From the Department of Physiology II, Kobe University
School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan and the ¶ Department of Biochemistry II, School of
Medicine, University of the Ryukyus, 207 Uehara, Nishihara-cho,
Okinawa 903-0215, Japan
Received for publication, September 12, 2000, and in revised form, October 3, 2000
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ABSTRACT |
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Phosphoinositide-specific phospholipase C
(PI-PLC) plays a pivotal role in regulation of intracellular signal
transduction from various receptor molecules. More than 10 members of
human PI-PLC isoforms have been identified and classified into three classes The hydrolysis of phosphatidylinositol 4,5-bisphosphate
(PIP2)1 by
phosphoinositide-specific phospholipase C (PI-PLC) is a key event
initiating intracellular signal transduction from various receptor
molecules at the plasma membrane (1). This reaction yields two
intracellular second messengers, diacylglycerol and inositol
1,4,5-trisphosphate, which induce activation of protein kinase C
and mobilization of Ca2+ from intracellular stores,
respectively. Concurrently, the reduction in the PIP2
concentration is likely an important signal because activities of
various actin-binding proteins and pleckstrin homology (PH)
domain-containing proteins are modulated through interaction with
PIP2 (2). PIP2 concentration at the membrane
has also been shown to be important in maintenance of the cell shape
through regulation of the actin cytoskeleton (3). More than 10 members of human PI-PLC isoforms have been identified and classified into three
classes, Ras proto-oncoproteins are small GTP-binding proteins that function as
molecular switches by cycling between the active GTP-bound state and
the inactive GDP-bound state (4). They regulate a variety of biological
responses including proliferation and differentiation of mammalian
cells. In mammalian cells, membrane-anchored GTP-bound Ras interacts
directly with the serine/threonine kinase Raf-1, leading to activation
of the mitogen-activated protein kinase cascade (5). In addition,
recent searches have identified a number of candidate Ras effectors
other than Raf-1 and its isoforms B-Raf and A-Raf in mammalian cells
(6). All these effector proteins exhibit a GTP-dependent
interaction with Ras, for which the intact effector region (residues
32-40 of mammalian Ras) is required (4-6). This interaction, when
made with post-translationally modified Ras, induces translocation of
the effectors from the cytosol to the plasma membrane, where their
substrates or activators are localized. The Ras-dependent
membrane translocation has been shown to be prerequisite to activation
of the Ras effectors, Raf-1 (7), phosphoinositide 3-kinase (8), and Ral
guanine nucleotide dissociation stimulator (RalGDS) (9). Some of the
candidate effectors of Ras, including RalGDS and AF-6/Afadin (10, 11), have been shown to possess homologous motifs of about 100 amino acids
in their Ras-associating (RA) regions (12). Recently, the RA domain of
RalGDS was reported to share a similar tertiary structure, the
ubiquitin superfold, with the Ras-binding domain of Raf-1, although
they exhibit no apparent homology in their amino acid sequences
(13, 14).
Rap1A, another member of Ras family small GTP-binding proteins,
possesses an identical effector region with that of Ras and associates
with almost all effector molecules of Ras (15). Although its
physiological functions remain to be investigated, the findings that
Rap1A can activate the Ras effector B-Raf and may cooperate with Ras in
regulation of B-Raf-mediated responses in some cell types (16, 17)
suggest that Rap1A may regulate the activity of other Ras effectors as
well. Recently, Rap1 has been implicated in integrin-mediated cell
adhesion signaling following stimulation of cell surface molecules,
such as the granulocyte colony-stimulating factor (18), CD31 (19), and
the T cell receptor (20).
In this report, we have identified a novel class of human PI-PLC, named
PLC Cloning of PLC In Vitro Binding and Two-hybrid Assays--
The
post-translationally modified forms of human Ha-Ras and Rap1A were
purified as described before (24-26). Residues 2094-2303 of PLC Adenylyl Cyclase Inhibition Assay--
Measurements of
Saccharomyces cerevisiae adenylyl cyclase activity dependent
on GTP Assay of PI-PLC Activity--
Full-length PLC Fluorescence Microscopy--
Full-length PLC Immunoprecipitation and Fractionation--
Full-length PLC Cloning of Human PLC Specific Binding of PLC
The interaction of the PLC Ras Induces Membrane Translocation of PLC
We further investigated whether the PIP2-hydrolyzing
activity of PLC Epidermal Growth Factor Stimulates Translocation of PLC We have discovered yet another class of PI-PLC, PLC The physiological function of PLC PLC,
, and
, which are regulated by distinct mechanisms. Here we report identification of a novel class of human PI-PLC, named
PLC
, which is characterized by the presence of a
Ras-associating domain at its C terminus and a CDC25-like domain
at its N terminus. The Ras-associating domain of PLC
specifically
binds to the GTP-bound forms of Ha-Ras and Rap1A. The dissociation
constant for Ha-Ras is estimated to be approximately 40 nM, comparable with those of other Ras effectors.
