From the Department of Urology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9110
Received for publication, October 17, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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DOC-2/DAB2 is a potent tumor suppressor in
many cancer types including prostate cancer. In prostate cancer,
expression of DOC-2/DAB2 can inhibit its growth. Our recent studies
demonstrate that DOC-2/DAB2 can suppress both protein kinase C and
peptide growth factor-elicited signal pathways via the
Ras-mitogen-activated protein kinase pathway. In this study, we further
showed that the proline-rich domain of DOC-2/DAB2 could also interact
with proteins containing the Src homology 3 domain, such as Src and Fgr. The binding of c-Src to DOC-2/DAB2 was enhanced in cells treated
with growth factor, and this interaction resulted in c-Src inactivation. The c-Src inactivation was evidenced by the decreased tyrosine 416 phosphorylation of c-Src and reduced downstream effector activation. It appears that DOC-2/DAB2 can bind to Src homology 3 domain of c-Src and maintain it in an inactive conformation. Thus, this
study provides a new mechanism for modulating c-Src in prostatic
epithelium and cancer.
DOC-2/DAB2 (differentially expressed in
ovarian
cancinoma-2/disabled-2)
is a gene that encodes a novel phosphoprotein involved in signal
transduction (1-7). The aberrant expression of DOC-2/DAB2 is often in
tumors such as ovarian, prostate, choriocarcinoma, and mammary tumors
(1, 3, 8-10). Increased expression of DOC-2/DAB2 inhibits the growth
of these tumors, indicating that DOC-2/DAB2 must play a key role in
controlling growth-related signal pathways.
The phosphorylation of DOC-2/DAB2 can be induced by several stimuli,
such as growth factors and protein kinase C
activator-TPA.1 We
demonstrate that the serine 24 phosphorylation in the N terminus of
DOC-2/DAB2 is required for its inhibitory effect on TPA-induced gene
transcription (5). Using yeast two-hybrid system, we further identify
an interactive protein (i.e. DIP1/2 or DAB2IP) associated with the N terminus of DOC-2/DAB2 protein (7). DIP1/2 protein is a new
member of the Ras-GAP family, and its activity can be enhanced by
interacting with DOC-2/DAB2 in prostate cancer (PCa) cells treated with
TPA (7), which results in inhibiting TPA-induced gene transcription and
cell growth. Therefore, we conclude that DIP1/2 is a key downstream
effector in DOC-2/DAB2-mediated signal cascade.
In addition, the C terminus of DOC-2/DAB2 contains unique
motifs such as three proline-rich domains (i.e. amino acid
619-627, 663-671, and 714-722). We demonstrated recently (6) that
one of these proline-rich domains (amino acid 663-671) can interact with Grb2, leading to the inhibition of both epidermal growth factor
(EGF)- and neurotropin (NT-3)-induced Erk activation and gene
transcription (6). Thus, DOC-2/DAB2 acts a negative feedback regulator
in the peptide growth factor-mediated Ras-mitogen-activated protein
kinase signal pathway.
In this study, we further dissected the role of other proline-rich
domains in the activity of DOC-2/DAB2. We found that the SH3 domain in
c-Src, a non-receptor tyrosine kinase, could interact with the first
proline-rich domain (amino acid 619-627) of DOC-2/DAB2, and the amount
of DOC-2/DAB2/c-Src complex was accumulated in PCa cells shortly
treated with EGF. Our results demonstrated that such interaction could
lead to the inhibition of tyrosine 416 phosphorylation of c-Src, a key
amino acid modulating its kinase activity, in PCa treated with EGF.
This interaction further resulted in the downstream effector-Erk
inactivation. Apparently, this is a new regulatory mechanism of Src
activity mediated by a potent negative factor, DOC-2/DAB2.
Cell Lines, Synthetic Peptides, and Plasmid
Constructs--
LNCaP, NbE, and COS cells were maintained in T medium
supplemented with 5% fetal bovine serum (3). The following peptides were synthesized according to amino acid sequence of DOC-2/DAB2: PPQ
(amino acid 619-627); LLQ (amino acid 619-627, proline to leucine);
PPL (amino acid 663-671); LLL (amino acid 663-671, proline to
leucine); and PPK (amino acid 714-722) (6). All DOC-2/DAB2 cDNA
constructs, pCI-neo-T7-p82 (p82) and pCI-neo-T7- Cell Transfection--
For transient transfection, cells were
plated 24 h prior to transfection using LipofectAMINE PLUS reagent
(Invitrogen). In each experiment, the control plasmid (pCI-neo) was
supplemented to make an equal amount of total DNA. Twenty-four h after
transfection, cells were switched to serum-free T medium for another
24 h prior to the treatment with 50 ng/ml EGF (Upstate Biotechnology).
