Received for publication, August 19, 2002, and in revised form, January 10, 2003
The epidermal growth factor (EGF) receptor has an
important role in cellular proliferation, and the enzymatic activity of phospholipase C (PLC)-
1 is regarded to be critical for EGF-induced mitogenesis. In this study, we report for the first time a
phospholipase complex composed of PLC-
1 and phospholipase D2 (PLD2).
PLC-
1 is co-immunoprecipitated with PLD2 in COS-7 cells. The results of in vitro binding analysis and co-immunoprecipitation
analysis in COS-7 cells show that the Src homology (SH) 3 domain of
PLC-
1 binds to the proline-rich motif within the Phox homology (PX) domain of PLD2. The interaction between PLC-
1 and PLD2 is EGF stimulation-dependent and potentiates EGF-induced inositol
1,4,5-trisphosphate (IP3) formation and Ca2+
increase. Mutating Pro-145 and Pro-148 within the PX domain of PLD2 to leucines disrupts the interaction between PLC-
1 and PLD2 and
fails to potentiate EGF-induced IP3 formation and
Ca2+ increase. However, neither PLD2 wild type nor PLD2
mutant affects the EGF-induced tyrosine phosphorylation of PLC-
1.
These findings suggest that, upon EGF stimulation, PLC-
1 directly
interacts with PLD2 and this interaction is important for PLC-
1 activity.
 |
INTRODUCTION |
The epidermal growth factor
(EGF)1 signaling pathways
have been implicated in cellular proliferation and cytoskeletal
reorganization (1). The enzymatic activity of phospholipase C
(PLC)-
1, a downstream signaling component of EGF, is required for
the EGF-induced cellular responses (2-4). PLC hydrolyzes
phosphatidylinositol 4,5-bisphosphate (PIP2), and one
product of this hydrolysis, inositol 1,4,5-trisphosphate
(IP3), mobilizes Ca2+ from the intracellular
stores, whereas the other product of the hydrolysis,
1,2-diacylglycerol, activates protein kinase C (PKC) (5). Until
recently, 11 mammalian PLC isozymes have been identified and classified
into four types (6-8). PLC-
, unlike the other PLC isozymes, has Src
homology (SH) domains: two SH2 and one SH3 domains (9). Tyrosine
phosphorylation of PLC-
1 by the EGF receptor is responsible for
inositol phosphate production (10-12). The SH2 domains of PLC-
1 are
important for binding to the EGF receptor and for phosphorylation by
the receptor, which is required for activation (13-15). However,
little is known about how phosphorylated PLC-
1 undergoes further
processes such as targeting to its PIP2 substrate molecules
in the membrane.
Phospholipase D is a membrane-associated enzyme, which hydrolyzes
phosphatidylcholine to generate phosphatidic acid (PA) and choline
(16). PA has been shown to be an intracellular lipid second messenger
involved in many physiological events such as the promotion of
mitogenesis, the secretory process, and actin cytoskeletal
reorganization (17-20). PLD activity is regulated by protein kinases,
small molecular weight G-proteins, and Ca2+ (21), and PKC
has been suggested to be a major mediator of PLD stimulation (22).
Moreover, PLD requires PIP2 for its enzymatic activity
(23). Two types of mammalian PLD isoforms, PLD1 and PLD2, have been
reported, which share 55% identity (24). EGF stimulation increases PLD
activity (25, 26), but the roles of the domains of PLD in EGF signaling
have not been identified.
It has been reported that the EGF receptor is highly enriched in the
plasma membrane substructure, caveolae (27, 28), and in our previous
study, we found that PLC-
1 translocates to caveolae upon EGF
stimulation (29). PLD isozymes exist both in the caveolae, and the
cellular distribution of PLD2, in particular, is largely restricted to
caveolae (25, 30). PIP2, the substrate of PLC-
1 and the
activator of PLD, is also highly enriched in caveolae (31). The
communication between PLC-
1 and PLD has been questioned. PKC, a
downstream signaling molecule of PLC-
1, is required for PLD
activation (26, 32). Mouse embryo fibroblast lacking PLC-
1 showed
markedly reduced PLD activity (33), and PA, the product of PLD action
in the membrane, increased in vitro PLC-
1 activity (34).
These reports suggest that the two phospholipases (PLC-
1 and PLD2)
may affect each other dynamically by changing the membrane
phospholipids. Nevertheless, the direct relationship between these
phospholipases in cells has not been studied.
In this report, we demonstrate for the first time the direct
association between two signaling phospholipases, PLC-
1 and PLD2. We
suggest that their interaction is important for the proper activation
of PLC-
1 after EGF stimulation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The enhanced chemiluminescence (ECL) kit was
purchased from Amersham Biosciences (Buckinghamshire, United Kingdom);
[3H]myristic acid and [3H]inositol
1,4,5-trisphosphate were obtained from PerkinElmer Life Sciences
(Boston, MA); Silica Gel 60 thin-layer chromatography plates were
obtained from MERK (Darmstadt, Germany); protein A-Sepharose was
obtained from RepliGen (Needham, MA); Dulbecco's modified Eagle's
medium and LipofectAMINE were obtained from Invitrogen (Grand Island,
NY); bovine calf serum was obtained from HyClone (Logan, UT); EGF was
obtained from the Daewoong Pharmaceutical Co. (Seoul, Republic of
Korea); cholic acid was obtained from USB (Cleveland, OH); and
horseradish peroxidase-conjugated goat anti-rabbit IgG and anti-mouse
IgG, IgM, and IgA were purchased from Kirkegaard and Perry
Laboratories, Inc. (Gaithersburg, MD). A polyclonal antibody (mSTP4)
that recognizes both PLD1 and PLD2 was produced as described previously
(35). Anti-actin monoclonal antibody was purchased from ICN
Pharmaceuticals (Costa Mesa, CA). Monoclonal antibodies for PLC-
1,
PLC-
1, and phosphotyrosine and DNAs of wild type human PLD2 in
pCDNA3.1, wild type rat PLC-
1 in pFLAG-CMV-2, and the SH3 domain
deleted rat PLC-
1 in pFLAG-CMV-2 were prepared as described
previously (36-39, 47). Full-length cDNAs of murine PLD2 and its
N-terminal deletion mutant (
1-308) were provided generously by Dr.