Co-expression of an activated Ha-Ras mutant with PLC
induces its
translocation from the cytosol to the plasma membrane. Upon stimulation
with epidermal growth factor, similar translocation of ectopically
expressed PLC
is observed, which is inhibited by co-expression of
dominant-negative Ha-Ras. Furthermore, using a liposome-based
reconstitution assay, it is shown that the phosphatidylinositol
4,5-bisphosphate-hydrolyzing activity of PLC
is stimulated in
vitro by Ha-Ras in a GTP-dependent manner. These
results indicate that Ras directly regulates phosphoinositide breakdown
through membrane targeting of PLC
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
, exemplified by 150-kDa PLC
1, 145-kDa PLC
1, and 85-kDa PLC
1, respectively (1). All the PI-PLC isoforms contain two regions of high sequence homology, designated X and Y,
which constitute the PLC catalytic domain. In addition, other accessory
modules, a PH domain, an EF-hand domain, and a C2 domain, are also
shared by all the mammalian PLC isoforms reported so far (see Fig.
1A). The three classes of PI-PLCs are linked to receptors by
distinct mechanisms (1): PLC
isozymes are activated by the
subunits of the Gq subfamily of heterotrimeric G proteins as well as by the G
dimer, whereas PLC
isozymes are activated by both receptor type and nonreceptor type protein tyrosine kinases through tyrosine phosphorylation.
, which is characterized by the presence of an RA domain, as a
human homolog of Caenorhabditis elegans PLC210 (21). We
demonstrate that PLC
is regulated through direct association with
Ras, adding another member to the growing list of Ras effectors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cDNA--
The BLAST search (22) of
GenBankTM entries identified a human EST clone zb59f12,
which had a striking homology to the C-terminal segment of C. elegans PLC210 (see Fig. 1A). To isolate cDNAs
coding for the upstream sequences, we performed 5'-RACE (23) by using a
cDNA library, synthesized from human fetal brain mRNA
(Invitrogen, San Diego, CA) by Marathon cDNA amplification
procedure, as a template according to the manufacturer's instruction
(CLONTECH, Palo Alto, CA). The nucleotide sequences
of the 5'-RACE-amplified cDNAs were confirmed by isolating and
sequencing multiple clones.
,
encompassing the RA domain, were expressed in Escherichia coli as an maltose-binding protein (MBP) fusion by using the
pMAL-c plasmid. The resulting MBP-PLC
(2094-2303) was examined for
in vitro association with Ha-Ras and Rap1A, which had been
loaded with GTP
S or GDP
S, as described before (25, 26).
Interaction of PLC
(2094-2303) with various effector region mutants
of Ha-Ras was examined by the yeast two-hybrid assay employing
pGAD-PLC
(2094-2303) and pGBT-Ha-RasG12V carrying the
mutations as described (27). The
-galactosidase activity was
measured by a filter assay as described (28)
S-loaded Ha-Ras and its inhibition by purified MBP-PLC
(2094-2303) were carried out as described before (29).
was expressed
with a FLAG epitope tag in Spodoptera frugiperda Sf9
cells by using a baculovirus vector pVL1393 (PharMingen, San Diego,
CA). FLAG-PLC
was affinity purified with resin conjugated with
anti-FLAG monoclonal antibody M2 (Sigma). The PI-PLC activities were
measured essentially as described (21). Briefly, the fusion proteins
were incubated in 50-µl reaction mixtures containing 50 mM Mes, pH 6.8, 10 µM Ca2+/EGTA,
100 mM NaCl, 0.2 mg/ml bovine serum albumin, 0.1 mM dithiothreitol, 90 µM
[3H]PIP2 (20,000 cpm), and 80 µM phosphatidylethanolamine at 30 °C for 30 min.
[3H]Inositol 1,4,5-trisphosphate produced was extracted
and quantitated by liquid scintillation counting. Various
EGTA/Ca2+ buffers giving different concentrations of free
Ca2+ were prepared as described (30). pGEX-PLC
1
(provided by Dr. Tadaomi Takenawa, University of Tokyo, Tokyo, Japan)
was used for expression of the full-length rat PLC
1 as a glutathione
S-transferase fusion in E. coli. For a
liposome-based reconstitution assay, liposomes were made similarly as
described (8) by sonication of dried lipids in buffer containing 20 mM Tris-HCl, pH 7.5, 1 mg/ml fatty acid-free bovine serum
albumin, 7 mM EDTA, and 5 mM MgCl2.
