COMMUNICATION
Interaction of the Small G Protein RhoA with the C Terminus of
Human Phospholipase D1*
Masakazu
Yamazaki
,
Yue
Zhang§,
Hiroshi
Watanabe
,
Takeaki
Yokozeki
¶,
Sigeo
Ohno
,
Kozo
Kaibuchi**,
Hideki
Shibata
,
Hideyuki
Mukai
,
Yoshitaka
Ono
,
Michael
A.
Frohman§, and
Yasunori
Kanaho
§§
From the
Department of Life Science, Tokyo
Institute of Technology, Yokohama 226-8501, the
Department
of Molecular Biology, Yokohama City University School of Medicine,
Yokohama 236-0004, the ** Division of Signal Transduction, Nara
Institute of Science and Technology, 8916-5 Takayama, Ikoma
630-0101, the 
Department of Biology,
Faculty of Science, Kobe University, Kobe 657-8501, Japan and the
§ Department of Pharmacological Sciences and the Institute
for Cell and Developmental Biology and the Program for Molecular and
Cellular Biology, State University of New York at Stony Brook,
Stony Brook, New York 11794-8651
 |
ABSTRACT |
Mammalian phosphatidylcholine-specific
phospholipase D1 (PLD1) is a signal transduction-activated enzyme
thought to function in multiple cell biological settings including the
regulation of membrane vesicular trafficking. PLD1 is activated by the
small G proteins, ADP-ribosylation factor (ARF) and RhoA, and by
protein kinase C-
(PKC-
). This stimulation has been proposed to
involve direct interaction and to take place at a distinct site in PLD1 for each activator. In the present study, we employed the yeast two-hybrid system to attempt to identify these sites. Successful interaction of ARF and PKC-
with PLD1 was not achieved, but a C-terminal fragment of human PLD1 (denoted "D4") interacted with the active mutant of RhoA, RhoAVal-14. Deletion of
the CAAX box from RhoAVal-14 decreased the
strength of the interaction, suggesting that lipid modification of RhoA
is important for efficient binding to PLD1. The specificity of the
interaction was validated by showing that the PLD1 D4 fragment
interacts with glutathione S-transferase-RhoA in
vitro in a GTP-dependent manner and that it
associates with RhoAVal-14 in COS-7 cells, whereas the
N-terminal two-thirds of PLD1 does not. Finally, we show that
recombinant D4 peptide inhibits RhoA-stimulated PLD1 activation but not
ARF- or PKC-
-stimulated PLD1 activation. These results conclusively
demonstrate that the C-terminal region of PLD1 contains the
RhoA-binding site and suggest that the ARF and PKC interactions occur
elsewhere in the protein.
 |
INTRODUCTION |
Phospholipase D (PLD)1
activity is present in many types of mammalian cells and tissues and is
up-regulated in response to a wide variety of agonists (1, 2). PLD
catalyzes the hydrolysis of phosphatidylcholine (PC) to yield
phosphatidic acid (PA) and choline (2). PA can be further metabolized
to diacylglycerol, the activator of protein kinase C (PKC), or to
lyso-PA, which acts on specific cell-surface receptors (3). PA itself
may play a role(s) as a second messenger; PA has been shown in
vitro to stimulate phosphatidylinositol 4-phosphate 5-kinase (4), PKC-
(5), and protein-tyrosine phosphatase (6). It has also been
reported that PA promotes coatomer binding (7) and enhances actin
polymerization (8).
Studies in recent years have identified several PLD activators,
including many members of the ADP-ribosylation factor (ARF) (9, 10) and
RhoA (11, 12) low molecular weight GTP-binding protein (small G
protein) families and PKC-
(13). In addition, phosphatidylinositol
4,5-bisphosphate (PIP2) has been demonstrated to be
absolutely required as a cofactor for such
activator-dependent PLD stimulation (9).