Michael A. Frohman (State University of New York, NY). Anti-FLAG M5
monoclonal antibody and all other chemicals were from Sigma (St. Louis, MO).
Cell Culture and Transfection--
COS-7 cells were cultured and
transfected using LipofectAMINE (Invitrogen) as described
previously (39, 40).
Expression and Purification of PLC-
1, PLC-
1, and
PLD2--
Recombinant rat PLC-
1, PLC-
1, and human PLD2 were
expressed in Sf9 cells and purified as described previously (41,
42).
Preparation of GST Fusion Proteins--
Glutathione
S-transferase (GST) fusion proteins containing the SH
domains of PLC-
1 and human PLD2 fragments were prepared as described
previously (38, 40, 43). GST fusion SH3 domains of Abl and CrkI were
kindly provided by Dr. Brian K. Kay (The University of Wisconsin,
Madison, WI) (44). GST fusion Phox homology (PX) domains of
p40phox and p47phox were kindly provided by Dr. Michael
B. Yaffe (Massachusetts Institute of Technology, MA) (45). After
harvesting the cells, the GST fusion proteins were purified by standard
methods (46) using glutathione-Sepharose 4B (Amersham Biosciences).
Immunoblot Analysis--
Proteins were denatured by boiling for
5 min at 95 °C in a Laemmli sample buffer (48), separated by
SDS-PAGE, and immunoblot analysis was performed as described previously
(39).
In Vitro Binding Analysis--
In vitro binding was
performed in 300 µl of binding buffer (20 mM Tris/HCl, pH
8.0, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 0.1% Triton X-100) at 4 °C for 3 h.
Immunoprecipitation--
Cells were lysed with IP buffer (20 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 1% Triton
X-100, 1% cholic acid) by sonication. Cell lysates were centrifuged at
100,000 × g at 4 °C for 30 min, and the
supernatants were incubated with an antibody immobilized to Protein
A-Sepharose beads.
Measurement of PLC Activity in Cells--
Cellular
IP3 concentration was determined by the
[3H]IP3 competition assay using
IP3 binding protein (49), which was prepared from bovine
adrenal cortex as described previously (50).
Measurement of PLD Activity in Cells--
PLD activity was
assayed by measuring phosphatidylbutanol formation in the
presence of 1-butanol as described previously (51).
Measurement of
[Ca2+]i--
[Ca2+]i
levels were determined using Grynkiewicz et al.'s
method with fura-2/AM in Ca2+-free Locke's solution (154 mM NaCl, 5.6 mM KCl, 1.2 mM
MgCl2, 5 mM HEPES, pH 7.3, 10 mM
glucose, and 0.2 mM EGTA) (52).
 |
RESULTS |
PLC-
1 Is Co-immunoprecipitated with PLD2 in Growing
Cells--
It has been reported that the EGF receptor, PLD2, and
PIP2 are enriched in the plasma membrane substructure,
caveolae, to which PLC-
1 translocates after EGF stimulation (25,
27-29, 31). PIP2, the substrate of PLC-
1, was reported
to be a cofactor of PLD activation (16). We checked the possibility of
the existence of PIP2-utilizing phospholipases in a
complex. The major isozyme of PLD in COS-7 cells is PLD2, and PLC-
1
was co-immunoprecipitated with PLD2 from COS-7 cells growing in media
containing 10% bovine calf serum (Fig.
1A). However, because the
amount of PLD2 in COS-7 cells is very small, the
co-immunoprecipitation could be detected after a long time exposure to
a film. By transfecting PLD2 into COS-7 cells, the
co-immunoprecipitation of PLC-
1 with PLD2 was more easily detected
(Fig. 1B). These results indicate that two phospholipases
exist in a complex.

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Fig. 1.
PLC- 1 is
co-immunoprecipitated with PLD2 in COS-7 cells. A,
COS-7 cells growing in media containing 10% bovine calf serum were
lysed (2 mg) with IP buffer, and immunoprecipitation with the indicated
antibodies was performed as described under "Experimental
Procedures." 40 µg of cell lysate was loaded for total lysate blot.
B, vector-transfected COS-7 cells and human PLD2-transfected
COS-7 cells growing in media containing 10% bovine calf serum were
lysed (2 mg) with IP buffer, and immunoprecipitation with anti-PLD
antibody (mSTP4) was performed as described under "Experimental
Procedures." 40 µg of cell lysate was loaded for total lysate blot.
Data are representative of two independent experiments.
|
|
PLD2 Increases EGF-induced PLC-
1 Activity, and the Interaction
between PLC-
1 and PLD2 Is EGF
Stimulation-dependent--
It could be deduced from
previous reports that EGF stimulation recruits PLC-
1 from the
cytosol to the plasma membrane where PLD2 is enriched (25, 29). We
observed that increasing the amount of PLD2 in COS-7 cells resulted in
increasing EGF-induced PLC-
1 activity, but PLD2 didn't affect
PLC-
1 activity without EGF stimulation or the time course of
PLC-
1 activity after EGF stimulation (Fig.
2, A and B). We
tested whether the interaction between PLC-
1 and PLD2 is dependent
on EGF stimulation. As shown in Fig. 2C, serum starvation
abolishes the interaction of PLC-
1 with PLD2, but as soon as cells
were stimulated with EGF, PLC-
1 binds to PLD2 and then dissociates
from PLD2. The interaction time course correlates with the activity
time course of PLC-
1 (Fig. 2B). These results suggest
that the interaction of PLC-
1 with PLD2 may contribute to increasing
EGF-induced PLC-
1 activity.

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Fig. 2.
PLD2 increases EGF-induced
PLC- 1 activity, and the interaction between
PLC- 1 and PLD2 is EGF
stimulation-dependent. A, COS-7 cells in
six-well plates were transfected with the indicated amounts of human
PLD2 DNA. The total amount of DNA was adjusted with vector DNA. After
serum starvation for 24 h, EGF stimulation (100 nM)
was performed for 30 s. IP3 generation was measured by
using a [3H]IP3 competition assay. Means ± S.E. from three independent assays are shown. B, COS-7
cells in six-well plates were transfected with the empty vector or
human PLD2 (1 µg). After serum starvation for 24 h, EGF was
treated at 100 nM for 0, 0.5, 1, 2, or 10 min.