The final lipid concentration was 640 µM
phosphatidylethanolamine, 600 µM phosphatidylserine, 280 µM phosphatidylcholine, 60 µM sphingomyelin and 160 nM [3H]PIP2 (5,000 cpm/pmol). Post-translationally modified Ha-Ras preloaded with either
GTP
S or GDP
S was added to the liposomes at a final concentration
of 35 nM and mixed at 4 °C for 90 min. Liposomes were
collected by centrifugation at 100,000 × g for 2 h and resuspended in the same volume of the buffer. The
PIP2-hydrolyzing activity of PLC
was measured as
described above by incubating 0.1 pmol of purified FLAG-PLC
with the
liposomes in a total volume of 50 µl.
was expressed in
COS-7 cells as an enhanced green fluorescent protein (EGFP) fusion.
COS-7 cells were cultured in a slide chamber in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and
transfected with pcDNA-EGFP-PLC
in combination with a pEFBOS-HA
vector carrying Ha-Ras or its mutants (0.5 µg each) by using
superfect transfection reagent (Qiagen). Cells were transferred to
serum-free medium 8 h post-transfection and further cultured for
18 h. After fixation with 3.7% formaldehyde and permeabilization
with 0.2% Triton X-100, cells were stained with mouse anti-influenza
virus HA monoclonal antibody (Roche Molecular Biochemicals) followed by
rhodamine-conjugated secondary antibody. Localizations of PLC
and
Ha-Ras were analyzed under a confocal laser microscope (MRC-1024;
Bio-Rad).
was expressed with a FLAG epitope tag using pFLAG-CMV (Sigma). COS-7
cells transfected with pFLAG-CMV-PLC
in combination with
pEFBOS-HA-Ha-RasG12V or
pEFBOS-HA-Ha-RasG12V,Y32F were subjected to serum
starvation for 18 h and lysed in buffer A (20 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM
MgCl2, 1% Triton X-100, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 1 µM leupeptin) and centrifuged at 100,000 × g for 60 min. HA-Ha-Ras was immunoprecipitated from the
supernatant with anti-HA monoclonal antibody. Ha-Ras and PLC
in the
immunoprecipitates were detected by immunoblotting with anti-HA
antibody and affinity purified rabbit polyclonal anti-PLC
antibody
raised against PLC
(2094-2303), respectively. For examination of
subcellular localization, COS-7 cells expressing the above proteins
were lysed in buffer A without 1% Triton X-100 by sonication. The
lysates were separated into the membrane and cytosolic fractions by
ultracentrifugation at 100,000 × g for 60 min.
FLAG-PLC
in the membrane and cytosolic fractions was detected with
anti-PLC
antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
Nucleotide sequence determination of
the human EST clone zb59f12 revealed a striking homology to the
C-terminal region of C. elegans PLC210 including the Y, C2,
and RA domains (Fig. 1, A and
B), suggesting that this may represent a human homolog of PLC210. We isolated cDNAs coding for the upstream sequences by the
5'-RACE procedure using a human fetal brain cDNA library as a
template. A putative initiator ATG was identified in the
5'-RACE-amplified cDNAs as that matched the Kozak consensus
sequence (31) and preceded by in-frame stop codons. A composite
cDNA was reconstructed by joining the 5'-RACE-amplified clones and
the EST clone zb59f12 and shown to encode the full-length protein with
the size of 2,303 amino acid residues. Comparison of the deduced amino
acid sequence with GenBankTM entries indicated that this
protein possessed the X, Y, and C2 domains, which were sandwiched
between an N-terminal CDC25-like domain and a C-terminal RA domain
(Fig. 1, A and B). The CDC25-like domain is
homologous to a family of guanine nucleotide exchange proteins for Ras,
represented by S. cerevisiae CDC25. All of these structural
elements are conserved with PLC210 except for a difference in the
number of the RA domains, confirming that we identified a human homolog
of PLC210. As observed with PLC210, this protein lacks a PH domain and
an EF-hand domain, both of which are invariably present in all the
known human PLC isoforms (Fig. 1A). The observations led us
to propose that this protein defines a novel class of human PI-PLC,
designated PLC
. The PLC
mRNA was 7.6 kb in size and detected
in various fetal and adult human tissues, such as brain, muscle, and
lung (data not shown). The PI-PLC activity was confirmed by using
recombinant full-length PLC
expressed in Sf9 insect cells.
Purified PLC
specifically hydrolyzed PIP2 with a
specific activity of 1.35 µmol/min/mg protein, which was comparable
with that of PLC
1, 304 nmol/min/mg protein, obtained under the same assay condition. Like most of other PI-PLC isoforms, the
PIP2-hydrolyzing activity of PLC
exhibited a dependence
on the Ca2+ concentration, and the maximal activity was
obtained around 10 µM Ca2+ (Fig.
