From the Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295
Received for publication, October 16, 2000, and in revised form, November 6, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rat brain phospholipase D1 (rPLD1) has two highly
conserved motifs (HXKX4D), denoted
HKD, located in the N- and C-terminal halves, which are required for
phospholipase D activity. The two halves of rPLD1 can associate
in vivo, and the association is essential for catalytic
activity and Ser/Thr phosphorylation of the enzyme. In this study, we
found that this association is also required for palmitoylation of
rPLD1, which occurs on cysteines 240 and 241. In addition,
palmitoylation of rPLD1 requires the N-terminal sequence but not the
conserved C-terminal sequence, since rPLD1 that lacks the first 168 amino acids is not palmitoylated in vivo, while the
inactive C-terminal deletion mutant is. Palmitoylation of rPLD1 is not
necessary for catalytic activity, since N-terminal truncation mutants
lacking the first 168 or 319 amino acids exhibit high basal activity
although they cannot be stimulated by protein kinase C (PKC). The lack
of response to PKC is not due to the lack of palmitoylation, since
mutation of both Cys240 and Cys241 to alanine
in full-length rPLD1 abolishes palmitoylation, but the mutant still
retains basal activity and responds to PKC. Palmitoylation-deficient rPLD1 can associate with crude membranes; however, the association is
weakened. Wild type rPLD1 remains membrane-associated when extracted
with 1 M NaCl or Na2CO3 (pH 11),
while rPLD1 mutants that lack palmitoylation are partially released. In
addition, we found that palmitoylation-deficient mutants are much less
modified by Ser/Thr phosphorylation compared with wild type rPLD1.
Characterization of the other cysteine mutations of rPLD1 showed that
mutation of cysteine 310 or 612 to alanine increased basal
phospholipase D activity 2- and 4-fold, respectively. In summary,
palmitoylation of rPLD1 requires interdomain association and the
presence of the N-terminal 168 amino acids. Mutations of cysteines 240 and 241 to alanine abolish the extensive Ser/Thr phosphorylation of the
enzyme and weaken its association with membranes.
Phospholipase D (PLD)1
is a ubiquitous enzyme found in bacteria, fungi, plants, and mammals
(1). It hydrolyzes phosphatidylcholine (PC) to phosphatidic acid and
choline. Phosphatidic acid is generally recognized as the signaling
product of PLD and functions as an effector in different physiological
processes. It can also be converted to diacylglycerol or to
lysophosphatidic acid. Diacylglycerol is a well characterized activator
for protein kinase C (PKC), while lysophosphatidic acid is a major
extracellular signal that acts on specific cell surface receptors. PLD
also catalyzes a phosphatidyl transfer reaction using primary alcohols
as nucleophilic acceptors to produce phosphatidylalcohols. This
reaction is used as a specific measure of PLD activity.
PLD activity has been found to be highly regulated (2). Different
factors, including protein-tyrosine kinases, PKC, heterotrimeric and
small G proteins, and intracellular Ca2+ regulate PLD
activity directly or indirectly (2-4). Based on the wide involvement
of PLD in signaling pathways and the actions of its products, multiple
functions of PLD have been proposed (2), which include signal
transduction, secretion, membrane trafficking, cytoskeleton
reorganization, and apoptosis (5).
Two isoforms of mammalian PLD (PLD1 and PLD2) have been cloned. These
isoforms share about 50% amino acid similarity but exhibit quite
different regulatory properties. PLD1 has a low basal activity and
responds strongly to PKC and to members of the Rho and ARF families of
small G proteins (6-9). The PKC interaction domain has been mapped to
the N-terminal part of the molecule (10-12), while the Rho interaction
domain has been localized to the C-terminal part of the enzyme (13).
PLD2, on the other hand, exhibits a high basal activity (14-16), and
some reports have shown that it is regulated by calcium, PKC, ARF, the
epidermal growth factor receptor, and PLD belongs to a superfamily defined by the motif,
HXKX4D, denoted "HKD" (22-24).
The enzymes within the family exhibit diverse functions and include
phospholipid synthases, poxvirus envelope proteins, a
Yersinia murine toxin, and the Nuc endonuclease. Despite the
distinct substrate specificities of the superfamily members, the
consensus HKD motif appears to be essential for their enzymatic activity. PLD contains two copies of the HKD motif located in the N-
and C-terminal halves of the molecule, respectively. Mutation of either
HKD motif inactivates human PLD1 and mouse PLD2 (12, 25). Biochemical
and structural studies of the Nuc endonuclease and Yersinia
toxin suggest that the histidine residue in the conserved motif is
directly involved in the catalytic reaction by forming a phosphoenzyme
intermediate (26-28).
Our studies on the rat PLD1 (rPLD1) isoform showed that the enzyme
could be split into two halves and that PLD activity could be restored
when the two fragments were coexpressed in COS7 cells. Coimmunoprecipitation experiments showed that the N- and C-terminal fragments could physically associate (11), and it was proposed that the
association brought the two HKD domains together to form a catalytic
center. Further studies showed that conserved amino acids in the HKD
domains were important for the interdomain association and that the
association was essential for the catalysis of the enzyme (29). In
addition, we found that rPLD1 can be modified by Ser/Thr
phosphorylation, and this modification is also required for the
association of the N- and C-terminal halves of rPLD1. We also found
that the phosphorylated rPLD1 localizes exclusively in the membrane
fraction (29). In the present study, we found that the interdomain
association was also required for the palmitoylation of rPLD1. We
investigated the role of this lipid modification and of cysteine amino
acids in the catalysis and properties of rPLD1.
Materials--
4 Plasmid Construction--
The N-terminal Xpress-tagged
full-length or truncated rPLD1 or the N- or C-terminal fragments of
rPLD1 with coding regions corresponding to amino acids 1-584 and
585-1036, respectively, were created by polymerase chain reaction
amplification and subcloned (11). The deletion or site-directed
mutations of rPLD1 were generated as described in the
QuickChangeTM Site-Directed Mutagenesis instruction manual
from Stratagene. All of the constructs were sequenced to verify the
coding regions of rPLD1. The oligonucleotide pairs from 5' to 3' for
site-directed mutagenesis were as follows: rPLD1(C79A),
GATCTACCTCTCTGGCGCTCCTGTAAAAGCAC and
GTGCTTTTACAGGAGCGCCAGAGAGGTAGATC; rPLD1(C240A),
GATACCAGGTGTGAATGCCTGTGGCCATGGAAGA and
TCTTCCATGGCCACAGGCATTCACACCTGGTATC; rPLD1(C241A),
CCAGGTGTGAATTGCGCTGGCCATGGAAGAGCC and
GGCTCTTCCATGGCCAGCGCAATTCACACCTGG; rPLD1(C240A,C241A),
GATACCAGGTGTGAATGCCGCTGGCCATGGAAGAGC and
GCTCTTCCATGGCCAGCGGCATTCACACCTGGTATC; rPLD1(C310A),
GACACTGATTTTAAAAGCTAACAGCTACAGACA and
TGCATGTCTGTAGCTGTTAGCTTTTAAAATCAGTGTC; rPLD1(C403A),
CGCTGGAGGCTGGACGCCATCCTCAAACGGAA and TTCCGTTTGAGGATGGCGTCCAGCCTCCAGCG;
rPLD1(C612A), CATGGGAAGGATTACGCCAACTTTGTCTTCAAG and CTTGAAGACAAAGTTGGCGTAATCCTTCCCATG.