IP3 generation was measured by using a
[3H]IP3 competition assay. Means ± S.E.
from three independent assays are shown. C, COS-7 cells were
transfected with the empty vector or human PLD2. After serum starvation
for 24 h, EGF was treated at 100 nM for the indicated
minutes. Cell lysates (2 mg) underwent immunoprecipitation with
anti-PLD antibody. 40 µg of cell lysate was loaded for total lysate
blot. Data are representative of two independent experiments.
|
|
PLC-
1 Interacts Directly with PLD2 through the SH3
Domain--
To test whether their interaction is direct or not,
in vitro binding analysis was performed with purified
PLC-
1 and PLD2. Purified PLC-
1, or PLC-
1 for comparison, was
incubated with purified PLD2 and immunoprecipitated with an anti-PLD
antibody. As shown in Fig. 3B,
PLC-
1, but not PLC-
1, was precipitated by PLD2. These results
indicate that PLC-
1 binds directly to PLD2. The major differences
between PLC-
1 and PLC-
1 lie in the linker region between the X
and Y domains. PLC-
1 has two SH2 domains and one SH3 domain, but
PLC-
1 has no SH domain (Fig. 3A). To identify which
domain of PLC-
1 is responsible for the interaction with PLD2,
in vitro binding analysis was performed with purified PLD2
and the GST fusion SH domains of PLC-
1. As shown in Fig.
3C, PLD2 bound to GST-SH223 and GST-SH3 but not to GST-SH2N
or GST-SH2C. This result indicates that the SH3 domain of PLC-
1
interacts with PLD2. For further confirmation, co-immunoprecipitation analysis was performed in cells growing in media containing 10% bovine
calf serum. PLD2 was co-transfected into COS-7 cells with PLC-
1 wild
type or PLC-
1 SH3 domain deletion mutant and immunoprecipitated with
an anti-PLD antibody. As shown in Fig. 3D, PLC-
1 wild
type co-immunoprecipitated with PLD2, whereas the PLC-
1 SH3 domain deletion mutant did not. These results indicate that the SH3 domain of
PLC-
1 is responsible for the interaction with PLD2.

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Fig. 3.
PLD2 binds to the SH3 domain of
PLC- 1. A, schematic depiction
of PLC- 1 and PLC- 1. PLC- 1 has two SH2 domains and one SH3
domain, which do not exist in PLC- 1. B, purified PLC- 1
or PLC- 1 was incubated with anti-PLD antibody (mSTP4)
alone or purified PLD2 immobilized to mSTP4. Incubated PLD2 was
detected by Ponceau staining. Data are representative of three
independent experiments. C, GST protein alone or GST fusion
proteins of PLC- 1 SH2 were incubated with purified PLD2. The amounts
of GST and GST fusion proteins were visualized on nitrocellulose
membrane by Ponceau staining (lower panel). Data are
representative of three independent experiments. D, COS-7
cells were co-transfected with FLAG-tagged PLC- 1 wild type
and human PLD2 wild type (PLC- 1 + PLD2) or FLAG-tagged PLC- 1 SH3
domain deletion mutant and human PLD2 wild type (PLC- 1 ( SH3) + PLD2). Cells growing in media containing 10% bovine calf serum were
lysed with IP buffer, and immunoprecipitation with anti-PLD antibody
was performed. Data are representative of two independent
experiments.
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|
Proline-rich Motif within the PX Domain of PLD2 Is Important for
the Interaction with PLC-
1--
To identify the region of PLD2
responsible for the interaction with PLC-
1, we constructed GST
fusion PLD2 fragments as shown in Fig.
4A (40). Fig. 4B
shows the result of in vitro binding analysis with purified
PLC-
1 and GST fusion PLD2 fragments. PLC-
1 was found to bind to
the F1 fragment (residues 1-314) of PLD2. For further confirmation,
co-immunoprecipitation analysis was performed. COS-7 cells were
transfected with the empty vector, PLD2 wild type, or the PLD2 deletion
mutant (
1-308), and immunoprecipitated with an anti-PLD antibody.
As shown in Fig. 4C, PLC-
1 was co-immunoprecipitated with
PLD2 wild type but not with the PLD2 deletion mutant (
1-308). These
results indicate that the N-terminal 1-308 residues of PLD2 are
important for interaction with PLC-
1. To determine the binding region more precisely, we prepared four GST fusion C-terminally deleted
constructs of the PLD2 F1 fragment (Fig. 4A). In
vitro binding analysis with purified PLC-
1 and each GST fusion
construct showed that PLC-
1 binds to f1, f1-1, and f1-2 but not to
f1-3 or f1-4, indicating that the residues from 114 to 166 of PLD2 are
responsible for the interaction (Fig. 4D). In the sequence from 114 to 166 of PLD2, there is a proline-rich motif (PSLP: residues
145-148) that can potentially interact with the SH3 domain. For
confirmation, two proline residues in the PSLP motif were mutated to
leucines, to produce PLD2 (P145L/P148L). This mutation did not change
the expression level, the subcellular localization, or the catalytic
properties of PLD2 in vitro (data not shown). COS-7 cells
were transfected with the empty vector, PLD2 wild type, or PLD2 mutant.
After immunoprecipitation with anti-PLD antibody and thorough washing,
immune complexes were incubated with purified PLC-
1. As shown in
Fig. 4E, PLC-
1 bound to PLD2 wild type but not to PLD2
mutant. These results strongly indicate that PLC-
1 binds to PSLP
motif within the PX domain of PLD2.

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Fig. 4.
PLC- 1 binds to the
proline-rich motif within the PX domain of PLD2. A,
schematic depiction of PLD2 fragments. B, GST protein alone
or the GST fusion proteins of PLD2 fragments were incubated with
purified PLC- 1. The amounts of GST and GST fusion proteins were
visualized on nitrocellulose membrane by Ponceau staining (lower
panel). Data are representative of three independent experiments.