1C).
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Fig. 1.
Cloning of human
PLC . A, schematic
representation of various functional domains of human PLC
, C. elegans PLC210, and human PLC
1, PLC
1, and PLC
1.
CDC25, CDC25-like domain; X, X domain;
Y, Y domain; C2, C2 domain; RA, RA
domain; PH, PH domain; SH2, SH2 domain;
SH3, SH3 domain. The ranges covered by the EST clone zb59f12
(EST zb59f12) are indicated below PLC
. B, the complete
amino acid sequence of PLC
deduced from the nucleotide sequence of
the cloned cDNA. Sequences in color indicate CDC25
(blue), X (yellow), Y (red), C2
(green), and RA (purple) domains.
C, Ca2+ dependence of the activity of PLC
.
Purified FLAG-PLC
(0.5 pmol) was examined for the
PIP2-hydrolyzing activity in the presence of various
concentrations of free Ca2+.
to Ha-Ras and Rap1A--
MBP-PLC
(2094-2303), encompassing the RA domain, was examined for association
with Ha-Ras and Rap1A, which had been loaded with GTP
S or GDP
S. A
dose-dependent association was observed with the GTP-bound
forms of Ha-Ras and Rap1A but not with their GDP-bound forms (Fig.
2A). The association with Ras
was further analyzed quantitatively by the yeast adenylyl cyclase
inhibition assay (29). A dose-dependent inhibition of
Ha-Ras-dependent adenylyl cyclase activity was observed by
purified PLC
(2094-2303) added into the reaction mixture (Fig.
2B). MBP-PLC
(2094-2303) had no effect on the
Mn2+-dependent adenylyl cyclase activity,
indicating that this protein exerted its effect by interacting with Ras
but not with adenylyl cyclase (data not shown). We carried out this
experiment in the presence of various concentrations of Ha-Ras and
MBP-PLC
(2094-2303) to prove the competitive nature of the
inhibition as described (29). At each point of Ha-Ras concentration in
the presence of MBP-PLC
(2094-2303), we obtained free Ha-Ras
concentration available for adenylyl cyclase activation as that
required for giving the same adenylyl cyclase activity in the absence
of MBP-PLC
(2094-2303). A difference between the original and the
free concentrations of Ha-Ras was regarded as that bound to MBP-PLC
(2094-2303), and a reciprocal of this value was plotted against a
reciprocal of the free Ha-Ras concentration (Fig. 2C). This
gave a series of straight lines for each value of MBP-PLC
(2094-2303), which converged on the horizontal axis. The data
indicated that MBP-PLC
(2094-2303) bound directly to Ha-Ras and
competitively sequestered it from adenylyl cyclase. The
Kd value for Ha-Ras was calculated from the point of
intersection with the horizontal axis and determined to be
approximately 40 nM, which is comparable with those of
other Ras effectors (27). The data also indicated that the maximal
Ha-Ras binding capacity of PLC
(2094-2303), calculated from the
point of intersection with the vertical axis (29), was about 0.5 pmol
of Ha-Ras bound to 1 pmol of MBP-PLC
(2094-2303).
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Fig. 2.
Specific association of
PLC with Ras and Rap1A. A,
in vitro association of the PLC
RA domain with Ha-Ras and
Rap1A. Various amounts of MBP-PLC
(2094-2033) was incubated with
Ha-Ras and Rap1A (10 pmol each) preloaded with either GTP
S
(lanes T) or GDP
S (lanes D). Ha-Ras and Rap1A
in the eluate were detected by immunoblotting with anti-Ras and
anti-Rap1A antibodies, respectively, along with one-tenth aliquots of
Ha-Ras and Rap1A used in the assay. B, inhibition of
Ras-dependent adenylyl cyclase activation by the PLC
RA
domain. Adenylyl cyclase activities dependent on various concentrations
of Ha-Ras were measured in the presence of various amounts of
MBP-PLC
(2094-2303) as follows: 0 (open square), 5 (closed circle), 10 (open circle), and 20 (closed square) pmol. One unit of activity is defined as 1 pmol of cAMP formed in 1 min of incubation with 1 mg of protein at
30 °C under standard assay conditions. C,
double-reciprocal plot analysis. The amounts of free and PLC
-bound
Ha-Ras were calculated as described (26). The symbols
correspond to those used in B. D, COS-7 cells expressing
HA-tagged Ha-Ras and FLAG-tagged PLC
were lysed, and Ha-Ras was
immunoprecipitated from total cell extract with anti-HA antibody. The
upper panel shows the co-immunoprecipitated PLC
detected
with anti-PLC
antibody, and the lower panel shows the
immunoprecipitated Ha-Ras detected with anti-HA antibody.