For peptide transfection, cells were plated in a 24-well plate with
serum-free medium for 24 h. ChariotTM reagent (Active
Motif) was mixed with 100 ng of different oligopeptides according to the manufacturer's protocol. One h after transfection, cells were treated with EGF (50 ng/ml), and cell lysate was prepared at
the indicated time.
GST Pull-down and Co-immunoprecipitation Assay--
For GST
pull-down assay, cells were exposed to 50 ng/ml of EGF, and cell lysate
was collected in 0.5 ml of lysis buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 5 mM EDTA supplemented with 1% Triton X-100, and a mixture of protease inhibitors) at the indicated time. After a low speed spin, 0.4 ml of supernatant was
separately incubated with either 30 µl of GST-glutathione-Sepharose or GST fusion protein-Sepharose overnight at 4 °C. The next day, the
pellet was washed twice with lysis buffer and dissolved in the sample
buffer and then subjected to Western blot analysis probed with
antibodies against DOC-2/DAB2 (
For co-immunoprecipitation, cell lysate was collected in 0.5 ml
of lysis buffer. After a low speed spin, 0.4 ml of supernatant was
incubated with 1 µg of antibody against Src (Oncogene Research Products) and then 40 µl of protein A-Sepharose (Amersham
Biosciences) overnight at 4 °C. The pellet was washed twice
with lysis buffer and dissolved in sample buffer and then subjected to
Western blot analysis detected by Detection of c-Src and Erk2 Protein Phosphorylation--
For
determining phosphorylation status of Src, cells were transfected with
Src expression vectors and exposed to 50 ng/ml EGF for 10 min. Cell
lysate was collected in 70 µl of phosphate-buffered saline (with 1%
Triton X-100 and a mixture of protease inhibitors). After a low speed
spin, 20 µl of supernatant was subjected to Western blot analysis.
The filter was probed with the antibody against phosphorylated Src
antibodies against either tyrosine 416 (
For determining phosphorylation status of Erk2 protein, cells were
transfected with hemagglutinin-Erk2 and exposed to 50 ng/ml EGF for 10 min. Cell lysate was collected in 0.5 ml of lysis buffer. After a low
speed spin, 0.4 ml of supernatant was immunoprecipitated with
hemagglutinin-Matrix (Covance). After washing twice with lysis buffer,
the pellets were added with sample buffer and subjected to Western blot
analysis. The filter was probed with the antibody against
phosphorylated extracellular signal-regulated kinase p44/42 ( DOC-2/DAB2 Interacts with Src through
SH3/Proline-rich Domain Interaction--
DOC-2/DAB2
contains three proline-rich domains indicating that the potential
interaction with other proteins containing SH3 domain, in addition to
Grb2 (4-6), should be expected. To screen the interaction with
proteins containing SH3 domain, we performed pull-down experiment using
several GST fusion proteins derived from three groups of proteins
containing SH3 domain with different functions. For adapter proteins,
Grb2 has been shown to interact with p82 (non-spliced DOC-2/DAB2
protein) previously (4-6); Crk did not show any detectable interaction
with p82 (Fig. 1A). For the
non-receptor Src tyrosine kinase family, both c-Src and Fgr could
interact with p82 (Fig. 1A). However, no detectable
interaction was shown between p82 and the SH3 domain of spectrin, an
actin-binding protein (Fig. 1A). Apparently, this
interaction required the C-terminal of DOC-2/DAB2, because the
N-terminal deletion of DOC-2/DAB2 (
We further mapped the specific site of proline-rich domain in
DOC-2/DAB2 associated with the Src protein; several proline-rich peptides were synthesized according to the three proline-rich domains
in DOC-2/DAB2 (6). As shown in Fig. 1B, peptide PPL corresponding to the second proline-rich domain of DOC-2/DAB2 could
interrupt the association of Grb2 with DOC-2/DAB2 in a
dose-dependent manner, whereas the control peptide LLL,
substitution of all the proline in PPL with leucine did not have any
effect (Fig. 1B, bottom panel). Also, the first
and third proline-rich peptides (PPQ and PPK) did not have any effect
on interaction of Grb2 with DOC-2/DAB2.