cDNAs encoding two mammalian PLDs, PLD1 (14-16) and PLD2 (15, 17),
have recently been cloned. Two alternatively spliced forms, termed
PLD1a and PLD1b, exist for PLD1 (18). PLD1a comprises 1074 amino acids,
whereas PLD1b is a shorter form lacking 38 amino acids residues
(585-622). PLD2 is ~50% identical to PLD1 and is constitutively
active in vitro and in vivo (15). In contrast, PLD1a and PLD1b exhibit a low basal activity and are stimulated by ARF,
RhoA, and PKC-
in the presence of PIP2. These findings were demonstrated using purified recombinant PLD1 and activators (18),
which led to the conclusion that PLD1 interacts directly with the lipid
cofactor PIP2 and the protein activators. Since the PKC-
and the small G protein activators elicit a synergistic activation when
combined, it was also proposed that they would mediate PLD1 stimulation
through interactions at separate sites (18). These sites of
interaction, however, have not yet been defined. We address this
question in the present study and show that the C-terminal region of
PLD1 is the RhoA-binding domain.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Phorbol 12-myristate 13-acetate (PMA) was
purchased from Sigma, and phosphatidylethanolamine (PE),
1,2-dipalmitoyl-PC (DPPC), PIP2,
[choline-methyl-3H]DPPC, and
GTP
S were from sources previously reported (12).
Yeast Two-hybrid Analysis--
A template for PCR to generate
cDNA fragments for truncation mutants of human (h) PLD1a was
prepared from total RNA of HL60 cells by reverse transcription with
random hexamer mixed primers, followed by PCR with a set of primers
designed to amplify the entire open reading frame of hPLD1a. The PCR
product was cloned into pT7Blue(R) vector (Novagen). cDNA fragments
encoding amino acid residues 1-415 (D1), 365-727 (D2), 663-1074
(D3), 712-1074 (D4), and 763-1074 (D5) of hPLD1a were generated by
PCR with primers containing 5'-BclI and 3'-NotI
sites, digested with BclI and NotI, and purified.
A cDNA fragment encoding amino acid residues 712-1015 (D6) of
hPLD1a was obtained from the D4 cDNA by digestion with PvuII. The cDNA fragments for D1-D6 were ligated into
the yeast expression vector pVP16 containing the VP16 transcription
activation domain (a generous gift of Dr. Stanley Fields).
The cDNAs encoding wild type and mutants of RhoA were ligated into
the vector pBTM116 containing LexA-binding domain as described previously (19). The expression plasmids were co-transfected into
Saccharomyces cerevisiae strain L40 cells and plated on
synthetic complete media lacking tryptophan and leucine. Interaction
was basically assessed by a filter assay for
-galactosidase activity (20).
Preparation of GST-RhoA and FLAG-tagged D4 Peptide--
Wild
type RhoA was expressed in Escherichia coli as a glutathione
S-transferase fusion protein (GST-RhoA) and purified by glutathione-Sepharose 4B column chromatography. After removal of free
glutathione by Q-Sepharose column chromatography, the purified GST-RhoA
was stored at
80 °C until use.
The D4 peptide was expressed in E. coli as a FLAG
epitope-tagged protein (FLAG-D4). cDNA of the D4 peptide was
amplified by PCR with a sense primer containing
5'-XhoI/EcoRI sites and an antisense primer
containing a 3'-BglII site using pT7Blue(R)-hPLD1a as a
template, digested with XhoI and BglII, and
inserted into the pFLAG MAC vector (Eastman Kodak Co.). FLAG-D4 was
inducibly expressed in E. coli strain BL21 cells at 30 °C
for 3 h with 0.3 mM
isopropyl-
-D-thiogalactopyranoside. After disruption of
cells by sonication and centrifugation (100,000 × g,
30 min), solubilized FLAG-D4 was purified with an anti-FLAG M2 antibody
affinity resin (Kodak) and stored at
80 °C until use.
In Vitro Association Assay--
Five µg of GST-RhoA was
incubated at 30 °C for 10 min with 5 µg of FLAG-D4 in the presence
of 40 µM GDP or GTP
S in a total volume of 50 µl.
GST-RhoA was then precipitated with a glutathione-Sepharose 4B resin in
the presence of 0.3%
n-octyl-
-D-glucopyranoside. FLAG-D4
co-precipitated with GST-RhoA was detected by immunoblotting with an
anti-FLAG M2 antibody (Kodak).