Cell Culture and Transfection--
COS-7 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with 4 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum in humidified 10%
CO2. Six-well plates were seeded with 2 × 105 COS7 cells/well, and 10-cm dishes were seeded with
6 × 105 cells and transfected with FuGENE6 according
to the manufacturer's instructions.
In Vivo PLD Assay--
After a 6-h transfection with FuGENE6,
COS7 cells in six-well plates were serum-starved (0.5% fetal bovine
serum in Dulbecco's modified Eagle's medium) in the presence of 1 µCi/ml [3H]myristic acid. After overnight starvation,
the cells were washed with phosphate-buffered saline (PBS) and
incubated in serum-free medium supplemented with 0.3% bovine serum
albumin for 50 min. PLD activity was then assayed as described (30).
Briefly, cells were incubated in 0.3% 1-butanol for 25 min. Cells were
then washed with ice-cold PBS and stopped with methanol. Lipids were
extracted, and the phosphatidylbutanol product was resolved by thin
layer chromatography. Bands comigrating with a phosphatidylbutanol
standard were quantitated by scintillation counting.
Subcellular Fractionation--
COS7 cells in 100-mm plates were
harvested after transfection with wild type or mutant rPLD1 constructs
and starved overnight as described above. For each rPLD1 construct, we
transfected six 100-mm plates of COS7 cells, and the cells were
combined and washed and harvested with ice-cold PBS buffer. They were
then centrifuged and resuspended in 750 µl of ice-cold lysis buffer
(25 mM Hepes, pH 7.2, 10% glycerol, 1 mM EDTA,
1 mM EGTA, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride and protease inhibitor mixture containing leupeptin, aprotinin, and pefabloc). The cells were passed through a
27-gauge needle seven times, and the cell lysate was centrifuged at
500 × g for 10 min to remove unbroken cells and
nuclei. The supernatant was then centrifuged at 120,000 × g for 45 min at 4 °C to separate the cytosolic and crude
membrane fractions. The particulate fraction was washed twice with the
lysis buffer and resuspended in 450 µl of this buffer by passing
through a 27-gauge needle until the pellet was resuspended. The
suspension (150 µg of protein) was then split into three parts and
supplemented with 100 µl of lysis buffer containing the appropriate
concentration of either NaCl, Na2CO3 (pH 11),
or Triton X-100 to make the final concentration of these reagents in
the buffer 1 M NaCl, 0.1 M Na2CO3 (pH 11), or 1% Triton X-100,
respectively. The mixtures were further incubated at 4 °C for 30 min
and then centrifuged at 120,000 × g for 45 min at
4 °C to separate the supernatant and pellet fraction. The pellet
fraction was rinsed with 750 µl of hypotonic lysis buffer two times
and then resuspended with 250 µl of lysis buffer. Equal volumes of
the cytosolic and membrane fractions were analyzed by Western blotting.
The intensity of the bands corresponding to rPLD1 in the cytosol or
membrane fractions was quantitated by densitometry. The amounts of the
indicated rPLD1 constructs in the cytosol or membrane fractions were
calculated as percentage of the total enzyme in both.
Western Blotting--
Protein samples were analyzed by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 8% gels and
transferred to polyvinylidene difluoride membranes (Immobilon-P,
Millipore Corp.). The blots were then blocked with 5% nonfat milk and
incubated with appropriate primary antibodies (1 mg/ml) (1:5000)
followed by incubation with horseradish peroxidase-conjugated secondary
antibody. Immunoreactive bands were detected using enhanced chemiluminescence.
Immunoprecipitation--
COS7 cells cultured on 6-well plates
were transfected and starved as described above. The cells (3 µg of
protein) were washed twice with ice-cold PBS and then resuspended in
300 µl of immunoprecipitation buffer containing 25 mM
Hepes, pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM
EGTA, 50 mM KC1, 0.5% Triton X-100, 10 mM NaF,
10 mM Na4P2O7, 1.2 mM Na3VO4, 1 µM
microcystin, and two tablets of protease mixture. The cell suspension
was then passed through a 27-gauge needle five times, and the resulting
cell lysate was centrifuged at 15,000 rpm in an Eppendorf
microcentrifuge for 10 min at 4 °C to pellet the unbroken cells. The
supernatant was then precleared by mixing it with 1 µg of
affinity-purified mouse IgG and 20 µl of a 1:1 slurry of protein A
beads for 1 h at 4 °C. The mixture was then centrifuged, and
the resulting supernatant was incubated with 2 µl of Xpress mouse
antibody (1 mg/ml) and 20 µl of protein A beads overnight. The
immunoprecipitates were washed four times with the lysis buffer and
then resuspended in SDS sample buffer and analyzed by Western blotting.
Palmitoylation Assay--
After 24 h of transfection, COS7
cells cultured on six-well plate's were preincubated for 1 h in serum-free medium supplemented with 0.3% bovine serum albumin and
then incubated with 0.2 mCi/ml [3H]palmitic acid for
4 h in the preincubation medium. [3H]palmitic acid
was dried under N2 and resuspended in ethanol, and the
final volume of ethanol was 1% of that of the incubation medium. After
the radiolabeling, cells were washed twice with cold PBS buffer and
then scraped with the immunoprecipitation buffer and immunoprecipitated
followed by washes as described above. The resulting immunoprecipitates
were analyzed by SDS-PAGE followed by Western blotting.
For fluorography, protein samples were separated by SDS-PAGE and then
incubated in fixing solution containing isopropyl
alcohol/H2O/acetic acid (25:65:10) for 30 min. The gel was
then treated with AmplifyTM for 30 min, dried under vacuum,
and exposed to Hyperfilm-MP at Palmitoylation of rPLD1--
Our study of the palmitoylation of
rPLD1 was initiated when we tried to examine the role of the conserved
amino acids at the C-terminal end in PLD catalysis. Mutagenesis studies
showed that an intact C-terminal end and the hydrophobicity of the last
four amino acids, EVWT, were essential for the catalytic activity of the enzyme (31). We investigated whether this hydrophobicity was
required for a potential palmitoylation of rPLD1, since human PLD1 had
been shown to be palmitoylated, and lack of this lipid modification was
reported to destroy the catalytic activity of the enzyme (32, 33). We
first transfected COS7 cells with either wild type rPLD1 or an inactive
C-terminal deletion mutant rPLD1-(1-1032) and radiolabeled the
cells with [3H]palmitic acid. All of the rPLD1 constructs
used in the studies were tagged with Xpress epitope if not specified.