On the other hand, both PPQ and PPL, but not control peptide LLL and
LLQ, could interrupt the interaction between c-Src and DOC-2/DAB2 (Fig.
1B, top panel). Also, PPK did not have any
significant inhibitory effect. These data indicated that the first and
second proline-rich domains of DOC-2/DAB2 could interact with c-Src
with a similar affinity (Fig. 1C).
EGF Enhances the Association of DOC-2/DAB2 with
c-Src--
To further verify the interaction of Src with DOC-2/DAB2
intracellularly, we performed co-immunoprecipitation assays. As shown in Fig. 2A, when both c-Src
and DOC-2/DAB2 were co-expressed in COS cells, the DOC-2/DAB2 was
detected in the immunocomplex precipitated with
We further determined whether EGF was able to increase the
affinity between c-Src and DOC-2/DAB2. We measured the dissociation constant between c-Src and DOC-2/DAB2 in the presence of PPQ. As shown
in Fig. 3, the increasing amount of PPQ
caused the dissociation between c-Src and DOC-2/DAB2. However, in the
presence of EGF, the IC50 of PPQ was 259 µM
compared with the IC50 of PPQ (168 µM) in the
absence of EGF. Taken together, these data indicated that EGF could
facilitate the interaction between c-Src and DOC-2/DAB2 because of the
increased the affinity between both proteins.
DOC-2/DAB2 Inhibits the Tyrosine Phosphorylation of
c-Src Protein--
It is known that c-Src has two major tyrosine
phosphorylation sites. The phosphorylation of tyrosine 416 (Tyr-416) in c-Src represents its activated status; in contrast,
the phosphorylation of tyrosine 527 (Tyr-527) in c-Src represents the
inactivate form of this protein (11-14). To further determine the
effect of DOC-2/DAB2 on the phosphorylation status of c-Src, we
examined the phosphorylation status, particularly Tyr-416 and Tyr-527,
of c-Src in LNCaP cells. As shown in Fig.
4A, the phosphorylation status
of c-Src was determined in LNCaP cells treated with EGF probed with
either
To avoid any transfection artifact, we decided to take another approach
by interrupting the interaction between DOC-2/DAB2 and c-Src in NbE
cells expressing endogenous DOC-2/DAB2 and c-Src proteins using
synthetic peptides. PPQ that inhibits the interaction between
DOC-2/DAB2 and c-Src (Fig. 2B) was transfected into NbE cells. LLQ with the substitution of proline with leucine was used as a
negative control. As shown in Fig.
5A, the phosphorylation of
c-Src was determined by using DOC-2/DAB2 Inhibits the c-Src-mediated Erk
Activation--
To understand the effect of the DOC-2/DAB2 on
c-Src-mediated signal transduction induced by EGF, the activation of
one of the downstream effectors, Erk2, was examined. As shown in Fig. 6A, EGF could stimulate Erk2
phosphorylation in LNCaP cells compared with control. In the presence
of c-Src protein, the levels of Erk2 phosphorylation further elevated
in LNCaP cells treated with EGF, indicating c-Src elicited an
alternative pathway to activate Erk2. Expression of DOC-2/DAB2
suppressed EGF alone induced Erk2 phosphorylation and c-Src-elicited
pathway leading to Erk2 phosphorylation. These data indicated that
DOC-2/DAB2 was able to inhibit c-Src activity.
The effect of DOC-2/DAB2 on the phosphorylation status of Erk induced
by c-Src was also studied in NbE cells. As shown in Fig. 6B,
EGF induced an approximately 4-fold elevation of Erk phosphorylation in
NbE cells in the absence of peptide. However, PPQ could further enhance
the EGF-mediated Erk2 phosphorylation (13-fold). Using the control
peptide LLQ, we observed a moderate induction of EGF-mediated Erk2
phosphorylation, because LLQ was a partial antagonist (Fig.
1B). These data provide further evidence that DOC-2/DAB2 is
involved in the negative feedback mechanism modulating c-Src activity
via the interaction between proline-rich domain and SH3 domain in
prostatic epithelia.
