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INTRODUCTION |
p62Dok, the rasGAP-binding protein, is a common target of
protein-tyrosine kinases
(PTKs).1 It is one of the
major tyrosine-phosphorylated molecules in v-Src-, v-Abl-, and
v-Fps-transformed cells and is also rapidly tyrosine-phosphorylated in
response to receptor tyrosine kinase activation in various cell systems
(1-5). These findings have underlined the importance of p62Dok in
cellular signaling.
Recent molecular cloning of Dok has shed more light on the mechanism by
which this protein may interact with PTK signaling pathways (4, 6). Dok
consists of an amino-terminal Pleckstrin homology (PH) domain, a
putative phosphotyrosine binding (PTB) domain, and a carboxyl-terminal
tail harboring at least seven potential tyrosine phosphorylation sites
(4, 6). The PH domain is likely to be necessary for targeting Dok to
the membrane, because PH domains preferentially bind phospholipids (7).
The putative PTB domain is most homologous to the IRS-1 and
FRS2/SNT-1 PTB domains, which recognize phosphotyrosine
(pY)-containing sequences (8-11). In the case of SNT-1, its PTB
domain can also bind distinct unphosphorylated sequences (11). It
remains to be determined whether the Dok PTB domain can bind
phosphopeptides and, if it does, whether it recognizes sequences
distinct from those recognized by the IRS-1 and SNT-1 PTB domains. The
multiple tyrosine residues in the Dok carboxyl-terminal tail are
candidate sites for tyrosine kinases. When phosphorylated, they become
potential docking sites for Src homology 2-containing proteins
such as p120 rasGAP and Nck (5, 12). Consistent with this notion, both
rasGAP and Nck have been shown to bind tyrosine-phosphorylated Dok (4, 13). Therefore, the carboxyl-terminal tail of Dok likely functions as a
molecular platform for signal complex assembly induced by activated
PTKs. However, the functional significance of the Dok PTB domain and
the carboxyl-terminal tail has yet to be addressed.
Additional Dok homologues such as Dok-L (or Dok-3) and Dok-R (or Dok-2
and FRIP) have been identified recently (14-18), indicating that Dok and its homologues may constitute a growing family of proteins
involved in a range of signaling pathways downstream of PTKs. However,
the physiological roles of Dok and its homologues remain to be
elucidated. Despite their structural similarities to the IRS-1 family
molecules, Dok family proteins have different PTB domains and
carboxyl-terminal tails that potentially mediate different signal
responses by recruiting distinct sets of Src homology 2-containing
signaling molecules. The mechanism by which Dok is phosphorylated and
primed to form specific signaling complexes thus becomes a key issue in
understanding Dok signaling. We have demonstrated here that the Dok PTB
domain is a functional phosphotyrosine binding module that facilitates
tyrosine phosphorylation and rasGAP binding of Dok. We have also found
that Dok can inhibit Src-induced cellular transformation. This
inhibitory effect depends on both the PTB domain and the
carboxyl-terminal tail of Dok. Furthermore, we have shown that Dok can
oligomerize via its PTB domain and Tyr146. This
oligomerization appears critical for the inhibition of v-Src-induced
transformation. These results suggest that the multiple domains of Dok
are required for Dok signaling.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Antibodies--
NIH3T3 cells were maintained in
Dulbecco's modified Eagle's media containing 10% calf serum
(Hyclone). The anti-hemagglutinin (HA) monoclonal antibody was
purchased from Berkeley Antibody Company. The anti-His-tag,
anti-rasGAP, and anti-phosphotyrosine monoclonal antibodies (4G10 and
PY20) were purchased from Santa Cruz Biotechnology. Anti-Ras monoclonal
antibody was from Upstate Biotechnology.
DNA Constructs--
The coding region of the murine Dok cDNA
(4) tagged with the HA epitope was cloned into the HpaI site
of the murine stem cell retroviral vector MSCVpuro (19). The HA epitope
was joined to the 5' end of Dok cDNA by polymerase chain reaction
(PCR) using the pfu polymerase (4). The carboxyl-terminal
truncated forms of Dok were similarly cloned. The Dok 277, 313, 336, and 363 constructs encode amino acids 1-277, 313, 336, and 363, respectively. The PTB domain mutant Dok-AA (Arg207 and
Arg208 to Ala) and the Y146F mutants (DokN-Y146F,
Dok313-Y146F, and Dok363-Y146F) were generated by site-directed
mutagenesis using PCR with the pfu polymerase. All
constructs were confirmed by DNA sequencing. Sequences encoding
wild-type (chicken c-Src) or activated (Src527F) Src were cloned into
the MSCV-neo vector as described previously (20).
Generation of Cell Lines Expressing Src and Dok
Variants--
The MSCV-based constructs encoding Src and HA-Dok and
their variants were transfected into the retrovirus packaging cell line BOSC23 (21). Retroviruses were harvested 2 days after transfection and
used to infect dividing NIH3T3 cells. Two days after infection, Dok- or
Src-expressing cells were selected in puromycin- or G418-containing media. Cells that coexpressed Dok and Src were generated by infecting Dok-expressing cells again with Src viruses followed by selection in
G418-containing media.