The exogenously transfected rPLD1 constructs were then
immunoprecipitated by anti-Xpress antibodies, and the
immunoprecipitates were analyzed by Western blotting (Fig.
1A) and fluorography (Fig.
1B) for the expression and the palmitoylation of the
constructs, respectively. As shown in Fig. 1B, tritium
incorporation was observed in a 120-kDa band that corresponded to the
size of both wild type and the C-terminal deleted rPLD1. The reduced
tritium incorporation observed in the rPLD1-(1-1032) is attributable
to the reduced level of this mutant in the immunoprecipitates (Fig.
1A). Thus, both wild type rPLD1 and the C-terminal deletion
mutant of the enzyme were palmitoylated in COS7 cells. The
incorporation of tritium was analyzed further by treating gels
containing tritium-labeled rPLD1 constructs with 1 M
Tris-HCl (pH 7.0) or 1 M hydroxylamine (pH 7.0). Only the hydroxylamine treatment released the tritium incorporated into wild
type rPLD1 and its C-terminal deletion mutant, confirming that the
palmitoylation of the enzyme is through a thioester linkage (data not
shown).
Palmitoylation of rPLD1 Requires the First 168 Amino
Acids--
Alanine mutagenesis of cysteine residues of rPLD1 showed
that the palmitoylation of rPLD1 occurs on Cys240 and
Cys241. As shown in Fig. 2,
wild type PLD1 was expressed (Fig. 2A, lane 1) and palmitoylated (Fig. 2B, lane
1) in COS7 cells labeled with [3H]palmitic
acid. However, no tritium incorporation was observed in the double
cysteine mutant, rPLD1(C240A,C241A) (Fig. 2B,
lane 2), although the constructs were expressed
and immunoprecipitated from COS7 cells (Fig. 2A,
lane 2). Mutation of the other cysteine residues
did not affect the palmitoylation of rPLD1 (see Fig. 4 or data not
shown). The finding that rPLD1 was palmitoylated on Cys240
and Cys241 was consistent with studies of human PLD1
(32).
Characterization of the palmitoylation-deficient human PLD1 had shown
that the lipid modification was required for efficient action of the
enzyme, since mutation of both of the palmitoylation sites abolished
most of the catalytic activity of the enzyme (32). This was surprising,
since our previous studies of N-terminal deletion mutants of rPLD1
showed that the basal catalytic activity of rPLD1-(320-1036) was not
decreased but was enhanced (10, 11). A possible explanation for this
discrepancy was that alternative cysteines were used for palmitoylation
in this N-terminal deletion mutation. We therefore investigated the
palmitoylation of this mutant along with that of two other rPLD1
mutants that lacked either the first 50 or the first 168 amino acids.
COS7 cells transiently transfected with the N-terminal deletion mutants
were labeled with [3H]palmitic acid, and the expressed
rPLD1 constructs were immunoprecipitated with anti-Xpress antibodies
and analyzed by Western blotting (Fig. 2A) and fluorography
(Fig. 2, B and C). As shown in Fig.
2B, rPLD1-(51-1036) was palmitoylated in COS7 cells, but no
apparent tritium incorporation was observed into rPLD1-(320-1036),
which lacks Cys240 and Cys241. Surprisingly,
rPLD1-(169-1036), which still retains Cys240 and
Cys241, also lacked detectable tritium incorporation. This
indicates that palmitoylation of rPLD1 requires the presence of the
N-terminal 168 amino acids. More importantly, the fact that the
N-terminal deletion mutants, rPLD1-(169-1036) and rPLD1-(320-1036),
that lacked palmitoylation still exhibited high basal activity (10, 11)
indicates that palmitoylation of rPLD1 is not necessary for its
catalytic activity. The same protein samples shown in Fig.
2A were also reanalyzed in Fig. 2A*, with the
difference that different amounts were loaded so that a comparable ECL
signal of the protein samples could be obtained by Western blotting.
Palmitoylation of rPLD1 Requires Interdomain Association between
the N- and C-terminal Fragments--
Our previous studies showed that
rPLD1 could be split into two halves and that the N- and C-terminal
halves associated in vivo. This interdomain association was
important for the catalytic activity of the enzyme and the Ser/Thr
phosphorylation of the rPLD1 (11, 29). We therefore investigated the
role of the interdomain association on the palmitoylation of rPLD1.
COS7 cells were cotransfected with wild type or mutated N- and
C-terminal halves of rPLD1 as indicated in Fig.
3. The N-terminal fragments were tagged
with V5 epitope, while the C-terminal fragment was tagged with Xpress
epitope. After 24 h of expression followed by 4 h of labeling
with [3H]palmitic acid, COS7 cells were harvested and
immunoprecipitated with both anti-Xpress and anti-V5 antibodies. The
immunoprecipitates were analyzed by Western blotting (Fig.
3A) with both anti-V5 and anti-Xpress antibodies to probe
the expression of the N- and C-terminal halves of rPLD1. The
palmitoylation was analyzed by fluorography to detect tritium
incorporation (Fig. 3B). As shown in Fig. 3B
(lane 4), tritium incorporation was detected in
the ~66-kDa band, which corresponds to the size of the N-terminal half of rPLD1 when COS7 cells were cotransfected with the wild type N-
and C-terminal halves. Mutation of Ile470 in the N-terminal
half or of Ile870 in the C-terminal half of rPLD1 has been
shown to disrupt the interdomain association (29). The interdomain
interaction was almost abolished when the Ser873 in the
C-terminal half was mutated to alanine (Fig. 3, C and D). When COS7 cells were transfected with the N- and
C-terminal fragments carrying these mutations, no apparent tritium
incorporation was detected in the N-terminal fragment of rPLD1 (Fig.
3B). Thus, the interdomain interaction between the N- and C-terminal
halves of rPLD1 appears to be required for the palmitoylation of the enzyme. In addition, we found that mutation of both Cys240
and Cys241 in the N-terminal half of rPLD1 also diminished
the tritium incorporation (Fig. 3B), which is consistent
with the finding with full-length rPLD1 (Fig. 2). However, single
mutation of Cys240 or Cys241 did not abolish
the palmitoylation (data not shown), suggesting that
palmitoyltransferase can act on either residue.