RA domain with Ha-Ras was also confirmed
by the yeast two-hybrid assay (Table I).
The mutations affecting Asn-26, Tyr-32, and Thr-35 specifically
abolished the interaction of Ha-Ras with PLC
(2094-2303).
Comparison with our past results obtained with other Ras effectors (21,
27) indicated that the binding specificity of PLC
was
indistinguishable from those of PLC210 and Schizosaccharomyces
pombe Byr2 but different from those of human Raf-1, RalGDS, and
AF-6/Afadin. PLC
(2094-2303) exhibited a positive interaction with
Rap1A but not with other small GTP-binding proteins such as R-Ras,
RalA, RhoA, Rac1, and Cdc42 by the two-hybrid assay (data not shown).
Furthermore, PLC
was co-immunoprecipitated with activated Ha-Ras,
Ha-RasG12V, but not with Ha-RasG12V,Y32F, from
the total cellular extract of COS-7 cells co-expressing them (Fig.
2D), demonstrating their in vivo association.
Interactions of Ha-Ras effector region mutants with PLC and
other effectors
, no interaction. The data on PLC210, Byr2,
Raf-1, RalGDS, and AF-6 are taken from Refs. 21, 27, 38, 39, and 40.
and Stimulates Its
PIP2-hydrolyzing Activity--
To examine whether the
association with Ras has a function to recruit PLC
to the plasma
membrane, we co-expressed FLAG-tagged PLC
with
Ha-RasG12V in serum-starved COS-7 cells and examined its
distribution in the membrane and cytosolic fractions. PLC
was
predominantly localized in the cytosolic fraction when expressed alone
(Fig. 3A). However, co-expression with Ha-RasG12V caused a gross increment of
the amount of PLC
in the membrane fraction. In contrast,
co-expression with Ha-RasG12V,Y32F failed to affect the
distribution of PLC
. Next, we took advantage of the GFP fusion and
examined the intracellular localization of PLC
under a confocal
laser microscope. GFP-PLC
was evenly distributed in the cytosol when
expressed alone in the serum-starved COS-7 cells (Fig. 3B,
top panels). However, GFP-PLC
became enriched at the
plasma membrane when co-expressed with Ha-RasG12V (Fig.
3B, middle panels). Again,
Ha-RasG12V,Y32F was found to be incompetent (Fig.
3B, bottom panels). Similar results were obtained
by using NIH3T3 cells (data not shown). These data taken together
indicate that Ras associates with PLC
and induces its translocation
from the cytosol to the plasma membrane.
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Fig. 3.
Effects of Ras on the membrane translocation
and the PIP2-hydrolyzing activity of
PLC . A, COS-7 cells expressing
the indicated proteins were subjected to serum starvation for 18 h
and lysed in buffer A without 1% Triton X-100 by sonication.
FLAG-PLC
in the membrane (middle panel) and cytosolic
(bottom panel) fractions was detected with anti-PLC
antibody. B, COS-7 cells expressing the indicated proteins
were examined for their subcellular localization under the confocal
laser microscope. The left panels show expressed Ha-Ras
stained with anti-HA antibody (red), the middle
panels show expressed GFP-PLC
(green), and the
right panels show the merged images. Co-localization of
PLC
with Ras is shown by yellow in the merged images.
C, GTP
S-loaded or GDP
S-loaded Ras was incorporated
into liposomes containing 2.4 pmol of
[3H]PIP2. Purified FLAG-PLC
was incubated
with the liposomes, and its PIP2-hydrolyzing activity was
presented as inositol 1,4,5-trisphosphate released.
is regulated through interaction with Ras. Because
simple mixing of GTP-bound Ha-Ras with purified PLC
had no effect on its activity (data not shown), we examined the effect of the
Ras-induced membrane translocation, which was in principle expected to
cause an increase of the PLC
activity by bringing it to the
proximity of its substrate PIP2. To this end, we employed
an in vitro reconstitution assay, in which
post-translationally modified Ha-Ras and
[3H]PIP2 were incorporated into liposomes and
incubated with purified PLC
. As shown in Fig. 3C,
GTP
S-loaded Ha-Ras stimulated the PIP2-hydrolyzing
activity of PLC
by approximately 2.4-fold. In contrast, no
stimulation was observed with the GDP
S-loaded form. This result
suggests that the Ras-dependent membrane translocation stimulates the PLC
activity.
through
Ras and Rap1A--
To further examine whether the
Ras-dependent translocation of PLC
could be induced by a
physiological stimulus, COS-7 cells expressing PLC
were stimulated
with EGF and analyzed for the distribution of PLC
by immunoblotting.