C, COS-7 cells were transiently transfected with the empty
vector (Vector), wild type murine PLD2 (PLD2), or
N-terminal 308-amino acid-truncated murine PLD2
(PLD2( 1-308)). Cells growing in media
containing 10% bovine calf serum were lysed with IP buffer, and
immunoprecipitation with anti-PLD antibody was performed. Data are
representative of two independent experiments. D, GST
protein alone or GST fusion proteins of PLD2 F1 fragment and
C-terminally deleted constructs of PLD2 F1 fragment were incubated with
purified PLC- 1. The amount of GST and GST fusion proteins were
visualized on the nitrocellulose membrane by Ponceau staining
(lower panel). Data are representative of three independent
experiments. E, COS-7 cells were transfected with the empty
vector, human PLD2 wild type, or human PLD2 mutant (PLD2
(P145L/P148L)). Cell lysates underwent
immunoprecipitation with anti-PLD antibody, and immune complexes were
washed three more times with binding buffer. In vitro
binding analysis with purified PLC- 1 and immune complexes were
performed. Data are representative of two independent
experiments.
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|
The SH3 Domain of PLC-
1 Specifically Binds to the PX Domain of
PLD2--
It is generally known that the selectivity of the SH3
domains for their ligands is marginal (53). We tested whether the interaction between the SH3 domain of PLC-
1 and the PX domain of
PLD2 was specific or not. First, we performed in vitro
binding analysis with purified PLD2 and GST fusion SH3 domain of
PLC-
1, Abl, and CrkI and found that PLD2 binds strongly to the SH3
domain of PLC-
1 but not to that of Abl or CrkI (Fig.
5A). Second, we performed
in vitro binding analysis with purified PLC-
1 and the GST
fusion PX domain of PLD2, p40phox, and p47phox and
found that PLC-
1 binds to the PX domain of PLD2 but not to the PX
domain of p40phox or p47phox (Fig. 5B).
These results indicate that the interaction between the SH3 domain of
PLC-
1 and the PX domain of PLD2 is specific.

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Fig. 5.
The SH3 domain of
PLC- 1 specifically binds to the PX domain of
PLD2. A, GST protein alone or the GST fusion proteins
of PLC- 1, Abl, and CrkI SH3 domain were incubated with purified
PLD2. The amounts of GST and GST fusion proteins were visualized on the
nitrocellulose membrane by Ponceau staining (lower panel).
Data are representative of three independent experiments. B,
GST protein alone or GST fusion proteins of PLD2, p40phox, and
p47phox PX domain were incubated with purified PLC- 1. The
amounts of GST and GST fusion proteins were visualized on the
nitrocellulose membrane by Ponceau staining (lower panel).
Data are representative of three independent experiments.
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|
Interaction Is Required for Increasing EGF-induced PLC-
1
Activity by PLD2--
To identify the role of their interaction in EGF
signaling, EGF-induced IP3 formation and Ca2+
increase were measured after transfecting PLD2 wild type or PLD2 mutant
(P145L/P148L) into COS-7 cells. EGF-induced IP3 formation and [Ca2+]i increase were potentiated in PLD2
wild type-transfected cells but not in PLD2 mutant-transfected cells
(Fig. 6, A and B).
The basal activities of PLD2 wild type and PLD2 mutant show no
difference, but the EGF-induced activity of PLD2 mutant decreased versus PLD2 wild type, indicating that PLC-
1 activation
is crucial for PLD2 activation in these cells (Fig. 6C), and
PLC-
1 bound only to PLD2 wild type and not to mutant after EGF
stimulation (Fig. 6D). These results indicate that the
interaction between PLC-
1 and PLD2 may be important for
EGF-induced IP3 formation and [Ca2+]i
increase.

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Fig. 6.
The interaction between
PLC- 1 and PLD2 potentiates EGF-induced
PLC- 1 activity. COS-7 cells were
transfected with the empty vector, human PLD2 wild type, or human PLD2
mutant (PLD2 (P145L/P148L)). A,
after serum starvation for 24 h, EGF stimulation was performed for
30 s. IP3 generation was measured by using a
[3H]IP3 competition assay. The open
bar without any treatment in vector cells was considered as
control. Means ± S.E. from three independent assays are shown.
B, after serum starvation for 24 h,
[Ca2+]i increase after 30 s of EGF
stimulation was measured. Means ± S.E. from two independent
assays are shown. C, after serum starvation for 24 h,
cells were treated with EGF. Phosphatidylbutanol accumulation
for 1 min was measured. Means ± S.E. from two independent assays
are shown. D, after serum starvation for 24 h, EGF
stimulation was performed for 30 s. Cell lysates underwent
immunoprecipitation with anti-PLD antibody. Data are representative of
two independent experiments.
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PLD2 Has No Effect on the EGF-induced Tyrosine Phosphorylation of
PLC-
1--
There remained a possibility that PLD2 might affect the
tyrosine phosphorylation of PLC-
1 to potentiate IP3
formation and [Ca2+]i increase. To test this
possibility, we checked the EGF-induced tyrosine phosphorylation of
PLC-
1 after transfecting PLD2 wild type or PLD2 mutant (P145L/P148L)
into COS-7 cells. PLD2 transfection was found to have no effect on the
EGF-induced tyrosine phosphorylation of PLC-
1 (Fig.
7). This result indicates that the
potentiation of IP3 formation and Ca2+ increase
is not due to the change of the tyrosine phosphorylation of
PLC-
1.

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Fig. 7.
PLD2 has no effect on the EGF-induced
tyrosine phosphorylation of PLC- 1. COS-7
cells were transfected with the empty vector, human PLD2 wild type, or
human PLD2 mutant (PLD2 (P145L/P148L)). After
serum starvation for 24 h, cells were preincubated with 0.1 mM sodium vanadate for 10 min, and then cells were
stimulated with EGF for 1 min. Cells were lysed with IP buffer
containing 1 mM sodium vanadate and underwent
immunoprecipitation with anti-PLC- 1 antibody. Data are
representative of two independent experiments.