In Vivo Association Assay--
For the expression of FLAG-D4 in
COS-7 cells, the D4 cDNA amplified by PCR as described above was
digested with EcoRI and BglII and cloned into
pTB701-FLAG vector (a generous gift of Drs. S. Kuroda and U. Kikkawa)
(21). To express a FLAG-tagged peptide (FLAG-D(1+2)) which complemented
the FLAG-D3/D4 peptides, a cDNA encoding amino acid residues 1-670
was generated. In brief, an allele of hPLD1b altered through the
insertion of a stop codon at amino acid 671 was used. The allele was
generated using a pentapeptide mutagenesis protocol (22) that will be
described in detail elsewhere. pTB701-FLAG-D(1+2) was constructed by
insertion of the 1.8-kilobase pair BglII-BamHI
fragment of pCGN-D(1+2) into the BglII-BglII gap
of pTB701-FLAG-hPLD1a (see below). pEF-BOS-HA-RhoA and
pEF-BOS-HA-RhoAVal-14 were prepared as described previously
(23). COS-7 cells were co-transfected with pTB701-FLAG-D4 or
pTB701-FLAG-D(1+2) and either pEF-BOS-HA-RhoA or
pEF-BOS-HA-RhoAVal-14 by electroporation. After 48 h,
the cells were lysed in a buffer containing 0.5% Nonidet P-40 and
centrifuged at 10,000 × g for 20 min. FLAG-D4 or
FLAG-D(1+2) was immunoprecipitated from the supernatant with the
anti-FLAG M2 antibody affinity resin, and co-immunoprecipitated
HA-tagged RhoA or RhoAVal-14 was detected by immunoblotting
with an anti-HA monoclonal antibody (16B12, BAbCO).
Preparation of FLAG-hPLD1a, RhoA, ARF, and PKC-
--
To
prepare FLAG-hPLD1a, the cDNA encoding the full-length of hPLD1a
was amplified by PCR with primers containing an MunI site using pT7Blue(R)-hPLD1a as a template, digested with MunI,
and inserted into the pTB701-FLAG vector. The plasmid was transfected into COS-7 cells by electroporation. After being cultured for 48 h, cells were lysed in a buffer containing 1% Nonidet P-40 and
centrifuged (50,000 × g, 30 min), and solubilized
FLAG-hPLD1a was purified with the anti-FLAG M2 antibody affinity resin.
For RhoA purification, bovine brain cytosol was subjected to the
DEAE-Toyopearl 650S and the phenyl-Toyopearl 650M column chromatography
(12). The major peak of RhoA eluted from the latter column (12) was
re-chromatographed on a phenyl-Toyopearl 650M column. To highly purify
ARF, the partially purified ARF from bovine brain cytosol by
DEAE-Sepharose and Sephacryl S-200 column chromatography (12) was then
sequentially subjected to chromatography on a heparin-Sepharose CL-6B
and a phenyl-Toyopearl 650M column. Both the purified small G proteins
were estimated to be greater than 90% pure by SDS-PAGE (data not
shown). Recombinant PKC-
was expressed in and purified from
Sf21 cells as previously reported (24).
PLD Assay--
FLAG-hPLD1a was reconstituted with the purified
RhoA or ARF or recombinant PKC-
in the presence of
PE/PIP2/[choline-methyl-3H]DPPC
vesicles (158.64 µM) in a molar ratio of 16:1.4:1 (9). The reaction also contained the indicated concentrations of the FLAG-D4
peptide. For the activation of FLAG-hPLD1a by PKC-
, 100 nM PMA was included in the reaction. The mixture was
incubated at 37 °C for 30 min in 45 mM Na-Hepes, pH 7.4, 3 mM EGTA, 150 mM KCl, 1 mM
dithiothreitol, 3 mM MgCl2, 2 mM
CaCl2, 40 µM GTP
S, and 1 mg/ml bovine
serum albumin, and then the production of [3H]choline was
determined (12).
Immunoblotting--
Proteins in samples were separated by
SDS-PAGE on 12% gel and transferred to polyvinylidene difluoride
membranes. The membrane was blocked and incubated with the first
antibodies and then with peroxidase-conjugated rabbit anti-mouse IgG
(12). Immunoreactive proteins were detected with an ECL immunoblotting
detection reagent (Amersham Pharmacia Biotech).