Cysteines 240 and 241 Are Required for Extensive Ser/Thr
Phosphorylation of rPLD1--
The preceding results (Fig. 3) showed
that the interdomain association between the N- and C-terminal
fragments is important for palmitoylation. We have previously shown
that this association is also required for Ser/Thr phosphorylation
modification of the enzyme (29), and the phosphorylation has been
reported to play roles in locating the enzymes and responding to PMA
activation (34). We therefore wondered whether these two types of
posttranslational modification of rPLD1 were interrelated. As shown in
the Western blot analysis in Figs. 2A* and 5B, we
found that rPLD1(C240A,C241A) that lacked palmitoylation also lacked
extensive Ser/Thr phosphorylation, as demonstrated by the disappearance
of the bands with slower electrophoretic mobility as compared with wild
type rPLD1 or those with single mutations. In addition, bands with
slower electrophoretic mobility were also not observed in the
N-terminal deletion mutants, rPLD1-(169-1036) and rPLD1-(320-1036),
which both lacked palmitoylation (Fig. 2A*). However,
rPLD1-(51-1036) that still retained palmitoylation retained extensive
Ser/Thr phosphorylation (Fig. 2A*). Furthermore, when wild
type and indicated mutant rPLD1 constructs (Fig.
4A) were analyzed by Western
blotting with anti-Thr(P) antibodies, we found similar results.
A 120-kDa band corresponding to the size of full-length rPLD1 was
detected by anti-Thr(P) antibodies in wild type or single Cys-mutated
rPLD1 molecules that still retained palmitoylation. However, no
apparent band was detected by the antibodies when the
palmitoylation-deficient mutant, rPLD1(C240A,C241A), was analyzed. In
summary, these results suggested that palmitoylation of rPLD1 is
required for extensive Ser/Thr phosphorylation of the enzyme. On the
other hand, we found that [3H]palmitate was incorporated
into rPLD1 molecules with either slow or fast electrophoretic mobility
(i.e. both upper and lower bands) when analyzed by Tritium
Screen (Fig. 4, B and C). Thus, extensive Ser/Thr
phosphorylation appeared not to be required for palmitoylation of
rPLD1.
Palmitoylation of rPLD1 Is Not Required for Its Response to PKC in
COS7 Cells--
We noticed that rPLD1-(169-1036) and
rPLD1-(320-1036), which lacked the palmitoylation and extensive
Ser/Thr phosphorylation, did not respond to PMA stimulation, which
activates PKC in COS7 cells. rPLD1-(51-1036), on the other hand, which
retained both of the posttranslational modifications, responded to PMA
stimulation. The lack of the response to PKC of the N-terminal
truncation mutants could be due to the lack of N-terminal amino acids
or the lack the posttranslational modifications. We therefore examined
whether full-length mutant rPLD1(C240A,C241A) that lacks these
posttranslational modifications could respond to PMA stimulation in
COS7 cells. The cells were transfected with wild type or the indicated
cysteine mutants (Fig. 5), and in
vivo PLD assays were carried out by measuring the
transphosphatidylation activity as described under "Experimental Procedures." As shown in Fig. 5A, although the basal
activity of rPLD1(C240A,C241A)was decreased by about 60% compared with wild type rPLD1, it still retained a significant response to PMA stimulation. The single Cys mutant, rPLD1(C241A), retained wild type
activity, while the basal activity and PMA response of of rPLD1(C240A)
were reduced by about 30%. The expression levels of these wild type
and mutant rPLD1 constructs in COS7 cells were comparable (Fig.
5B). Thus, palmitoylation (Fig. 2) and extensive Ser/Thr
phosphorylation of rPLD1 (Fig. 5B) are not absolutely required for PKC stimulation of the enzyme. The finding that extensive Ser/Thr phosphorylation is not required for the rPLD1 response to PKC
in vivo is consistent with our studies in vitro
(29).
Palmitoylation of rPLD1 Affects the Association Affinity of the
Enzyme with Membranes--
Palmitoylation has been shown to play a
role in membrane association of proteins (35). Here we utilized
biochemical fractionation to examine the effect of the palmitoylation
of rPLD1 on its membrane association. As described under the
"Experimental Procedures," membrane fractions of COS7 cells
expressing either wild type or mutant rPLD1 were first isolated under
the hypotonic conditions, where no salt or detergent was added to the
extraction buffer. The resulting membrane fractions were resuspended
and incubated with the extraction buffer containing 1 M
NaCl, 0.1 M Na2CO3 (pH 11), or 1%
Triton X-100 at 4 °C for 30 min and then centrifuged to separate the
membrane and supernatant fractions. As shown in Fig.
6, wild type rPLD1 remained
membrane-associated when extracted with hypotonic buffer, 1 M NaCl, or 0.1 M
Na2CO3 (pH 11). About 10% of wild type rPLD1
was released into supernatant when the membrane fractions were further
extracted with 1% Triton X-100. rPLD1 mutants that carried a single
cysteine mutation at either position 240 or 241 also remained
associated with the membrane fraction when extracted with hypotonic
buffer (Fig. 6A, lanes 7 and
10) and were minimally released when the membranes
were extracted with either 1 M NaCl, 0.1 M Na2CO3 (pH 11) or 1% Triton X-100 (Fig. 6, A-C, lanes 8 and
11). However, when palmitoylation-deficient rPLD1(C240A,C241A) was analyzed, we found that ~10% of the protein was present in the cytosolic fraction (Fig. 6A,
lane 4). When the membrane fractions were further
extracted, we found an additional 20-30% of rPLD1(C240A,C241A) was
released by 1 M NaCl or 1% of Triton X-100 (Fig. 6,
A and C, lane 5). However,
when the membrane fraction was further extracted with 0.1 M
Na2CO3 (pH 11), an additional 60% was released
(Fig. 6B, lane 5).
Na2CO3 (pH 11), at a concentration of 0.1 M, primarily acts to denature protein structure without disrupting the organization of the bilayer and, thus, often dissociates peripheral membrane proteins from membranes (36). These results indicate that although the palmitoylation-deficient rPLD1 can associate
with membranes, its association is weakened compared with wild type
enzyme.
Alanine Mutation of Cysteine 310 or 612 Dramatically Elevates Basal
rPLD1 Activity in Vivo--
During our investigation of the role of
palmitoylation of rPLD1, we also generated multiple Cys mutations that
did not affect palmitoylation of the enzyme. We transfected these
mutants into COS7 cells and examined the effect of the mutations on the
basal PLD activity and the response to PMA stimulation in
vivo. We found that mutation of Cys247 or
Cys403 to Ala did not affect the basal activity of rPLD1 or
its response to PMA. Mutation of Cys79 to Ala increased the
basal PLD activity ~40%. When Cys310 or
Cys612 was mutated to Ala, the basal PLD activity was
increased ~2- and 4-fold, respectively (Fig.