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|
 |
DISCUSSION |
In this study, we report for the first time upon the phospholipase
complex of PLC-
1 and PLD2. The communication between PLC-
1 and
PLD has been questioned because EGF stimulation activates PLC-
1 and
PLD2 both and because in each signaling pathway there are common
molecules such as PKC and PIP2 (22, 23). PIP2, the substrate of PLC-
1, was reported to be a cofactor for PLD activation (16). It was also reported that PLC-
1 influenced PLD
activity through PKC, suggesting that PLC-
1 might be an upstream regulator of PLD (26, 33, 54). PA, the product of PLD action, increased
PLC-
1 activity in vitro (34). However, these were an
indirect communications. In the present study, we found that PLC-
1
binds directly to PLD2, which may provide a new perspective on the
communication between PLC-
1 and PLD. This interaction may increase
the efficacy of their communication via other cellular components such
as PIP2 and PKC, and the interaction itself may have an
important role. In this report, we propose that PLD2 may function as an
adaptor in the redistributing of PLC-
1 to the membrane region. This
is an as yet unidentified function of PLD2, and we suggest that PLD2
can function as an upstream regulator of PLC-
1 in EGF signaling.
Upon EGF stimulation, PLC-
1 is recruited from the cytosol to the
plasma membrane where EGF receptor is activated (29), but how PLC-
1
has access to PIP2 is not fully understood. In the
recruitment of PLC-
1, two SH2 domains of PLC-
1 have reported to
play critical roles (15). After being phosphorylated at tyrosine residues, PLC-
1 is redistributed to its substrate-enriched region. We suggest that the SH3 domain of PLC-
1 may contribute to the redistribution process. Little is known of the roles of the PLC-
1 SH3 domain in EGF-induced PIP2 hydrolysis. Our findings
suggest that the SH3 domain of PLC-
1 may increase its substrate
accessibility by bringing PLC-
1 into the vicinity of
PIP2. It was also reported that a PLC-
1 mutant
lacking the SH3 domain showed reduced membrane association and activity
(55). The binding partner of the PLC-
1 SH3 domain in the membrane,
however, has not been identified. We found that PLD2 binds to the SH3
domain of PLC-
1. PLD2 is a membrane protein and requires
PIP2 for its activity, which means that PLD2 should be in
the membrane region where PIP2 resides. Several reports
show that PLD2 is mainly localized to the membrane substructure where
PIP2 is enriched (25, 56, 57). We suggest that PLD2 is the
binding partner of the PLC-
1 SH3 domain in its redistribution.
Nevertheless, the SH3 domain may not be the only domain responsible for
this redistribution, because the deletion of the SH3 domain did not
result in the complete inhibition of PLC-
1 activity (55). Membrane
association was also facilitated by the interaction between the C-SH2
or the pleckstrin homology (PH) domain of PLC-
1 and PIP3
(58, 59). Prevention of PIP3 generation by inhibiting
phosphatidylinositol 3-kinase with wortmannin or LY294002 resulted in
an ~40% decrease in PLC activity (60). Considering our findings and
those of previous studies, we suggest that the SH3 domain of PLC-
1,
together with the C-SH2 and the PH domain, play an important role in
the redistribution of PLC-
1 to increase its substrate accessibility.
PLD2 has a region consisting of the PX and the PH domains whose exact
roles have not been clarified. Our results demonstrate that PLC-
1
binds directly to the PX domain of PLD2. This finding suggests that the
PX domain of PLD2 serves as an adaptor for other proteins. In the
53-amino acid region responsible for PLC-
1 binding (Fig.
4D), there is a proline-rich, canonical SH3 domain binding motif. Mutating prolines in this motif to leucines abrogates
interaction with PLC-
1 (Fig. 4E). Although it cannot be
said that this mutation has no effect on the conformation of PLD2 PX,
it does not change the expression level, subcellular localization, or
catalytic properties of PLD2 in vitro (data not shown).
These results suggest that the effect of this mutation on the PLD2
structure, if any, is neither dramatic nor significant for the PLD2
function except for the PLC-
1 interaction. Other proteins containing
the SH3 domain may also use the PX domain of PLD2 as an adaptor, but it seems likely that the interaction is most specific to the PLC-
1 SH3
domain (Fig. 5A). The specificity of the PX domains to bind PLC-
1 has also been proven to be unique (Fig. 5B).
Recently, it was reported that the PX domain of p47phox binds
to its own C-terminal SH3 domain (69). Karathanassis et al.
(70) suggested that p47phox-PX would have to undergo a
conformational change to interact with its C-SH3 domain. However, this
prediction may not fit with PLD2-PX because PLC-
1 does not interact
with the p47phox-PX (Fig. 5B). The PX domain
structure of PLD2 may not be exactly the same as that of
p47phox. It is also known that PX domains bind to
phosphoinositides and play critical roles in the intracellular
localization of a variety of cell-signaling proteins, including
p47phox and p40phox (61). Because PLD binds to
PIP2, requires PIP2 for enzymatic activity, and
localizes in PIP2-enriched membranes (16, 25), the PX
domain of PLD may exist in proximity to PIP2. Considering our findings and previous studies, we suggest that the PX domain of
PLD2 contributes to increasing the substrate accessibility of PLC-
1
by functioning as an adaptor for its SH3 domain.
The mechanism of the interaction between PLC-
1 and PLD2 is the issue
at question. We found that the interaction between PLC-
1 and PLD2 is
dependent on EGF stimulation and is transient (Fig. 2C). In
the COS-7 cells, it was estimated that ~0.5% of the total PLC-
1
translocates to the caveolae after EGF stimulation (data not shown).
This increases to 1% after PLD2 transfection. The co-immunoprecipitated PLC-
1 almost equals this percentage. Without EGF stimulation, PLC-
1 showed no interaction with PLD2; however, after EGF stimulation, PLC-
1 bound to PLD2, and this interaction disappeared 1 min after EGF stimulation. We observed that the activity
of PLC-
1 after EGF stimulation was highest at around the time when
PLC-
1 interacts with PLD2 (Fig. 2B). Because the overexpression of PLD2 in COS-7 cells potentiates the EGF-induced IP3 formation and [Ca2+]i increase
(Fig. 6, A and B), it is possible that PLD2 augments PLC-
1 recruitment and its tyrosine phosphorylation. However, as shown in Fig. 7, PLD2 dose not affect PLC-
1 tyrosine phosphorylation levels. PLD2 may contribute to bring PLC-
1 to the
proximity of PIP2, and PLC-
1 may be redistributed to the membrane by binding with PLD2. Our findings show that the interaction between the SH3 domain of PLC-
1 and the PX domain of PLD2 is important for EGF-induced Ca2+ signaling. These results
also suggest that the signaling pathways of PLC-
1 and PLD2 may
cooperatively propagate.