Protein Assay--
Protein concentration was determined by the
method of Bradford (25) using bovine serum albumin as a standard.
 |
RESULTS AND DISCUSSION |
RhoA Interacts with the C Terminus of PLD1--
To identify the
PLD1 domains mediating interaction with each activator, we employed the
LexA version of the yeast two-hybrid system (26). Since reporter gene
activation in this system requires import of the fusion proteins into
the nucleus and this nuclear import is often unsuccessful for large
proteins (26), fragments of hPLD1 and the activators (RhoA, ARF, and
PKC-
) were fused to the binding domain of LexA and the herpes
VP16/GAL4 activation domain, respectively. Consistent with our previous
report (27), RhoAVal-14, the active form of RhoA,
successfully interacted with the C terminus of PLD1. However, this was
the only combination that succeeded (data not shown). The
RhoAVal-14/PLD1 C terminus interaction appears likely to be
specific, since the corresponding C-terminal regions of mammalian PLD2
and yeast PLD, which are related in sequence to mammalian PLD1 but do
not respond to RhoA (15), did not activate the yeast reporter genes when co-expressed with RhoAVal-14 (data not shown).
To begin to validate the RhoA-PLD1 interaction, we switched the PLD
fragments to the VP16 activation vector and RhoA to the LexA DNA
binding vector and attempted to subdivide the interacting PLD1 region
(Fig. 1A). Confirming the
results described above, the N-terminal (D1) and central (D2) fragments
did not interact with RhoAVal-14, but the C terminus (D3,
amino acid residues 663-1074, and D4, amino acid residues 712-1074)
did. Further truncation of the D4 fragment from either end (D5, amino
acid residues 763-1074, and D6, amino acid residues 712-1015)
eliminated the interaction. These results demonstrate that a 362-amino
acid C-terminal fragment of hPLD1 constitutes essentially the minimal
fragment capable of interacting with RhoAVal-14 in the
yeast two-hybrid system.

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Fig. 1.
Dissection of the PLD1 interaction with
RhoAVal-14. Yeast L40 cells were co-transfected with
expression vectors encoding fragments of hPLD1a fused to the VP16
activation domain and wild type or mutants of RhoA fused to the LexA
DNA-binding domain. Interaction was assessed using a filter assay for
-galactosidase activity. A, confirmation and refinement
of the RhoA-binding region in hPLD1 using RhoAVal-14 and a
series of hPLD1a peptides. Hatched boxes
(I-IV) in PLD1a (top line) indicate
the regions highly conserved in the PLD superfamily (43). B,
interaction of the hPLD1a D4 fragment with wild type and mutant RhoA.
The results shown are from a single experiment representative of
seven.
|
|
The C-terminal motif CAAX (C, cysteine; A, aliphatic amino
acid; X, any amino acid) found in RhoA family members is
post-translationally modified by geranylgeranylation on the cysteine
residue, which leads to subsequent proteolysis and carboxylmethylation
(28, 29). These post-translational modifications of the Rho GTPases are
important for promoting interaction with their GDP/GTP exchange proteins and effectors (30-32). To examine the role of C-terminal modification of RhoA in the interaction with PLD1 in the yeast two-hybrid system, four types of RhoA (wild type, the CAAX
deletion mutant RhoACLVL
, the active mutant
RhoAVal-14, and the CAAX-deleted active mutant
RhoAVal-14CLVL
) were tested (Fig.
1B). Wild type RhoA and RhoACLVL
both failed
to interact, confirming that PLD1 specifically interacts with the
active form of RhoA. RhoAVal-14CLVL
interacted with PLD1, but the interaction was clearly weaker than that
observed with RhoAVal-14, suggesting that
geranylgeranylation of RhoA strengthens the interaction with PLD1.
RhoA Interacts with the D4 Fragment in Vitro--
An important
control for two-hybrid interactions is to demonstrate successful
association in vitro. This was addressed by mixing
bacterially expressed GST-RhoA with FLAG-D4 in the presence of GDP or
GTP
S and precipitating the GST-RhoA using glutathione-Sepharose (Fig. 2). In the presence of GTP
S,
FLAG-D4 was readily co-precipitated (lane 4), whereas very
little FLAG-D4 was co-precipitated in the presence of GDP (lane
3). GST itself did not interact with FLAG-D4 at all (lanes
1 and 2). Thus, the D4 fragment specifically interacts only with the active form of RhoA in vitro as well.