7A). These two mutants still
responded to PMA stimulation, but the -fold increase was lower compared with wild type rPLD1 (Fig. 7B). The high basal activity of
these mutants was not due to their higher expression level in COS7
cells (Fig. 7C). These data suggest that Cys310
and Cys612 play roles in regulating the catalytic activity
of rPLD1, but the detailed mechanisms need further investigation.
Our previous studies of rPLD1 showed that the N- and C-terminal
halves of the enzyme could associate in vivo to restore PLD activity when cotransfected (11). The interdomain association between
the N- and C-terminal halves of rPLD1 is also required for Ser/Thr
phosphorylation of the enzyme (29). In this study, we found that rPLD1
can be modified by palmitoylation, and this lipid acylation also
requires the association of the N- and C-terminal halves of
rPLD1.2 A previous study of
the palmitoylation of human PLD1 showed that mutation of
Ser911 to alanine abolished the lipid modification of the
enzyme (33); however, the mechanism was not elucidated. Here, we found
that mutation of the corresponding Ser873 in rPLD1 to
alanine disrupted the interdomain association between the N- and
C-terminal halves (Fig. 3D), which is the likely reason for
the diminished palmitoylation of this mutant enzyme (Fig. 3A). Examination of the palmitoylation of the N- and
C-terminal halves showed that the lipid modification occurred on the
N-terminal half. We mutated all of the Cys residues in the N-terminal
half except Cys55, since this residue is not conserved
between human and rat PLD1. Studies of all the Cys mutants in the
N-terminal half of rPLD1 showed that only mutation of both
Cys240 and Cys241 abolished the palmitoylation
of rPLD1. Thus, Cys240 and Cys241 are the
probable sites for the palmitoylation of rPLD1 (Fig. 2 and 4).
An important finding in the present study is that palmitoylation
of rPLD1 is not necessary for its catalytic activity. This is because
the N-terminal deletion mutants, rPLD1-(169-1036) and rPLD1-(320-1036), that lack the palmitoylation modification (Fig. 2)
exhibited higher basal activity compared with wild type enzyme (10,
11). These results suggested that palmitoylation is not required for
catalytic activity of rPLD1. However, the palmitoylation-deficient mutant, rPLD1(C240A,C241A), showed a reduced basal catalytic activity compared with wild type enzyme (Fig. 5), which is in agreement with the
in vivo finding of Sugars et al. (32). One
explanation for this difference may be the presence of the N-terminal
sequence in the full-length palmitoylation-deficient mutant. The
N-terminal 168 amino acids have been shown to be suppressive to the
basal activity but are required for the PKC activation of rPLD1 (10, 11). It is possible that in the full-length enzyme, palmitoylation of
rPLD1 may relieve part of the suppressive effect mediated by the
N-terminal sequence in vivo, and this effect is not seen
when the inhibitory N-terminal fragment of rPLD1 is deleted. Proof of
this hypothesis will require detailed studies on how the N-terminal sequence regulates the activity of PLD1. In addition, the finding that
the palmitoylation-deficient full-length human PLD1 had wild type
activity when assayed in vitro (32) again suggests that this
lipid modification is not essential for the catalysis of PLD1 but may
play some regulatory role in vivo. It is also possible that
lack of Cys residues instead of lack of palmitoylation is partly
responsible for the changes in the properties of the PLD mutant, such
as the dramatically reduced basal activity of the double Cys mutant.
The fact that rPLD1-(320-1036), which lacks both Cys240
and Cys241, still exhibits high basal activity (Refs. 10
and 11) suggests that lack of the Cys residues may not be solely
responsible for the changes in PLD activity, although the possibility
still exists that lack of Cys240 and Cys241 may
have different effects depending on the presence or absence of the
N-terminal amino acid. A related question is whether or not
palmitoylation of rPLD1 requires an active enzyme. In Fig. 1, we showed
that a C-terminal deletion mutant of rPLD1 that lacks the last four
amino acids, EVWT, is palmitoylated. However, it is completely inactive
when assayed in vivo or in vitro for
transphosphatidylation or choline release activity (31).
It is interesting to find that rPLD1 can be modified by both
Ser/Thr phosphorylation and palmitoylation and that these two post-translational modifications are interrelated (Ref. 29 and this
study). Both of the modifications require the interdomain association
between the N- and C-terminal halves of rPLD1 and occur in the
N-terminal half of the enzyme (Fig. 3 and Ref. 11). Correlation of
palmitoylation and phosphorylation has been documented in the studies
of the rat bradykinin B2 receptor in Chinese hamster ovary cells (37),
although palmitoylation and phosphorylation of the receptor were found
to be mutually exclusive. In the case with rPLD1, we found that
palmitoylation of rPLD1 is, on the contrary, required for an extensive
Ser/Thr phosphorylation of the enzyme (Fig. 4). N-terminally truncated
rPLD1 or full-length rPLD1(C240A,C241A) that lacked palmitoylation also
lacked extensive Ser/Thr phosphorylation. However, analysis of wild
type and various other rPLD1 constructs showed that extensive Ser/Thr
phosphorylation of the enzyme is not necessary for its palmitoylation
(Fig. 4). These results suggest that palmitoylation and phosphorylation
of rPLD1 are carried out sequentially. Palmitoylation of rPLD1 may help
to specify its location in a membrane fraction, where the kinase is
available for phosphorylation of the enzyme. Our previous finding that
the Ser/Thr-phosphorylated rPLD1 is detected exclusively in the
membrane fraction (29) is consistent with our current findings and presumptions.
It was surprising to find that rPLD1 that lacked the N-terminal 168 amino acids was not palmitoylated in COS7 cells, since both
Cys240 and Cys241 are present in the molecule.
rPLD1 that lacks the first 50 amino acids exhibits wild type enzyme
properties (Refs. 10 and 11 and this study). Therefore, it is likely
that amino acids 50-168 are important for the lipid modification.
These amino acids have also been proposed to play important roles in
regulating the properties of rPLD1 (Refs. 10 and 11 and Fig. 2 of this
study), which include inhibition of the basal activity, mediation of
the activation by PKC, and Ser/Thr phosphorylation of the enzyme. The
N-terminal 168 amino acids may be involved in maintaining a structural
feature that is important for the regulation; thus, truncation of these amino acids could cause a conformational change that makes the phosphorylation or palmitoylation sites inaccessible to the kinase or
palmitoyltransferase. It is also possible that these amino acids may be
directly involved in recruiting the enzymes that modify rPLD1, since a
potential PX domain has been defined for human PLD1 within these amino
acids, which is usually involved in protein-protein interactions (12).