Cell proliferation plays a fundamental role in the development and
maintenance of organisms. EGF is known to influence cell proliferation,
and the EGF receptor is the receptor most often found to be
up-regulated in a wide variety of human tumors (62). Cell
proliferation, however, requires the triggering of numerous downstream
signaling pathways. These pathways include those that involve PLC-
1
and its downstream Ca2+- and PKC-mediated cascades and PLD.
In many tumors there is no increase in the number of EGF receptors but,
rather, its signaling is up-regulated (63). The enzymatic activity of
PLC-
1 is required for EGF-induced cell cycle progression into the S
phase (2). Furthermore, PLC-
1 signaling is regarded as a convergence
point for a number of motility- and/or invasion-inducing pathways in cancers (64). Evidence shows that PLC-
1 and PLD2 are overexpressed in many cancers (65-68), and, in the present study, we found that the
direct interaction between PLC-
1 and PLD2 is important for EGF
signaling. The possibility of the involvement of the coordination between PLC-
1 and PLD2 in tumor cell mitogenesis or movement is an
important issue for future studies.
The abbreviations used are:
EGF, epidermal
growth factor;
PLC, phospholipase C;
PLD, phospholipase D;
PIP2, phosphatidylinositol 4,5-bisphosphate;
IP3, inositol 1,4,5-trisphosphate;
PKC, protein kinase C;
SH, Src homology;
PH, pleckstrin homology;
PX, Phox homology;
PA, phosphatidic acid;
ECL, enhanced chemiluminescence;
GST, glutathione
S-transferase.
1.
|
Ciardiello, F.
(2000)
Drugs
60,
25-32[Medline]
[Order article via Infotrieve]
|
2.
|
Wang, Z.,
Glück, S.,
Zhang, L.,
and Morgan, M. F.
(1998)
Mol. Cell. Biol.
18,
590-597[Abstract/Free Full Text]
|
3.
|
Pei, Z.-D.,
and Williamson, J. R.
(1998)
FEBS Lett.
423,
53-56[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Yu, H.,
Fukami, K.,
Itoh, T.,
and Takenawa, T.
(1998)
Exp. Cell Res.
243,
113-122[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Berridge, M. J.
(1984)
Biochem. J.
220,
345-360[Medline]
[Order article via Infotrieve]
|
6.
|
Rhee, S. G.,
Suh, P.-G.,
Ryu, S. H.,
and Lee, S. Y.
(1989)
Science
244,
546-550[Medline]
[Order article via Infotrieve]
|
7.
|
Rhee, S. G.,
and Bae, Y. S.
(1997)
J. Biol. Chem.
272,
15045-15048[Free Full Text]
|
8.
|
Song, C.,
Hu, C.-D.,
Masago, M.,
Kariya, K.-I.,
Yamawaki-Kataoka, Y.,
Shibatohge, M.,
Wu, D.,
Satoh, T.,
and Kataoka, T.
(2001)
J. Biol. Chem.
276,
2752-2757[Abstract/Free Full Text]
|
9.
|
Suh, P.-G.,
Ryu, S. H.,
Moon, K. H.,
Suh, H. W.,
and Rhee, S. G.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
5419-5423[Abstract]
|
10.
|
Rhee, S. G.
(1991)
Trends Biochem. Sci.
16,
297-301[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Martin, T. F. J.
(1991)
Pharmacol. Ther.
49,
329-345[Medline]
[Order article via Infotrieve]
|
12.
|
Kim, U. H.,
Kim, H. S.,
and Rhee, S. G.
(1990)
FEBS Lett.
270,
33-36[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Gergel, J. R.,
McNamara, D. J.,
Dobrusin, E. M.,
Zhu, G.,
Saltiel, A. R.,
and Miller, W. T.
(1994)
Biochemistry
33,
14671-14678[Medline]
[Order article via Infotrieve]
|
14.
|
Anderson, D.,
Koch, C. A.,
Grey, L.,
Ellis, C.,
Moran, M. F.,
and Pawson, T.
(1990)
Science
250,
979-982[Medline]
[Order article via Infotrieve]
|
15.
|
Chattopadhyay, A.,
Vecchi, M.,
Ji, Q.-S.,
Mernaugh, R.,
and Carpenter, G.
(1999)
J. Biol. Chem.
274,
26091-26097[Abstract/Free Full Text]
|
16.
|
Frohman, M. A.,
Sung, T. C.,
and Morris, A. J.
(1999)
Biochim. Biophys. Acta
1439,
175-186[Medline]
[Order article via Infotrieve]
|
17.
|
Jones, D.,
Morgan, C.,
and Cockcroft, S.
(1999)
Biochim. Biophys. Acta
1439,
229-244[Medline]
[Order article via Infotrieve]
|
18.
|
Danniel, L. W.,
Sciorra, V. A.,
and Ghosh, S.
(1999)
Biochim. Biophys. Acta
1439,
265-276[Medline]
[Order article via Infotrieve]
|
19.
|
Cross, M. J.,
Roberts, S.,
Ridley, A. J.,
Hodgkin, M. N.,
Stewart, A.,
Claesson-Welsh, L.,
and Wakelam, M. J. O.
(1996)
Curr. Biol.
6,
588-597[Medline]
[Order article via Infotrieve]
|
20.
|
Rizzo, M. A.,
Shome, K.,
Vasudevan, C.,
Stolz, D. B.,
Sung, T. C.,
Frohman, M. A.,
Watkins, S. C.,
and Romero, G.
(1999)
J. Biol. Chem.
274,
1131-1139[Abstract/Free Full Text]
|
21.
|
Exton, J. H.
(1999)
Biochim. Biophys. Acta
1439,
121-133[Medline]
[Order article via Infotrieve]
|
22.
|
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[Abstract/Free Full Text]
|
23.
|
Sciorra, V. A.,
Rudge, S. A.,
Prestwich, G. D.,
Frohman, M. A.,
Engebrecht, J.,
and Morris, A. J.