From our previous studies (5-7), apparently, DOC-2/DAB2 protein
can suppress the signal cascade elicited by mitogens via multiple
pathways. To delineate the mechanism of action of DOC-2/DAB2 in more
detail, we analyzed its interactive protein in this study. From a
pull-down assay (Fig. 1A), in addition to Grb2, we further found that Src family proteins were able to interact with the C
terminus, but not the N terminus, of DOC-2/DAB2. However, it is
interesting to notice that the N terminus of DOC-2/DAB2 contains a
disabled motif as in DAB1 phosphorylated by c-Src on its tyrosine residue (15, 16). The phosphorylated DAB1 protein serves as a binding
site for SH2 domain of c-Src (15, 16). There is no detectable tyrosine
phosphorylation in DOC-2/DAB2 by c-Src (data not
show).2 These data
suggest a functional difference between DAB1 and DOC-2/DAB2.
Src is a member of a family of non-receptor tyrosine kinases involved
in protein receptor tyrosine kinase-mediated pathways (17). In human
colon cancer, there is a strong correlation between c-Src protein
kinase activity and tumor stage (18). In PCa, c-Src signal transduction
is involved in increased migration capacity of these cells (19). We
also noticed that almost all PCa cell lines tested in our laboratory
expressed c-Src protein, some with absent DOC-2/DAB2 expression (data
not shown). These data prompted us to investigate the role of
interaction between c-Src and DOC-2/DAB2 in PCa cells under the
stimulation of EGF. Our data demonstrated (Fig. 6A) that
increased expression of c-Src in LNCaP cells enhanced Erk2
phosphorylation, indicating that the involvement of c-Src in the
mitogen-activated protein kinase pathway in PCa cells. In the presence
of DOC-2/DAB2, EGF-induced Erk2 phosphorylation was suppressed (Fig.
6A). This could be, at least partially, attributed to
the interaction between DOC-2/DAB2 and Grb2 (6). However, DOC-2/DAB2
could also inhibit c-Src-mediated Erk2 phosphorylation (Fig.
6A), implying that DOC-2/DAB2 had an effect on c-Src
activity. Data from co-immunoprecipitation assay (Fig. 2A)
clearly indicated the DOC-2/DAB2 complex contained c-Src and the major
interactive site located in the first proline-rich domain in
DOC-2/DAB2. Using a specific peptide, PPQ, the Erk activation can be
further enhanced in a prostatic epithelium, expressing endogenous
DOC-2/DAB2, treated with EGF (Fig. 6B). These data support
the multiple inhibitory roles of DOC-2/DAB2 in EGF-mediated signal cascade.
It is known that there are two major tyrosine phosphorylation sites
(Tyr-416 and Tyr-527) in c-Src protein (Fig.
7). However, tyrosine phosphorylation in
c-Src has a different impact on its kinase activity (11-14). Tyr-416
phosphorylation, located in the activation loop of Src, is closely
correlated with c-Src kinase activity (13, 14, 20). In contrast,
Tyr-527 phosphorylation, located in the C terminus of c-Src, associates
with the inactive form of c-Src (11-14). X-ray crystallography
analyses demonstrate (13, 14, 20-25) that the intramolecular
interactions, the binding between SH2 domain and the phosphorylated
Tyr-527 and the binding SH3 domain between the kinase linker, are
critical for maintaining the inactive status of c-Src. Presumably,
changing from a "closed conformation" (inactive) to an
"open conformation" (active) signifies c-Src activation. Therefore,
deletion or mutation of Tyr-527, often seen in v-Src, leads to
constitutive activation of Src kinase activity. Disruption of either
intramolecular interaction will lead to activation of c-Src. For
example, displacement of SH3 domain with other SH3 binding protein
leads to a more profound activation of c-Src activity (26, 27). Also,
mutations in SH3 domain have been found in some v-Src proteins
(28-30), indicating the key role of SH3 domain in modulating c-Src
activity. However, in this study, we demonstrated that DOC-2/DAB2 could
interact with the SH3 domain of c-Src and perhaps stabilize its closed conformation. This appears to be a novel mechanism in the modulation of
c-Src activity. Because DOC-2/DAB2 acts a potent negative regulator for
cell growth, the association of DOC-2/DAB2 with c-Src provides an
additional mechanism for its action.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N (
N), and
GST-Grb2 have been described previously (6).
p96) (Transduction Laboratories) or
against T7 tag (
T7) (Novagen).