Transformation Assay--
The ability of Src to induce cellular
transformation was scored by the focus formation assay in NIH3T3 cells.
Parental and Dok variant-expressing cells were infected by low titer
(1 × 104/ml) MSCV-c-Src or MSCV-Src527F viruses.
These cells were maintained in Dulbecco's modified Eagle's media
containing 10% calf serum for 7-10 days for focus formation. The
number of foci were then quantitated to determine transformation
activities. Three replicate dishes were plated for each sample, and
each experiment was repeated three times.
GST Fusion Proteins and Phosphopeptide Library--
To generate
the glutathione S-transferase (GST) fusion protein construct
containing only the amino-terminal PH and PTB domains of Dok
(GST-DokN), the DNA fragment encompassing residues 1-277 of murine Dok
was PCR-amplified and cloned into the SmaI site of the
pGEX4T-1 vector (Amersham Pharmacia Biotech). Similarly, to generate
the GST-PTB and GST-PTB-AA constructs, the DNA fragment encoding
residues 125-264 was PCR-amplified using wild-type Dok or Dok-AA as a
template. These fragments were subsequently cloned into the
BamHI site of pGEX4T-1. These constructs were used to transform Escherichia coli to produce GST fusion proteins
that were purified using glutathione-agarose beads.
To study the binding specificity of the Dok PTB domain, 100-200 µg
of fusion proteins that were immobilized on glutathione-agarose beads
were incubated with 1 mg of peptide library. The peptide library had a
sequence of MAXXXNXXpYXAKKK, where
X indicates any amino acid except Cys and Trp. This
particular library was designed to examine PTB domain specificities,
because PTB domains prefer turn-forming sequences near pY and
hydrophobic residues at 5-8 positions amino-terminal to pY (22). The
mixture of peptides bound to the fusion proteins was eluted with acid
after washing. This peptide mixture was then sequenced on an ABI477
machine. The specificity of binding was then determined by comparing
the sequence of bound mixture with that of the mock experiment using GST alone (12).
His-tagged Fusion Proteins and Src Phosphorylation--
To
generate poly-His-tagged Dok (His-DokN) fusion proteins, sequences
encoding residues 1-264 of Dok were PCR-amplified and cloned into the
BamHI site of the pRSET vector (Invitrogen). His-DokN-Y146F was constructed in the same manner, except that Tyr146 was
mutated to Phe. His-tagged proteins were purified from BL21 cells using
nitrilotriacetic acid-agarose (Qiagen) and eluted with
immidazole. To phosphorylate these fusion proteins, they were incubated
with 2 µg of purified recombinant c-Src (a generous gift from Dr.
Wenqing Xu, University of Washington) in the presence of 1 mM ATP and 20 mM MgCl2 for 1 h
at 30 °C.
Immunoprecipitation and Western Blot Analysis--
Cells were
washed once with PBS, lysed in the lysis buffer (20 mM
Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 10%
glycerol, 1% Nonidet P-40, 1 mM phenlymethylsulfonyl
fluoride, 1 µM pepstatin A, 1 µM aprotinin,
1 µM leupeptin, 2 mM
-glyerolphosphate,
500 µM sodium vanadate, and 1 mM
dithiothreitol), and centrifuged at 10,000 × g at
4 °C. The supernatants were subsequently collected for immunobloting
and immunoprecipitation. For immunobloting,
of the
supernatants was mixed with SDS-loading buffer, boiled, and analyzed by
SDS-polyacrylamide gel electrophoresis. For immunoprecipitation, the
supernatants were incubated with antibodies at 4 °C for 90 min.
Protein A/G-agarose beads were then added, and the mixture was
incubated at 4 °C for another 90 min. The beads were subsequently
washed four times with lysis buffer, resuspended in SDS-loading buffer,
and boiled, and the eluents were analyzed by SDS-polyacrylamide gel
electrophoresis. SDS-polyacrylamide gel electrophoresis and Western
blot analyses were performed as described previously (4).
Ras Activation Assay--
For analyzing the effect of Dok
expression on Ras GTP loading in Src-transformed cells, GST-Raf Ras
binding domain-agarose beads (Ras Activation Assay kit, Upstate
Biotechnology) were used to precipitate GTP-bound Ras as described in
the manufacturer's manual. A monoclonal antibody against Ras was also
supplied in the kit and used to probe for endogenous Ras.
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RESULTS |
Dok Can Inhibit Src-induced Cellular Transformation--
Although
tyrosine phosphorylation of Dok occurs concurrently with PTK
activation, the exact role of Dok in cell signaling remains ambiguous.
To address the question of whether Dok facilitates or inhibits Src
transformation, we examined the effect of Dok expression on Src-induced
transformation in NIH3T3 cells. Dok and Src retroviruses (see
"Experimental Procedures") were used to infect NIH3T3 cells. As
shown in Fig. 1, A and
B, expression of c-Src or activated Src (Src527F) readily
induced focus formation within 1 week. However, coexpression of Dok and
Src strongly inhibited focus formation induced by Src. Although Dok is
commonly phosphorylated by oncogenic tyrosine kinases, this result
indicates that the physiological effect of DOK is to inhibit cellular
transformation by the Src-tyrosine kinase.