EGF induced transient membrane translocation of PLC
, starting from 5 min after the stimulation and terminating at 40 min (Fig.
4A, left panels). The EGF-stimulated membrane translocation was efficiently blocked by
co-expression of the dominant-negative Ha-Ras mutant
Ha-RasS17N (Fig. 4A, right panels).
Further, translocation of GFP-PLC
to the plasma membrane upon EGF
stimulation was observed (Fig. 4B). In addition, an
enrichment of GFP-PLC
in the perinuclear region was also noticed
(Fig. 4B). We speculated that this perinuclear enrichment
was mediated by Rap1A because Rap1A was reported to be activated upon
EGF stimulation (32) and because Rap1A was found to be associated with
PLC
(Fig. 2A). In fact, we observed co-localization of
GFP-PLC
with Rap1AG12V in this region (Fig.
4C). Moreover, following EGF stimulation, GFP-PLC
was
translocated to the plasma membrane in cells overexpressing wild type
Ha-Ras, whereas in wild type Rap1A-overexpressing cells, GFP-PLC
was
translocated to the perinuclear region (Fig. 4D). Taken
together, EGF may direct translocation of PLC
to different subcellular regions through Ras or Rap1A.
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Fig. 4.
EGF-induced translocation of
PLC . A, COS-7 cells were
transfected with pcDNA-EGFP-PLC
alone or in combination with
pEFBOS-HA-Ha-RasS17N (0.5 µg each) by using superfect
transfection reagent. The cells were transferred to serum-free medium
8 h post-transfection and further cultured for 18 h before
stimulation with EGF (100 ng/ml) for the indicated periods. PLC
in
the membrane fraction was detected with anti-PLC
antibody.
B, subcellular localization of GFP-PLC
was examined after
stimulation with EGF (100 ng/ml) for the indicated periods under the
confocal laser microscope. The arrowhead indicates the
plasma membrane enrichment of GFP-PLC
, and the arrow
indicates the perinuclear enrichment. C,
HA-Rap1AG12V (left) and GFP-PLC
(middle) co-expressed in COS-7 cells were examined for their
subcellular localization as described in Fig. 3B. D, COS-7
cells expressing GFP-PLC
in combination with either wild type Ha-Ras
(upper panels) or wild type Rap1A (lower panels)
were left unstimulated (left panels) or stimulated with EGF
(100 ng/ml) for 5 min (right panels).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which is
characterized by the presence of the CDC25-like domain and the RA
domain as well as by the absence of PH and EF-hand domains. As
predicted from the close homology at the X, Y, and C2 domains with the
known PLC isoforms, PLC
exhibited a
Ca2+-dependent PI-PLC activity with the maximal
activity observed at 10 µM Ca2+. PLC
possessed the RA domain, which exhibited a specific binding activity to
GTP-bound Ha-Ras with an affinity comparable with those of other
Ras-effector proteins (27). This binding was abolished by specific
mutations in the effector region, which constitutes a major interface
for interaction with Ras effectors (4). Further, it was shown that Ras
could induce translocation of PLC
to the plasma membrane and
stimulate its PIP2-hydrolyzing activity in the liposome
reconstitution assay. These observations indicate that PLC
functions
as a direct downstream effector of Ras. Considering that full-length
PLC
expressed in Sf9 cells by itself possessed a higher
specific activity than PLC
and could not be activated in
vitro by mixing with Ras, the association with Ras is likely to
induce activation of PLC
through its recruitment to the plasma
membrane, where its substrate PIP2 exists. This mechanism
is similar to those observed for other Ras effectors, phosphoinositide
3-kinase (8) and RalGDS (9). However, we cannot exclude the possibility
that Ras has another role of directly activating PLC
as observed for
Raf-1 (7).
remains to be elucidated. The
observed membrane recruitment of PLC
by EGF treatment suggests that
PLC
may be involved in signal transduction from growth factor receptors. In mammalian cells, an increase in PI-PLC activity upon
treatment with growth factors has been largely attributed to the
activation of PLC
(1). However, the observation that the rate of
phosphoinositide turnover in Ras-transformed NIH3T3 cells was three
times that in untransformed cells implied a persistent stimulation of
PI-PLC in these cells (33). Further, injection of anti-PI-PLC antibody
has been shown to inhibit Ras-induced mitogenesis (34). These
observations are consistent with the possibility that an as yet
unidentified species of PI-PLC regulated by Ras may play a role in
mammalian cell proliferation, although they might be explained by
accessory events accompanying the Ras-induced transformation such as
autocrine stimulation of receptors coupled to the known PI-PLCs. Our
present findings suggest that they might be accounted for by
PLC
.
binds not only to Ras but also to Rap1A as shown in Fig.
