CrossRef][Medline] [Order article via Infotrieve] |
4. | Houle, M. G., and Bourgoin, S. (1999) Biochim. Biophys. Acta 1439, 135-149[Medline] [Order article via Infotrieve] |
5. | McPhail, L. C., Waite, K. A., Regier, D. S., Nixon, J. B., Qualliotine-Mann, D., Zhang, W. X., Wallin, R., and Sergeant, S. (1999) Biochim. Biophys. Acta 1439, 277-290[Medline] [Order article via Infotrieve] |
6. | Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C., and Sternweis, P. C (1993) Cell 75, 1137-1144[Medline] [Order article via Infotrieve] |
7. | Brown, H. A., and Sternweis, P. C (1995) Methods Enzymol. 257, 313-324[Medline] [Order article via Infotrieve] |
8. |
Hammond, S. M.,
Altshuller, Y. M.,
Sung, T. C.,
Rudge, S. A.,
Rose, K.,
Engebrecht, J.,
Morris, A. J.,
and Frohman, M. A
(1995)
J. Biol. Chem.
270,
29640-29643 |
9. | Colley, W. C., Sung, T. C., Roll, R., Jenco, J., Hammond, S. M., Altshuller, Y., Bar-Sagi, D., Morris, A. J., and Frohman, M. A (1997) Curr. Biol. 7, 191-201[Medline] [Order article via Infotrieve] |
10. |
Kodaki, T.,
and Yamashita, S.
(1997)
J. Biol. Chem.
272,
11408-11413 |
11. |
Liscovitch, M.,
Chalifa, V.,
Pertile, P.,
Chen, C. S.,
and Cantley, L. C
(1994)
J. Biol. Chem.
269,
21403-21406 |
12. |
Brown, H. A.,
Gutowski, S.,
Kahn, R. A.,
and Sternweis, P. C
(1995)
J. Biol. Chem.
270,
14935-14943 |
13. | Rose, K., Rudge, S. A., Frohman, M. A., Morris, A. J., and Engebrecht, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12151-12155[Abstract] |
14. | Cockcroft, S., Thomas, G. M., Fensome, A., Geny, B., Cunningham, E., Gout, I., Hiles, I., Totty, N. F., Truong, O., and Hsuan, J. J (1994) Science 263, 523-526[Medline] [Order article via Infotrieve] |
15. |
Malcolm, K. C.,
Ross, A. H.,
Qiu, R. G.,
Symons, M.,
and Exton, J. H
(1994)
J. Biol. Chem.
269,
25951-25954 |
16. |
Singer, W. D.,
Brown, H. A.,
Bokoch, G. M.,
and Sternweis, P. C
(1995)
J. Biol. Chem.
270,
14944-14950 |
17. |
Kwak, J. Y.,
Lopez, I.,
Uhlinger, D. J.,
Ryu, S. H.,
and Lambeth, J. D
(1995)
J. Biol. Chem.
270,
27093-27098 |
18. |
Balboa, M. A.,
and Insel, P. A
(1995)
J. Biol. Chem.
270,
29843-29847 |
19. |
Kuribara, H.,
Tago, K.,
Yokozeki, T.,
Sasaki, T.,
Takai, Y.,
Morii, N.,
Narumiya, S.,
Katada, T.,
and Kanaho, Y.
(1995)
J. Biol. Chem.
270,
25667-25671 |
20. |
Singer, W. D.,
Brown, H. A.,
Jiang, X.,
and Sternweis, P. C
(1996)
J. Biol. Chem.
271,
4504-4510 |
21. |
Conricode, K. M.,
Brewer, K. A.,
and Exton, J. H
(1992)
J. Biol. Chem.
267,
7199-7202 |
22. |
Hammond, S. M.,
Jenco, J. M.,
Nakashima, S.,
Cadwallader, K.,
Gu, Q.,
Cook, S.,
Nozawa, Y.,
Prestwich, G. D.,
Frohman, M. A.,
and Morris, A. J
(1997)