T7.
pSrc416)
(Upstate Biotechnology) or tyrosine 527 (
pSrc527) (Cell
Signaling), and the same filter was stripped and reprobed with the
antibody against total Src (
Src) (Oncogene).
pErk)
(Cell Signaling), and the same filter was stripped and reprobed with
antibody against either total extracellular signal-regulated kinase 1/2
(
Erk) or p42 (
Erk2) (Cell Signaling).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N) (5) had much stronger
interaction compared with p82 (Fig. 1A, lower
panel).
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Fig. 1.
Differential interaction between the
proline-rich domain of DOC-2/DAB2 and several proteins containing SH3
domain. A, COS cells (4 × 105/60-mm
dish) were transfected with either p82 (top panel) or N
(bottom panel) for 48 h. An equal amount of cell lysate
was incubated with the indicated GST/SH3 domain fusion proteins. A
pull-down assay was performed, and the Western blot was analyzed with
T7. B, cell lysate prepared from NbE cells was subjected
to a pull-down assay using GST-SH3 (Src) (top panel) or
GST-SH3 (Grb2) (bottom panel) with increasing amounts of
peptides. The amount of DOC-2/DAB2 from each lane was
determined by Western analysis using
p96. The number
underneath each lane represented the relative level of DOC-2/DAB2
in each treatment compared with that in control (= 1).
C, a schematic representation of the SH3 domain in
DOC-2/DAB2 interacting with either Grb2 or c-Src.
Src. The amount of
DOC-2/DAB2 in the immunocomplex was substantially increased when the
cells were treated with EGF. Similarly, in LNCaP cells, the association
of c-Src with DOC-2/DAB2 is promptly elevated in a
time-dependent manner after EGF treatment (Fig. 2B). The interaction peaked at 20 min after EGF treatment
and remained unchanged for 60 min, indicating that c-Src had a
prolonged interaction with DOC-2/DAB2.
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Fig. 2.
Increased association of DOC-2/DAB2 with
c-Src in cells treated with EGF. A, COS cells (6 × 105/100-mm dish) were co-transfected with c-Src and p82
vectors. Twenty min after EGF treatment, cells lysate was subjected to
a co-immunoprecipitation assay. B, LNCaP cells (8 × 105/100-mm dish) were treated with EGF, and cell lysate was
collected at the indicated time. The pull-down was performed using
GST-Src and then Western blot was performed using p96 (top
panel). Equal amounts of cell lysate from each sample were
analyzed by Western blot using
p96 (bottom panel).
C, the profile of the DOC-2/DAB2/c-Src complex in LNCaP
cells after EGF treatment. Data (mean ± S.E.) were calculated
from experiments performed in duplicate.
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Fig. 3.
The effect of EGF effect on the interaction
of c-Src and DOC-2/DAB2. COS cells (6 × 105/100-mm dish) were co-transfected with both c-Src and
DOC-2/DAB2 vectors. Cells were collected 20 min after EGF treatment.
The co-immunoprecipitation was performed using Src in the presence
of increasing amounts of PPQ and then detected with
p96. The amount
of DOC-2/DAB2 was quantified by densitometry, and each data point was
calculated based on the ratio between the amount of DOC-2/DAB2 with and
without PPQ treatment (= 100%). The IC50 was determined as
the amount of PPQ needed for 50% of inhibition.
pSrc416 or
pSrc527, respectively,
and the total protein levels of c-Src and DOC-2/DAB2 were also
determined. In the presence of EGF (Fig. 4B), the
phosphorylation of Tyr-416 gradually increased and reached the plateau
after 20 min. Concurrently, the phosphorylation of Tyr-527
decreased after 10 min and then rebounded after 20 min (Fig.
4C). Forty min after EGF treatment, both Tyr-416 and Tyr-527
phosphorylation returned to the basal levels (Fig. 4, B and
C). In contrast, the presence of DOC-2/DAB2 inhibited the
elevated phosphorylation of Tyr-416 after EGF treatment (Fig.
4B). On the other hand, DOC-2/DAB2 did not have any effect
on Tyr-527 phosphorylation (Fig. 4C).
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Fig. 4.