2A. Furthermore, Rap1A may mediate translocation of PLC
to the perinuclear region in response to EGF (Fig. 4). Considering that
Rap1A (35, 36) and protein kinase C (37) were reported to be localized
at the Golgi apparatus, PLC
, downstream of Rap1A, may have some role
in the regulation of Golgi functions mediated by protein kinase C. Further analysis of the function of PLC
may reveal its unique role
in some cellular phenomena.
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FOOTNOTES |
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* This work was supported by Ministry of Education, Science, Sports and Culture of Japan Grants 11470034, 12215098, 12670116, and 12670136 and by funds from the Sankyo Foundation of Life Science, Hyogo Science and Technology Association, and Kobe University.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF190642.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
81-78-382-5380; Fax: 81-78-382-5399; E-mail:
kataoka@kobe-u.ac.jp.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M008324200
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ABBREVIATIONS |
---|
The abbreviations used are:
PIP2, phosphatidylinositol 4,5-bisphosphate;
PLC, phospholipase C;
PI-PLC, phosphoinositide-specific PLC;
PH, pleckstrin homology;
RA, Ras-associating;
RalGDS, Ral guanine nucleotide dissociation
stimulator;
MBP, maltose-binding protein;
GTPS, guanosine
5'-O-(3-thiotriphosphate);
GDP
S, guanosine
5'-O-(2-thiodiphosphate);
EGFP, enhanced green fluorescent
protein;
EGF, epidermal growth factor;
HA, influenza virus
hemagglutinin;
RACE, rapid amplification of cDNA ends;
EST, expressed sequence tag;
Mes, 2-(N-morpholino)ethanesulfonic
acid.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Rhee, S. G.,
and Bae, Y. S.
(1997)
J. Biol. Chem.
272,
15045-15048 |
2. | Toker, A. (1998) Curr. Opin. Cell Biol. 10, 254-261[CrossRef][Medline] [Order article via Infotrieve] |
3. | Czech, M. P. (2000) Cell 100, 603-606[Medline] [Order article via Infotrieve] |
4. | Marshall, M. S. (1993) Trends Biochem. Sci. 18, 250-254[CrossRef][Medline] [Order article via Infotrieve] |
5. | Daum, G., Eisenmann-Tappe, I., Fries, H.-W., Troppmair, J., and Rapp, U. R. (1994) Trends Biochem. Sci. 19, 474-480[CrossRef][Medline] [Order article via Infotrieve] |
6. | Katz, M. E., and McCormick, F. (1997) Curr. Opin. Genet. Dev. 7, 75-79[CrossRef][Medline] [Order article via Infotrieve] |
7. | Morrison, D. K., and Cutler, R. E., Jr. (1997) Curr. Opin. Cell Biol. 9, 174-179[CrossRef][Medline] [Order article via Infotrieve] |
8. | Rodriguez-Viciana, P., Warne, P. H., Vanhaesebroeck, B., Waterfield, M. D., and Downward, J. (1996) EMBO J. 15, 2442-2551[Abstract] |
9. | Kishida, S., Koyama, S., Matsubara, K., Kishida, M., Matsuura, Y., and Kikuchi, A. (1997) Oncogene 15, 2899-2907[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Kuriyama, M.,
Harada, N.,
Kuroda, S.,
Yamamoto, T.,
Nakafuku, M.,
Iwamatsu, A.,
Yamamoto, D.,
Prasad, R.,
Croce, C.,
Canaani, E.,
and Kaibuchi, K.
(1996)
J. Biol. Chem.
271,
607-610 |
11. | Watari, Y., Kariya, K., Shibatohge, M., Liao, Y., Hu, C.-D., Goshima, M., Tamada, M., Kikuchi, A., and Kataoka, T. (1998) Gene (Amst.) 224, 53-58[CrossRef][Medline] [Order article via Infotrieve] |
12. | Ponting, C. P., and Benjamin, D. R. (1996) Trends Biochem. Sci. 21, 422-425[CrossRef][Medline] [Order article via Infotrieve] |
13. | Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) Nature 375, 554-560[CrossRef][Medline] [Order article via Infotrieve] |
14. | Huang, L., Weng, X., Hofer, F., Martin, G. S., and Kim, S. H. (1997) Nat. Struct. Biol. 4, 609-614[Medline] [Order article via Infotrieve] |
15. |
Bos, J. L.
(1998)
EMBO J.
17,
6776-6782 |
16. | Vossler, M. R., Yao, H., York, R. D., Pan, M. G., Rim, C. S., and Stork, P. J. S. (1997) Cell 89, 73-82[Medline] [Order article via Infotrieve] |
17. | York, R. D., Yao, H., Dillon, T., Ellig, C. L., Eckert, S. P., McCleskey, E. W., and Stork, P. J. S. (1998) Nature 392, 622-626[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Tsukamoto, N.,
Hattori, M.,
Yang, H.,
Bos, J. L.,
and Minato, N.