The possibility also exists that the N-terminal Xpress epitope tag is
close to the palmitoylation sites and thus suppresses the lipid modification.
Characterization of both the phosphorylation and palmitoylation
suggests that both of the posttranslational modifications are not
required for the catalytic activity of the enzyme. However, these
modifications may play roles in membrane association of the enzyme. Our
previous studies showed that Ser/Thr-phosphorylated rPLD1 locates
exclusively in the membrane fraction (29). In this study, we found that
although the palmitoylation-deficient rPLD1(C240A,C241A) can associate
with the membrane, the association is weakened compared with wild type
enzyme. The palmitate group by itself has been shown to promote more
stable membrane association of a peptide (38). It is possible that
palmitoylation of rPLD1 plays a role in tethering the enzyme to
membranes by increasing the hydrophobicity of the enzyme. The fact that
palmitoylation-deficient rPLD1(C240A,C241A) is partially released from
the membrane fraction by 1 M NaCl treatment suggests that
in addition to hydrophobic interactions, ionic interactions may
contribute to the association of rPLD1 with membranes. We also found
that rPLD1(C240A,C241A) was very sensitive to treatment with 0.1 M Na2CO3 (pH 11), and less than
30% of this double cysteine mutant remained membrane-associated after
this treatment (Fig. 6B). Na2CO3 (pH
11) at a concentration of 0.1 M primarily acts to denature
protein structure without disrupting the organization of the bilayer
and, thus, often dissociates peripheral membrane proteins from
membranes (36). Since wild type rPLD1 remains membrane-associated when
treated with 0.1 M Na2CO3 (pH 11)
(Fig. 6B), it seems that palmitoylation may play a role in
membrane association of the enzyme. This function of palmitoylation has
been documented for PSD-95 (postsynaptic
density-95), a protein enriched in brain that mediates
synaptic plasticity. Wild type PSD-95 has been shown to partition as an
integral membrane protein, and this membrane association of PSD-95
requires palmitoylation at N-terminal cysteines (39).
In addition to its role in membrane association, palmitoylation helps
specify the membrane location of many proteins (35). PLD activity has
been detected in many subcellular membranes, including the nuclear
envelope, endoplasmic reticulum, Golgi apparatus, secretory vesicles,
plasma membrane, and a specific subdomain on plasma membranes, the
caveolae (40). Studies of palmitoylation-deficient human PLD1 showed
that the double Cys mutant was located at the plasma membrane, while
the wild type PLD1 was located on intracellular membranes (32). It is
possible that, although palmitoylation and Ser/Thr phosphorylation may
not be essential for the catalytic activity of rPLD1, they may help to
specify its location within the cell. Interestingly, like N-terminally
truncated rPLD1, N-terminally truncated SPO14, a yeast PLD isoform,
also lacks a Ser/Thr phosphorylation modification, while the wild type
SPO14 is Ser/Thr-phosphorylated (41). This N-terminally deleted SPO14
retains catalytic activity; however, the activity is mislocalized, and
the yeast strain carrying this SPO14 mutant cannot sporulate
(21).
In summary, we have found that rPLD1 is palmitoylated at
Cys240 and Cys241 and that this lipid acylation
requires the presence of the N-terminal 168 amino acids and the
interdomain association of the N- and C-terminal halves of rPLD1.
Although palmitoylation of rPLD1 is not essential for its catalytic
activity or its response to PKC, it is required for an extensive
Ser/Thr phosphorylation modification of the enzyme and its stable
association with membranes. In addition, we found that the cysteine
residues, Cys310 and Cys612, play important
roles in regulating the enzymatic activity of rPLD1. However, the
biochemical mechanisms of these effects and the physiological functions
of palmitoylation and Ser/Thr phosphorylation of rPLD1 require further investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin (16-20).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Phorbol 12-myristate 13-acetate,
phosphatidylinositol 4,5-bisphosphate, bovine serum albumin, Triton
X-100, and microcystin were from Sigma. Phosphatidylethanolamine,
PC, and phosphatidylbutanol standard were from Avanti Polar
Lipids Corp. Dipalmitoyl[2-palmitoyl-9,10-3H]PC,
dipalmitoyl[choline-methyl-3H]PC,
[3H]myristic acid, and [3H]palmitic acid
were from PerkinElmer Life Sciences. Protein A-agarose beads,
Dulbecco's modified Eagle's medium, penicillin, streptomycin, and
fetal bovine serum were from Life Technologies, Inc. The transfection reagent FuGENE6 and the protease inhibitor mixture were from Roche Molecular Biochemicals. COS7 cells were from the American Type Culture
Collection. SDS-polyacrylamide gels were from Novex. The PcDNA3
vectors and the monoclonal antibodies against the V5 and Xpress epitope
tags were from Invitrogen. Anti-mouse antibodies conjugated with
horseradish peroxidase were from Vector Laboratories. The
AmplifyTM and Hyperfilm-MP were from Amersham Pharmacia Biotech.
80 °C. For hydroxylamine treatment,
duplicate gels were treated with either 1 M Tris-HCl (pH
7.0) or 1 M hydroxylamine (pH 7.0) overnight at room
temperature and then subjected to fluorography as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (30K):
[in a new window]
Fig. 1.
Palmitoylation of rPLD1. Wild type or
C-terminally truncated rPLD1 was transfected into COS7 cells as
indicated in the figure. After 24 h of expression, COS7
cells were labeled with [3H]palmitic acid for 4 h
and harvested and immunoprecipitated by anti-Xpress antibodies. The
immunoprecipitates were then analyzed either by SDS-PAGE followed by
Western blotting with anti-Xpress antibodies (A) or by
fluorography (exposed at 80 °C for 30 days) (B) as
described under "Experimental Procedures."
View larger version (29K):
[in a new window]
Fig. 2.
Requirement of the first 168 amino acids for
palmitoylation of rPLD1. Wild type or site-directed or deletion
mutants of rPLD1 were transfected into COS7 cells as indicated in the
figure. After 24 h of expression, COS7 cells were
labeled with [3H]palmitic acid for 4 h and harvested
and immunoprecipitated by anti-Xpress antibodies. A, the
immunoprecipitates were analyzed by SDS-PAGE followed by Western
blotting with anti-Xpress antibodies. A*, to obtain a
comparable ECL signal so that single and double bands of different
constructs could be visualized, the same samples in A were
loaded at different amounts and reanalyzed by SDS-PAGE followed by
Western blotting with anti-Xpress antibodies. B, after the
immunoprecipitates were analyzed by SDS-PAGE, the gel was first
incubated with 1 M Tris-HCl (pH 7.0) overnight and then
analyzed by fluorography (exposed at 80 °C for 40 days).