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Fig. 2.
In vitro interaction of the PLD1 C
terminus with the active form of RhoA. FLAG-D4 expressed in and
purified from E. coli was incubated with GST or GST-RhoA in
the presence of 40 µM GDP or GTP S, following which the
GST-RhoA was precipitated as described under "Experimental
Procedures." The FLAG-D1 and -D2 peptides were expressed poorly in
E. coli, which prevented us from confirming that these
peptides failed to interact with RhoA in vitro. The amount
of FLAG-D4 co-precipitated with GST or the GST-RhoA was detected by
immunoblotting with an anti-FLAG M2 antibody. The results shown are
from a single experiment representative of six performed with
independent preparations of FLAG-D4 and GST-RhoA.
|
|
We previously reported that non-geranylgeranylated RhoA does not
activate rat brain PLD, whereas geranylgeranylated RhoA does (12),
which was recently confirmed by Bae et al. (33). The results
shown in Figs. 1B and 2 nonetheless provide evidence that non-geranylgeranylated RhoA interacts with hPLD1a, although seemingly not as well as geranylgeranylated RhoA (Fig. 1B). These
observations, taken together, suggest that the post-translational
modification of RhoA plays a role not only in the interaction with PLD1
but also, as a separate event, in the subsequent activation of PLD1.
RhoA Interacts with the D4 Fragment in COS-7 Cells--
Another
important control for two-hybrid interactions is to demonstrate
association in a physiological setting. We addressed this by examining
interaction of the D4 peptide with RhoA in COS-7 cells. When FLAG-D4
was co-expressed with HA-RhoA or -RhoAVal-14 in COS-7 cells
and immunoprecipitated, HA-RhoAVal-14 readily
co-immunoprecipitated, whereas there was very little evidence for
interaction of FLAG-D4 with wild type RhoA (Fig. 3A, lanes 5 and
6 in the upper panel). Equivalent
amounts of FLAG-D4 and of RhoA were expressed in each sample, ruling
out differences in protein stability or expression as an explanation
for the finding (Fig. 3A, middle and
lower panels, respectively). The expression levels of FLAG-D1 and -D2 in COS-7 cells were extremely low as compared
with FLAG-D4, which prevented subsequent analysis of their interaction
or lack of interaction with RhoA. However, FLAG-D(1+2), which encodes
amino acids 1-670 of hPLD1b and is thus complementary to FLAG-D3/D4,
was expressed in COS-7 cells to a level comparable with that of FLAG-D4
(Fig. 3B, middle panel). As
anticipated, FLAG-D(1+2) failed to interact with both wild RhoA and
RhoAVal-14 (Fig. 3B, upper
panel). These results in a physiological setting strengthen
the premise that the PLD1 D4 fragment specifically interacts with the
active form of RhoA.

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Fig. 3.
In vivo association of the PLD1 C
terminus with the active form of RhoA. FLAG-D4 (A) and
FLAG-D(1+2) (B) were co-expressed with HA-RhoA or
-RhoAVal-14 in COS-7 cells. After immunoprecipitation of
FLAG-D4 and FLAG-D(1+2) using an anti-FLAG M2 affinity resin, the
amount of RhoA co-immunoprecipitated was detected by immunoblotting
with an anti-HA antibody (upper panel). As a
control, the amounts of FLAG-D4 and FLAG-D(1+2) immunoprecipitated were
visualized in a parallel blot using an anti-FLAG M2 antibody
(middle panel), and the amounts of wild type RhoA
and RhoAVal-14 expressed in the cell lysates were detected
by immunoblotting using an anti-HA antibody (lower
panel). The results shown are from a single experiment
representative of three.
|
|
The D4 Peptide Blocks RhoA Stimulation of PLD1 in
Vitro--
Although the results presented thus far conclusively
demonstrated an interaction between the D4 peptide and the active form of RhoA, they did not indicate whether ARF or PKC-
might also activate PLD1 through interaction with the C terminus. We addressed this by examining the effects of the D4 peptide on PLD1 stimulation by
ARF, RhoA, and PKC-
. The D4 peptide inhibited RhoA-stimulated PLD1
activation in a dose-dependent manner, but not ARF- and
PKC-
-stimulated activation (Fig. 4).