(1999)
J. Biol. Chem.
274,
18463-18469 |
19. |
Reedquist, K. A.,
Ross, E.,
Koop, E. A.,
Wolthuis, R. M.,
Zwartkruis, F. J.,
van Kooyk, Y.,
Salmon, M.,
Buckley, C. D.,
and Bos, J. L.
(2000)
J. Cell Biol.
148,
1151-1158 |
20. |
Katagiri, K.,
Hattori, M.,
Minato, N.,
Irie, S.,
Takatsu, K.,
and Kinashi, T.
(2000)
Mol. Cell. Biol.
20,
1956-1969 |
21. |
Shibatohge, M.,
Kariya, K.,
Liao, Y.,
Hu, C.-D.,
Watari, Y.,
Goshima, M.,
Shima, F.,
and Kataoka, T.
(1998)
J. Biol. Chem.
273,
6218-6222 |
22. | Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve] |
23. | Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8998-9002[Abstract] |
24. | Kuroda, Y., Suzuki, N., and Kataoka, T. (1993) Science 259, 683-686[Medline] [Order article via Infotrieve] |
25. |
Hu, C.-D.,
Kariya, K.,
Tamada, M.,
Akasaka, K.,
Shirouzu, M.,
Yokoyama, S.,
and Kataoka, T.
(1995)
J. Biol. Chem.
270,
30274-30277 |
26. |
Hu, C.-D.,
Kariya, K.,
Kotani, G.,
Shirouzu, M.,
Yokoyama, S.,
and Kataoka, T.
(1997)
J. Biol. Chem.
272,
11702-11705 |
27. |
Akasaka, K.,
Tamada, M.,
Wang, F.,
Kariya, K.,
Shima, F.,
Kikuchi, A.,
Yamamoto, M.,
Shirouzu, M.,
Yokoyama, S.,
and Kataoka, T.
(1996)
J. Biol. Chem.
271,
5353-5360 |
28. | Bartel, P. L., Chien, C.-T., Sternglanz, R., and Fields, S. (1993) in Cellular Interactions in Development: A Practical Approach (Hartley, D. A., ed) , pp. 153-179, Oxford University Press, Oxford |
29. |
Minato, T.,
Wang, J.,
Akasaka, K.,
Okada, T.,
Suzuki, N.,
and Kataoka, T.
(1994)
J. Biol. Chem.
269,
20845-20851 |
30. | Homma, Y., and Emori, Y. (1997) in Signaling by Inositides (Shears, S., ed) , pp. 99-116, Oxford University Press, Oxford |
31. | Kozak, M. (1986) Cell 44, 283-292[Medline] [Order article via Infotrieve] |
32. |
Zwartkruis, F. J. T.,
Wolthuis, R. M. F.,
Nabben, N. M. J. M.,
Franke, B.,
and Bos, J. L.
(1998)
EMBO J.
17,
5905-5912 |
33. | Fleischman, L. F., Chahwala, S. B., and Cantley, L. (1986) Science 231, 407-410[Medline] [Order article via Infotrieve] |
34. | Smith, M. R., Liu, Y. L., Kim, H., Rhee, S. G., and Kung, H. F. (1990) Science 247, 1074-1077[Medline] [Order article via Infotrieve] |
35. | Beranger, F., Goud, B., Tavitian, A., and de Gunzburg, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1606-1610[Abstract] |
36. | Matsubara, K., Kishida, S., Matsuura, Y., Kitayama, H., Noda, M., and Kikuchi, A. (1999) Oncogene 18, 1303-1312[CrossRef][Medline] [Order article via Infotrieve] |
37. | De Matteis, M. A., Santini, G., Kahn, R. A., Tullio, G. D., and Luini, A. (1993) Nature 364, 818-821[CrossRef][Medline] [Order article via Infotrieve] |
38. | White, M. A., Nicolette, C., Minden, A., Polverino, A., Van Aelst, L., Karin, M., and Wigler, M. H. (1995) Cell 80, 533-541[Medline] [Order article via Infotrieve] |
39. |
White, M. A.,
Vale, T.,
Camonis, J. H.,
Schaefer, E.,
and Wigler, M. H.
(1996)
J. Biol. Chem.
271,
16439-16442 |
40. | Khosravi-Far, R., White, M. A., Westwick, J. K., Solski, P. A., Chrzanowska-Wodnicka, M., Van Aelst, L., Wigler, M. H., and Der, C. J. (1996) Mol. Cell. Biol. 16, 3923-3933[Abstract] |