The effect of DOC-2/DAB2 on the tyrosine
phosphorylation of c-Src proteins. A, LNCaP cells
(4 × 105/60-mm dish) were co-transfected with c-Src
and p82 vectors. Shortly after EGF treatment, cell lysate was collected
at the indicated time. Western blot analyses were carried out using a
variety of antibodies. B, the intensity of phosphorylation
of Tyr-416 was quantified and normalized with c-Src. C, the
intensity of phosphorylation of Tyr-527 was quantified and normalized
with c-Src. Data (mean ± S.D.) were calculated from experiments
performed in triplicate.
pSrc416 in NbE cells after
EGF treatment. The intensity of Tyr-416 phosphorylation was measured
and normalized with the total Src (Fig. 5B). In the absence
of blocking peptide, EGF induced Tyr-416 phosphorylation (~3-fold).
Apparently, PPQ could antagonize the interaction between DOC-2/DAB2 and
c-Src and significantly increased the Tyr-416 phosphorylation (~4- to
6-fold) in NbE cells without or with EGF treatment. Although LLQ
slightly increased Tyr-416 phosphorylation (~2-fold), maybe because
of its partial inhibitory activity (Fig. 1B), however, no
further elevation in Tyr-416 phosphorylation (~2-fold) was detected
in NbE cells after EGF treatment. Taken together, these data indicated
that DOC-2/DAB2 could directly suppress the Tyr-416 phosphorylation,
but not Tyr-527 phosphorylation, of c-Src via binding to the SH3 domain
of c-Src.
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Fig. 5.
The effect of proline-rich peptide on the
phosphorylation of Src. A, NbE cells (2 × 104/24-well plate) were transfected with each peptide at
100 ng/well. Twenty min after EGF treatment, cell lysate from each
sample was subjected to Western blot analyses using both
pSrc416 and
Src. B, the amount of
phosphorylated Src and the total Src was measured by densitometry. The
-fold induction was calculated based on the ratio between
phosphoprotein and total protein. Data (mean ± S.D.) were
calculated from experiments performed in triplicate.
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Fig. 6.
The inhibitory effect of DOC-2/DAB2 on the
c-Src-mediated Erk activation. A, LNCaP cells (4 × 105/60-mm dish) were co-transfected with plasmid(s) as
indicated. Twenty min after EGF treatment, cell lysate from each sample
was subjected to Western blot analyses using a variety of antibodies.
B, NbE cells (2 × 104/24-well plate) were
transfected with each peptide at 100 ng/well. Twenty min after EGF
treatment, cell lysate from each sample was subjected to Western blot
analyses using both pErk1/2 and
Erk2. The amount of each protein
was measured by densitometry. The -fold induction was calculated based
on the ratio between phosphoprotein and total protein. Data (mean ± S.D.) were calculated from experiments performed in
triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
The role of DOC-2/DAB2 in modulating Src
activity. The inactive Src is illustrated by the presence of
non-phosphorylated Tyr-416, phosphorylated Tyr-527, and two
intramolecular interactions critical for maintaining the closed
conformation. The SH3 domain binds to the kinase linker connecting the
SH2 domain to the kinase domain. The SH2 domain binds to phosphorylated
Tyr-527 (C terminus). The active Src is illustrated by the presence of
phosphorylated Tyr-416, non-phosphorylated Tyr-527, and both relaxed
intramolecular interactions. The proline-rich domain of DOC-2/DAB2 can
interlock the SH3 domain of Src that leads to the suppression of Src
kinase activity. Y, non-phosphorylated tyrosine;
*Y, phosphorylated tyrosine.
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ACKNOWLEDGEMENTS |
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We thank Gina Hernandez for excellent technical assistant, Andrew Webb for editorial assistance, and Dr. Koeneman for reading this manuscript. All GST-SH3 fusion plasmids and Src expression vector were kindly provided by Dr. Bing Wang.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK 47657 and by United States Army Grant PC970259.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of Texas
Southwestern Medical Center, Department of Urology, 5323 Harry Hines
Blvd., Dallas, TX 75390-9110. Tel.: 214-648-3988; Fax: 214-648-8786; E-mail: JT.Hsieh@UTSouthwestern.edu.
Published, JBC Papers in Press, December 8, 2002, DOI 10.1074/jbc.M210628200
2 Personal communication with Dr. Jonathan A. Cooper.
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ABBREVIATIONS |
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The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; PCa, prostate cancer; EGF, epidermal growth factor; SH, Src homology; GST, glutathione S-transferase.
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