These results suggest that ARF and PKC-
interact with other regions
of PLD1 and also rule out possible sources of nonspecific inhibition
that might otherwise represent alternative mechanisms through which the
D4 peptide could be causing inhibition of RhoA-mediated activation of
PLD1.

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Fig. 4.
Effects of the D4 peptide on activation of
hPLD1a by RhoA, ARF, and PKC- . Purified
FLAG-hPLD1a (8 nM) was incubated at 37 °C for 30 min
with 0.14 µM RhoA, 0.3 µM ARF, or 5.3 nM PKC- in the presence of the indicated concentrations
of the FLAG-D4 peptide, and PLD activity was determined as described
under "Experimental Procedures." The PLD activities stimulated by
RhoA, ARF, and PKC- were 84.12, 38.31, and 12.12 pmol/30 min/assay,
respectively. The PLD activities are presented as a percentage of the
activity stimulated by each activator in the absence of the FLAG-D4
peptide. The error bars represent the differences
of duplicate determinations. The results shown are from a single
experiment representative of three.
|
|
Various proteins, including Rhofillin, PKN, Rhotekin, PRK2, citron, Rho
kinase/p160ROCK/ROK, p140mDia, and myosin-binding subunit, have been
identified as downstream effectors for RhoA (34). The amino acid
sequences of the RhoA-binding domains of Rhofillin, PKN, Rhotekin, and
PRK2 share about 50% homology and have therefore been denoted as the
RhoA effector motif class 1 (REM-1) (35). The RhoA-binding motif of the
other effectors, termed REM-2, does not share homology with REM-1 (36).
Thus, two RhoA-binding motifs have thus far been identified. The nature of the interaction of PLD1 with the Rho family GTPases seems to be
different from those of the already reported Rho-binding effectors. PLD1 appears to be able to interact with many subsets of Rho family members at the same site, as is inferred from the observation that Rac1
and Cdc42 also activate hPLD1a, and their effects are not synergistic
with RhoA or with each other (18). On the other hand, citron and PRK2
interact with RhoA and Rac1, but not with Cdc42 (34, 37, 38), and other
RhoA target proteins, PKN, Rhotekin, p160ROCK, and p140Dia, interact
only with RhoA (35, 39-41). These observations suggest that the
mechanism employed by the PLD1 RhoA-binding motif is different from
that used by REM-1 and REM-2. Not surprisingly, computer analysis using
the CLUSTAL W program (42) reveals that the amino acid sequence of the
RhoA-binding region in PLD1 defined in this study is not significantly
similar to either REM-1 or REM-2 (data not shown), confirming that the
PLD1 interacting site is unique.
 |
ACKNOWLEDGEMENTS |
We are grateful to Drs. Shun'ichi Kuroda and
Ushio Kikkawa for providing the pTB701-FLAG vector and to Dr. Stanley
Fields for providing the pVP16 vector. We also thank Dr. Akira Matsuura for helpful discussions.
 |
FOOTNOTES |
*
This work was supported in part by research grants from the
Ministry of Education, Science, Sports and Culture, Japan, the ONO
Medical Research Foundation, and the Sankyo Foundation of Life Science
(to Y. K.) and by National Institutes of Health Grant GM54813 (to
M. A. F.).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.
¶
Research Fellow of the Japan Society for the Promotion of Science.
§§
To whom correspondence should be addressed. Tel.: 81-45-924-5717;
Fax: 81-45-924-5774; E-mail: ykanaho{at}bio.titech.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
PLD, phospholipase
D;
PC, phosphatidylcholine;
DPPC, 1,2-dipalmitoyl-PC;
PE, phosphatidylethanolamine;
PIP2, phosphatidylinositol
4,5-bisphosphate;
PA, phosphatidic acid;
PMA, phorbol 12-myristate
13-acetate;
small G proteins, low molecular weight GTP-binding
proteins;
ARF, ADP-ribosylation factor;
PKC, protein kinase C;
PCR, polymerase chain reaction;
GTP
S, guanosine
5'-O-(3-thiotriphosphate);
PAGE, polyacrylamide gel
electrophoresis;
REM-1 and REM-2, Rho effector motif class 1 and 2;
GST, glutathione S-transferase;
HA, hemagglutinin.
 |
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