(1999)
EMBO J.
18,
5911-5921[Abstract/Free Full Text]
|
24.
|
Kim, Y.,
Han, J. M.,
Park, J. B.,
Lee, S. D.,
Oh, Y. S.,
Chung, C.,
Lee, T. G.,
Kim, J. H.,
Park, S.-K.,
Yoo, J.-S.,
Suh, P.-G.,
and Ryu, S. H.
(1999)
Biochemistry
38,
10344-10351[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Xu, L.,
Shen, Y.,
Joseph, T.,
Bryant, A.,
Luo, J.-O.,
Frankel, P.,
Rotunda, T.,
and Foster, D. A.
(2000)
Biochem. Biophys. Res. Commun.
273,
77-83[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Hornia, A.,
Lu, Z.,
Sukezane, T.,
Zhong, M.,
Joseph, T.,
Frankel, P.,
and Foster, D. A.
(1999)
Mol. Cell. Biol.
19,
7672-7680[Abstract/Free Full Text]
|
27.
|
Couet, J.,
Sargiacomo, M.,
and Lisanti, M. P.
(1997)
J. Biol. Chem.
272,
30429-30438[Abstract/Free Full Text]
|
28.
|
Pike, L. J.,
and Miller, J. M.
(1998)
J. Biol. Chem.
273,
22298-22304[Abstract/Free Full Text]
|
29.
|
Jang, I.-H.,
Kim, J. H.,
Lee, B. D.,
Bae, S. S.,
Park, M. H.,
Suh, P.-G.,
and Ryu, S. H.
(2001)
FEBS Lett.
491,
4-8[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Kim, J. H.,
Han, J. M.,
Lee, S.,
Kim, Y.,
Lee, T. G.,
Park, J. B.,
Lee, S. D.,
Suh, P.-G.,
and Ryu, S. H.
(1999)
Biochemistry
38,
3763-3769[CrossRef][Medline]
[Order article via Infotrieve]
|
31.
|
Pike, L. J.,
and Casey, L.
(1996)
J. Biol. Chem.
271,
26453-26456[Abstract/Free Full Text]
|
32.
|
Voss, M.,
Weernink, P. A.,
Haupenthal, S.,
Moler, U.,
Cool, R. H.,
Bauer, B.,
Camonis, J. H.,
Jakobs, K. H.,
and Schmidt, M.
(1999)
J. Biol. Chem.
274,
34691-34698[Abstract/Free Full Text]
|
33.
|
Hess, J. A.,
Ji, Q.-S.,
Carpenter, G.,
and Exton, J. H.
(1998)
J. Biol. Chem.
273,
20517-20524[Abstract/Free Full Text]
|
34.
|
Jones, G. A.,
and Carpenter, G.
(1993)
J. Biol. Chem.
268,
20845-20850[Abstract/Free Full Text]
|
35.
|
Kim, J. H.,
Kim, Y.,
Lee, S. D.,
Lopez, I.,
Arnold, R. S.,
Lambeth, J. D.,
Suh, P.-G.,
and Ryu, S. H.
(1999)
FEBS Lett.
454,
42-46[CrossRef][Medline]
[Order article via Infotrieve]
|
36.
|
Bae, S. S.,
Lee, Y. H.,
Chang, J.-S.,
Galadari, S. H.,
Kim, Y. S.,
Ryu, S. H.,
and Suh, P.-G.
(1998)
J. Neurochem.
71,
178-185[Medline]
[Order article via Infotrieve]
|
37.
|
Bahk, Y. Y.,
Lee, Y. H.,
Lee, T. G.,
Seo, J.,
Ryu, S. H.,
and Suh, P.-G.
(1994)
J. Biol. Chem.
269,
8240-8245[Abstract/Free Full Text]
|
38.
|
Kim, M. J.,
Chang, J.-S.,
Park, S. K.,
Hwang, J.-I.,
Ryu, S. H.,
and Suh, P.-G.
(2000)
Biochemistry
39,
8674-8682[CrossRef][Medline]
[Order article via Infotrieve]
|
39.
|
Lee, S.,
Kim, J. H.,
Lee, C. S.,
Kim, J. H.,
Kim, Y.,
Heo, K.,
Ihara, Y.,
Goshima, Y.,
Suh, P.-G.,
and Ryu, S. H.
(2002)
J. Biol. Chem.
277,
6542-6549[Abstract/Free Full Text]
|
40.
|
Lee, S.,
Park, J. B.,
Kim, J. H.,
Kim, Y.,
Kim, J. H.,
Shin, K.-J.,
Lee, J. S.,
Ha, S. H.,
Suh, P.-G.,
and Ryu, S. H.
(2001)
J. Biol. Chem.
276,
28252-28260[Abstract/Free Full Text]
|
41.
|
Bae, S. S.,
Perry, D. K.,
Oh, Y. S.,
Choi, J. H.,
Galadari, S. H.,
Ghayur, T.,
Ryu, S. H.,
Hannun, Y. A.,
and Suh, P.-G.
(2000)
FASEB J.
14,
1083-1092[Abstract/Free Full Text]
|
42.
|
Kim, J. H.,
Lee, S. D.,
Han, J. M.,
Lee, T. G.,
Kim, Y.,
Park, J. B.,
Lambeth, J. D.,
Suh, P.-G.,
and Ryu, S. H.
(1998)
FEBS Lett.
430,
231-235[CrossRef][Medline]
[Order article via Infotrieve]
|
43.
|
Kim, J. H.,
Lee, S.,
Kim, J. H.,
Lee, T. G.,
Hirata, M.,
Suh, P.-G.,
and Ryu, S. H.
(2002)
Biochemistry
41,
3414-3421[CrossRef][Medline]
[Order article via Infotrieve]
|
44.
|
Sparks, A. B.,
Hoffman, N. G.,
McConnell, S. J.,
Fowlkes, D. M.,
and Kay, B. K.
(1996)
Nat. Biotechnol.
14,
741-744[Medline]
[Order article via Infotrieve]
|
45.
|
Kanai, F.,
Liu, H.,
Field, S. J.,
Akbary, H.,
Matsuo, T.,
Brown, G. E.,
Cantley, L. C.,
and Yaffe, M. B.