J. Biol. Chem.
272,
3860-3868 |
23. |
Lopez, I.,
Arnold, R. S.,
and Lambeth, J. D
(1998)
J. Biol. Chem.
273,
12846-12852 |
24. |
Sung, T. C.,
Altshuller, Y. M.,
Morris, A. J.,
and Frohman, M. A
(1999)
J. Biol. Chem.
274,
494-502 |
25. | Singer, W. D., Brown, H. A., and Sternweis, P. C (1997) Annu. Rev. Biochem. 66, 475-509[CrossRef][Medline] [Order article via Infotrieve] |
26. | Frohman, M. A., Sung, T. C., and Morris, A. J (1999) Biochim. Biophys. Acta 1439, 175-186[Medline] [Order article via Infotrieve] |
27. | Exton, J. H (1999) Biochim. Biophys. Acta 1439, 121-133[Medline] [Order article via Infotrieve] |
28. | Park, S. K., Min, D. S., and Exton, J. H (1998) Biochem. Biophys. Res. Commun. 244, 364-367[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Sung, T. C.,
Zhang, Y.,
Morris, A. J.,
and Frohman, M. A
(1999)
J. Biol. Chem.
274,
3659-3666 |
30. |
Zhang, Y.,
Altshuller, Y. M.,
Hammond, S. M.,
Hayes, F.,
Morris, A. J.,
and Frohman, M. A
(1999)
EMBO J.
18,
6339-6348 |
31. |
Yamazaki, M.,
Zhang, Y.,
Watanabe, H.,
Yokozeki, T.,
Ohno, S.,
Kaibuchi, K.,
Shibata, H.,
Mukai, H.,
Ono, Y.,
Frohman, M. A.,
and Kanaho, Y.
(1999)
J. Biol. Chem.
274,
6035-6038 |
32. |
Sung, T. C.,
Roper, R. L.,
Zhang, Y.,
Rudge, S. A.,
Temel, R.,
Hammond, S. M.,
Morris, A. J.,
Moss, B.,
Engebrecht, J.,
and Frohman, M. A
(1997)
EMBO J.
16,
4519-4530 |
33. |
Ponting, C. P.,
and Kerr, I. D
(1996)
Protein Sci.
5,
914-922 |
34. | Lachmann, P. J., Strangeways, L., Vyakarnam, A., and Evan, G. (1986) Ciba Found. Symp. 119, 25-57[Medline] [Order article via Infotrieve] |
35. | Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 265-269[Abstract] |
36. | Randazzo, P. A., Weiss, O., and Kahn, R. A (1995) Methods Enzymol. 257, 128-135[Medline] [Order article via Infotrieve] |
37. | Laemmli, U. K (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
38. | Schaffner, W., and Weissmann, C. (1973) Anal. Biochem. 56, 502-514[Medline] [Order article via Infotrieve] |
39. | Waite, M. (1999) Biochim. Biophys. Acta 1439, 187-197[Medline] [Order article via Infotrieve] |
40. | Stuckey, J. A., and Dixon, J. E (1999) Nat. Struct. Biol. 6, 278-284[CrossRef][Medline] [Order article via Infotrieve] |
41. | Leiros, I., Secundo, F., Zambonelli, C., Servi, S., and Hough, E. (2000) Struct. Fold. Des. 8, 655-667[Medline] [Order article via Infotrieve] |
42. |
Xie, Z.,
Ho, W. T.,
and Exton, J. H
(1998)
J. Biol. Chem.
273,
34679-34682 |
43. |
Siddiqi, A. R.,
Smith, J. L.,
Ross, A. H.,
Qiu, R. G.,
Symons, M.,
and Exton, J. H
(1995)
J. Biol. Chem.
270,
8466-8473 |
44. |
Ohguchi, K.,
Banno, Y.,
Nakashima, S.,
and Nozawa, Y.
(1996)
J. Biol. Chem.
271,
4366-4372 |