C, after the immunoprecipitates were analyzed by SDS-PAGE,
the gel was incubated with 1 M hydroxylamine (pH 7.0)
overnight and then analyzed by fluorography as described under
"Experimental Procedures."
View larger version (36K):
[in a new window]
Fig. 3.
Requirement of interdomain association for
palmitoylation of rPLD1. Wild type or the indicated mutant N- and
C-terminal halves of rPLD1 were cotransfected into COS7 cells. The
N-terminal half of rPLD1 was tagged with a V5 epitope, while the
C-terminal half was tagged with an Xpress epitope. After 24 h of
expression, COS7 cells were labeled with [3H]palmitic
acid for 4 h and harvested and immunoprecipitated by anti-Xpress
and anti-V5 antibodies. A, the immunoprecipitates were then
analyzed by SDS-PAGE followed by Western blotting with anti-V5 and
anti-Xpress antibodies. B, the immunoprecipitates were
analyzed by SDS-PAGE and then analyzed by fluorography (exposed at
80 °C for 60 days). The C-terminally V5-tagged N-terminal fragment
of rPLD1-(1-584) was coexpressed either with
-galactosidase tagged
with Xpress epitope at the N terminus (LacZ) (lane
1), with the N-terminally Xpress-tagged C-terminal fragment
of rPLD1 (lane 2), or with the C-terminal
fragment of rPLD1 carrying an S873A mutation (lane
3) in COS7 cells. C, the cell lysate was analyzed
by SDS-PAGE and Western blotted with both anti-V5 and anti-Xpress
antibodies. D, the immunoprecipitates by monoclonal
anti-Xpress antibodies were analyzed by SDS-PAGE followed by Western
blotting with anti-V5 antibodies. A*, a darker exposure of
part of the Western blot shown in A, so that the
modification of the wild type N-terminal fragment could be visualized.
NT, C-terminally V5-tagged N-terminal fragment of
rPLD1-(1-584). CT, N-terminally Xpress-tagged C-terminal
fragment of rPLD1-(585-1036).
View larger version (38K):
[in a new window]
Fig. 4.
Palmitoylation of rPLD1 is required for
extensive Ser/Thr phosphorylation of the enzyme. Wild type rPLD1
or the indicated cysteine mutants were transfected into COS7 cells.
After 24 h of expression, the cells were harvested and
immunoprecipitated with anti-Xpress antibodies. The immunoprecipitates
were then analyzed by SDS-PAGE followed by Western blotting with
antiphospho-Thr antibodies (A) or anti-Xpress antibodies
(C). D, after Western blotting and
enhanced chemiluminescence detection, the blot from C
was rinsed with PBS, air-dried, and exposed to Tritium Screen at
room temperature for 10 days and scanned by the
StormTM 860 detection system from Molecular Dynamics, Inc.
(Sunnyvale, CA).
View larger version (21K):
[in a new window]
Fig. 5.
Palmitoylation of rPLD1 is not required for
its activation by PKC. Wild type rPLD1 or the indicated cysteine
mutants were transfected into COS7 cells, and the PLD activity
(A) was measured in the absence or presence of 100 nM PMA treatment for 20 min as described under
"Experimental Procedures." The PLD activity is represented by the
percentage of the phosphatidylbutanol synthesized versus the
total tritium incorporated into the lipid of the COS7 cells. The
results are representative of three experiments performed in
triplicate. Mean values ± S.E. are shown. The gray and
black bars represent the PLD activity in the
absence and presence of PMA treatment, respectively. The corresponding
cell lysate was analyzed by Western blotting (B) with
anti-Xpress antibodies to examine the expression level of wild type or
mutant rPLD1 constructs in COS7 cells.
View larger version (43K):
[in a new window]
Fig. 6.
Membrane association of
palmitoylation-deficient rPLD1. Wild type rPLD1 or the indicated
cysteine mutants were transfected into COS7 cells. After 24 h of
expression, the cells were harvested, fractionated by hypotonic buffer,
and ultracentrifuged to separate the cytosol and crude membrane
fractions. The crude membrane fractions were then further extracted by
incubation with either 1 M NaCl (A), 0.1 M Na2CO3 (B), or 1%
Triton X-100 (C) for 30 min at 4 °C and ultracentrifuged
to separate the supernatant and pellet fractions. Equal volumes of the
cytosol and membrane fractions were analyzed by Western blotting with
anti-Xpress antibodies as described under "Experimental
Procedures." The intensity of the bands corresponding to rPLD1 in the
cytosol or membrane fractions was quantitated by densitometry. The
amounts of the indicated rPLD1 constructs in the cytosol or membrane
fractions were calculated as percentage of the total enzyme in both.
C1, the cytosol fraction obtained from the first
fractionation using the hypotonic buffer; C2, the
supernatant fraction obtained from the additional extraction with
indicated salt or detergent. M, the resulting membrane
fraction after the two consecutive fractionations with hypotonic buffer
and the indicated salt or detergent.
View larger version (17K):
[in a new window]
Fig. 7.
Effect of alanine mutation of
Cys310 and Cys612 of rPLD1 on Its Catalytic
Activity. Wild type rPLD1 or indicated cysteine mutants were
transfected into COS7 cells, and the PLD activity was measured in the
absence or presence of 100 nM PMA as described under
"Experimental Procedures." The PLD activity is represented by the
percentage of the phosphatidylbutanol synthesized versus the
total tritium incorporated into the lipid of the COS7 cells. The
results are representative of three experiments performed in
triplicate. Mean values ± S.E. are shown. A, the PLD
activity in the absence of stimulation. B, the PLD activity
in the absence (gray bars) or presence
(black bars) of PMA treatment for 20 min.
C, the corresponding cell lysate was analyzed by Western
blotting with anti-Xpress antibodies to examine the expression level of
wild type or mutant rPLD1 constructs in COS7 cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENT |
---|
We thank Judy Nixon for typing the manuscript.
![]() |
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.
Investigator of the Howard Hughes Medical Institute. To whom all
correspondence should be addressed. Tel.: 615-322-6494; Fax: 615-322-4381; E-mail: john.exton@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M009425200
2 Since palmitic acid can be converted to other fatty acids and metabolites in cells, the identity of the acyl group(s) incorporated into rPLD1 cannot be stated with certainty in the absence of further studies requiring a large amount of enzyme protein. The involvement of cysteine residues and the susceptibility to hydroxylamine indicate a thioester linkage, and the rapid labeling of human PLD1 in COS cells incubated with [3H]palmitic acid (33) suggests that the fatty acid may be incorporated intact.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PLD, phospholipase D; rPLD, rat PLD; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PMA, phorbol 12-myristate 13-acetate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Exton, J. H. (1998) Biochim. Biophys. Acta 1436, 105-115[Medline] [Order article via Infotrieve] |
2. |
Exton, J. H.