(2001)
Nat. Cell Biol.
3,
675-678[CrossRef][Medline]
[Order article via Infotrieve]
|
46.
|
Lee, C.,
Kim, S. R.,
Chung, J. K.,
Frohman, M. A.,
Kilimann, M. W.,
and Rhee, S. G.
(2000)
J. Biol. Chem.
275,
18751-18758[Abstract/Free Full Text]
|
47.
|
Ho, S. N.,
Hunt, H. D.,
Horton, R. M.,
Pullen, J. K.,
and Pease, L. R.
(1989)
Gene (Amst.)
77,
51-59[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
|
49.
|
Suh, B. C.,
Chae, H. D.,
Chung, J. H.,
and Kim, K. T.
(1999)
Br. J. Pharmacol.
126,
399-406[Abstract/Free Full Text]
|
50.
|
Challiss, R. A.,
Chilvers, E. R.,
Willcocks, A. L.,
and Nahorski, S. R.
(1990)
Biochem. J.
265,
421-427[Medline]
[Order article via Infotrieve]
|
51.
|
Kim, J. H.,
Lee, B. D.,
Kim, Y.,
Lee, S. D.,
Suh, P.-G.,
and Ryu, S. H.
(1999)
J. Immunol.
163,
5462-5470[Abstract/Free Full Text]
|
52.
|
Grynkiewicz, G.,
Poenie, M.,
and Tsien, R. Y.
(1985)
J. Biol. Chem.
260,
3440-3450[Abstract]
|
53.
|
Mayer, B. J.
(2001)
J. Cell Sci.
114,
1253-1263[Abstract/Free Full Text]
|
54.
|
Slaaby, R.,
Du, G.,
Altshuller, Y. M.,
Frohman, M. A.,
and Seedorf, K.
(2000)
Biochem. J.
351,
613-619[CrossRef][Medline]
[Order article via Infotrieve]
|
55.
|
Debell, K. E.,
Stoica, B. A.,
Veri, M.-C.,
Baldassarre, A. D.,
Miscia, S.,
Graham, L. J.,
Rellahan, B. L.,
Ishiai, M.,
Kurosaki, T.,
and Bonvini, E.
(1999)
Mol. Cell. Biol.
19,
7388-7398[Abstract/Free Full Text]
|
56.
|
Czarny, M.,
Lavie, Y.,
Fiucci, G.,
and Liscovitch, M.
(1999)
J. Biol. Chem.
274,
2717-2724[Abstract/Free Full Text]
|
57.
|
Liscovitch, M.,
Czarny, M.,
Fiucci, G.,
Lavie, Y.,
and Tang, X.
(1999)
Biochim. Biophys. Acta
1439,
245-263[Medline]
[Order article via Infotrieve]
|
58.
|
Falasca, M.,
Logan, S. K.,
Lehto, V. P.,
Baccante, G.,
Lemmon, M. A.,
and Schlessinger, J.
(1998)
EMBO J.
17,
414-422[Abstract/Free Full Text]
|
59.
|
Bae, Y. S.,
Cantley, L. G.,
Chen, C. S.,
Kim, S. R.,
Kwon, K. S.,
and Rhee, S. G.
(1998)
J. Biol. Chem.
273,
4465-4469[Abstract/Free Full Text]
|
60.
|
Rameh, L. E.,
Rhee, S. G.,
Spokes, K.,
Kazlauskas, A.,
Cantley, L. C.,
and Cantley, L. G.
(1998)
J. Biol. Chem.
273,
23750-23757[Abstract/Free Full Text]
|
61.
|
Sato, T. K.,
Overduin, M.,
and Emr, S. D.
(2001)
Science
294,
1881-1885[Abstract/Free Full Text]
|
62.
|
Mendelsohn, J.
(2001)
Endocr. Relat. Cancer
8,
3-9[Abstract/Free Full Text]
|
63.
|
Wells, A.
(1999)
Int. J. Biochem. Cell Biol.
31,
637-643[CrossRef][Medline]
[Order article via Infotrieve]
|
64.
|
Kassis, J.,
Lauffenburger, D. A.,
Turner, T.,
and Wells, A.
(2001)
Semin. Caner Biol.
11,
105-117[CrossRef]
|
65.
|
Noh, D. Y.,
Lee, Y. H.,
Kim, S. S.,
Kim, Y. I.,
Ryu, S. H.,
Suh, P.-G.,
and Park, J.-G.
(1994)
Cancer
73,
36-41[Medline]
[Order article via Infotrieve]
|
66.
|
Chang, J.-S.,
Noh, D. Y.,
Park, I. A.,
Kim, M. J.,
Song, H.,
Ryu, S. H.,
and Suh, P.-G.
(1997)
Cancer Res.
57,
5465-5468[Abstract]
|
67.
|
Zhao, Y.,
Ehara, H.,
Akao, Y.,
Shamoto, M.,
Nakagawa, Y.,
Banno, Y.,
Deguchi, T.,
Ohishi, N.,
Yagi, K.,
and Nozawa, Y.
(2000)
Biochem. Biophys. Res. Commun.
278,
140-143[CrossRef][Medline]
[Order article via Infotrieve]
|
68.
|
Min, D. S.,
Kwon, T. K.,
Park, W.-S.,
Chang, J.-S.,
Park, S.-K.,
Ahn, B.-H.,
Ryoo, Z.-Y.,
Lee, Y. H.,
Lee, Y. S.,
Rhie, D.-J.,
Yoon, S.-H.,
Hahn, S. J.,
Kim, M.-S.,
and Jo, Y.-H.
(2001)
Carcinogenesis
22,
1641-1647[Abstract/Free Full Text]
|
69.
|
Hiroaki, H.,
Ago, T.,
Ito, T.,
Sumimoto, H.,
and Kohda, D.
(2001)
Nat. Struct. Biol.
8,
526-530[CrossRef][Medline]
[Order article via Infotrieve]
|
70.
|
Karathanassis, D.,
Stahelin, R.,
Bravo, J.,
Perisic, O.,
Pacold, C. M.,
Cho, W.,
and Williams, R. L.
(2002)
EMBO J.
21,
5057-5068[Abstract/Free Full Text]
|