(1997)
Physiol. Rev.
77,
303-320 |
3. | Houle, M. G., and Bourgoin, S. (1999) Biochim. Biophys. Acta 1439, 135-150[Medline] [Order article via Infotrieve] |
4. | Exton, J. H. (1999) Biochim. Biophys. Acta 1439, 121-133[Medline] [Order article via Infotrieve] |
5. |
Zhang, Y.,
Redina, O.,
Altshuller, Y. M.,
Yamazaki, M.,
Ramos, J.,
Chneiweiss, H.,
Kanaho, Y.,
and Frohman, M. A.
(2000)
J. Biol. Chem.
275,
35224-35232 |
6. |
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 |
7. |
Hammond, S. M.,
Jenco, J. M.,
Nakashima, S.,
Cadwallader, K.,
Gu, G.,
Cook, S.,
Nozawa, Y.,
Prestwich, G. D.,
Frohman, M. A.,
and Morris, A. J.
(1997)
J. Biol. Chem.
272,
3860-3868 |
8. | Park, S.-K., Provost, J. P., Bae, C. D., Ho, W.-T., and Exton, J. H. (1997) J. Biol. Chem. 272, 29268-29271 |
9. | Colley, W. C., Altshuller, Y. M., Sue-Ling, C. K., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Branch, K. D., Tsirka, S. E., Bollag, R. J., Bollag, W. B., and Frohman, M. A. (1997) Biochem. J. 326, 745-753[Medline] [Order article via Infotrieve] |
10. | Park, S.-K., Min, D. S., and Exton, J. H. (1998) Biochem. Biophys. Res. Commun. 244, 364-367[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Xie, Z.,
Ho, W.-T.,
and Exton, J. H.
(1998)
J. Biol. Chem.
273,
34679-34682 |
12. |
Sung, T. C.,
Zhang, Y.,
Morris, A. J.,
and Frohman, M. A.
(1999)
J. Biol. Chem.
274,
3659-3666 |
13. |
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 |
14. |
Kodaki, T.,
and Yamashita, S.
(1997)
J. Biol. Chem.
272,
11408-11413 |
15. | Colley, W. C., Sung, T.-C., Roll, R., Jenco, J., Hamond, 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] |
16. |
Lopez, I.,
Arnold, R. S.,
and Lambeth, J. D.
(1998)
J. Biol. Chem.
273,
12846-12852 |
17. | Siddiqi, A. R., Srajer, G. E., and Leslie, C. C. (2000) Biochim. Biophys. Acta 1497, 103-114[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Park, J. B.,
Kim, J. H.,
Kim, Y.,
Ha, S. H.,
Yoo, J. S.,
Du, G.,
Frohman, M. A.,
Suh, P. G.,
and Ryu, S. H.
(2000)
J. Biol. Chem.
275,
21295-21301 |
19. | Watanabe, H., and Kanaho, Y. (2000) Biochim. Biophys. Acta 1495, 121-124[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Slaaby, R.,
Jensen, T.,
Hansen, H. S.,
Frohman, M. A.,
and Seedorf, K.
(1998)
J. Biol. Chem.
273,
33722-33727 |
21. |
Rudge, S. A.,
Morris, A. J.,
and Engebrecht, J.
(1998)
J. Cell Biol.
140,
81-90 |
22. | Morris, A. J., Engebrecht, J., and Frohman, M. A. (1996) Trends Pharmacol. Sci. 17, 182-185[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Ponting, C. P.,
and Kerr, I. D.
(1996)
Protein Sci.
5,
914-922 |
24. | Koonin, E. G. (1996) Trends Biochem. Sci. 21, 242-243[CrossRef][Medline] [Order article via Infotrieve] |
25. |
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 |
26. |
Rudolph, A. E.,
Stuckey, J. A.,
Zhao, Y.,
Matthews, H. R.,
Patton, W. A.,
Moss, J.,
and Dixon, J. E.
(1999)
J. Biol. Chem.
274,
11824-11831 |
27. | Stuckey, J. A., and Dixon, J. E. (1999) Nat. Struct. Biol. 6, 278-284[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Gottlin, E. B.,
Rudolph, A. E.,
Zhao, Y.,
Matthews, H. R.,
and Dixon, J. E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9202-9207 |
29. |
Xie, Z.,
Ho, W.-T.,
and Exton, J. H.
(2000)
J. Biol. Chem.
275,
24962-24969 |
30. |
Malcolm, K. C.,
Elliott, C. M.,
and Exton, J. H.
(1996)
J. Biol. Chem.
271,
13135-13139 |
31. |
Xie, Z.,
Ho, W.-T.,
and Exton, J. H.
(2000)
Eur. J. Biochem.
267,
7138-7146 |
32. |
Sugars, J. M.,
Cellek, S.,
Manifava, M.,
Coadwell, J.,
and Ktistakis, N. T.
(1999)
J. Biol. Chem.
274,
30023-30027 |
33. |
Manifava, M.,
Sugars, J.,
and Ktistakis, N. T.
(1999)
J. Biol. Chem.
274,
1072-1077 |
34. |
Kim, Y.,
Han, J. M.,
Han, B. R.,
Lee, K. A.,
Kim, J. H.,
Lee, B. D.,
Jang, I. H.,
Suh, P. G.,
and Ryu, S. H.
(2000)
J. Biol. Chem.
275,
13621-13627 |
35. | Dunphy, J. T., and Linder, M. E. (1998) Biochim. Biophys. Acta 1436, 245-261[Medline] [Order article via Infotrieve] |
36. | Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P. B. (1982) J. Cell Biol. 93, 97-102[Abstract] |
37. |
Soskic, V.,
Nyakatura, E.,
Roos, M.,
Muller-Esterl, W.,
and Godovac-Zimmermann, J.
(1999)
J. Biol. Chem.
274,
8539-8545 |
38. | Peitzsch, R. M., and McLaughlin, S. (1993) Biochemistry 32, 10436-10443[Medline] [Order article via Infotrieve] |
39. | Topinka, J. R., and Bret, D. S. (1998) Neuron 20, 125-134[Medline] [Order article via Infotrieve] |
40. | Liscovitch, M., Czarny, M., Fiucci, G., Lavie, Y., and Tang, X. (1999) Biochim. Biophys. Acta 1439, 245-263[Medline] [Order article via Infotrieve] |
41. | Rudge, S. A., and Engebrecht, J. (1999) Biochim. Biophys. Acta 1439, 167-174[Medline] [Order article via Infotrieve] |