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Fig. 1.
Expression of Dok can inhibit Src
transformation. Parental and HA-tagged Dok-expressing NIH3T3
fibroblast cells were infected with c-Src or Src527F retroviruses. The
effect of various Dok mutants on Src transformation was studied using a
focus formation assay in these cells. A, culture morphology
of cells expressing c-Src alone (left) or both c-Src and Dok
(right). B, comparison of the focus-forming
abilities of parental NIH3T3 cells or those that coexpressed Dok
variants with either c-Src (left) or Src527F
(right). The numbers indicate the numbers of foci
formed per 104 Src viral particles. C, diagram
of different Dok deletion mutants and mapping of domains that are
required for Dok inhibitory function. +, inhibition; , no
inhibition.
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The Dok PTB Domain and Carboxyl-terminal Region Are Necessary for
Its Inhibitory Effects--
Because Dok consists of multiple domains,
we went on to map the functional domains in Dok that are responsible
for its inhibitory effects. Mutant Dok molecules that were HA
epitope-tagged were generated and coexpressed with Src in NIH3T3 cells.
We postulated that the multiple potential tyrosine phosphorylation
sites within the Dok carboxyl terminus might contribute to Dok
inhibition of Src transformation. Consistent with this notion, deletion
of the carboxyl-terminal tail (Dok277) abolished the inhibitory
activity of Dok (Fig. 1B). Using a panel of
carboxyl-terminal deletion constructs, we further defined the regions
within the Dok carboxyl terminus necessary for transformation
inhibition. Although Dok363 still blocked Src transformation, Dok313
and Dok336 no longer retained the inhibitory abilities (Fig.
1B). These data indicate that the residues between 336 and
363 constitute a functional domain for the inhibitory action of Dok.
To determine the role of the amino-terminal portion, mutations were
made in the putative PTB domain of Dok. On the basis of sequence
homology with the IRS-1 PTB domain (4), amino acids Arg207
and Arg222 of Dok are predicted to coordinate
phosphotyrosine binding. We therefore reasoned that mutation of
Arg207 might block phosphotyrosine binding and thereby
affect Dok function. Supporting our hypothesis, mutation of
Arg207 and Arg208 to Ala residues (Dok-AA)
eliminated the inhibitory function of Dok (Fig. 1B). These
results strongly suggest that the Dok amino-terminal PTB domain
represents a distinct regulatory domain of Dok that may associate with
tyrosine-phosphorylated proteins.
Tyrosine Phosphorylation of Dok Mutant Proteins--
Because
tyrosine phosphorylation of Dok may be critical for its in
vivo activities, we examined tyrosine phosphorylation of various
HA-tagged Dok mutants in Src527F-transformed NIH3T3 fibroblasts. The
different Dok proteins including the PTB domain mutant (Dok-AA) and all
carboxyl-terminal deletion mutants (Dok277, -313, -336, and -363) were
expressed (Fig. 2, A and
B). Western blots of whole cell lysates indicated that they
were all tyrosine-phosphorylated (data not shown). Surprisingly,
Western blots of anti-HA immunoprecipitates that were probed with
anti-phosphotyrosine antibodies showed that even Dok277 (which lacks
the carboxyl-terminal region with its multiple potential
phosphorylation sites) was still tyrosine-phosphorylated (Fig.
2B), suggesting that a major tyrosine phosphorylation site is located in the amino-terminal domain of Dok. Therefore, changes in
the gross tyrosine phosphorylation levels of various Dok mutants induced by the Src PTK may not account for the differences in their
inhibitory abilities. However, phosphorylation of specific tyrosine
sites on Dok may be necessary for inhibition of Src-mediated transfromation.

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Fig. 2.
Expression and tyrosine phosphorylation of
wild-type and mutant Dok proteins. Lysates from cells that
coexpressed various HA-tagged Dok constructs with Src527F were
immunoprecipitated and Western blotted. A, expression of
wild-type and mutant HA-tagged Dok proteins in Src527F-transformed
cells. Equal amounts of whole cell lysates were resolved by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes. The membranes were probed with anti-HA
antibodies. B, comparison of tyrosine phosphorylation of
HA-Dok, Dok-AA, and Dok277 proteins. Dok proteins were
immunoprecipitated (IP) using anti-HA antibodies and probed
with an anti-phosphotyrosine antibody. p-Dok, phosphorylated
Dok; p-Dok277, phosphorylated Dok277.
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Dok PTB Domain Binds to Specific Phosphopeptide Sequences--
We
have shown here that the Dok PTB domain (residues 125-264) is
functional and necessary for Dok to inhibit Src-induced transformation, possibly through its interaction with phosphotyrosine-containing proteins. However, whether the Dok PTB domain does indeed bind specific
phosphotyrosine-containing sequences remains to be determined. To this
end, we used a combinatorial peptide library approach (see
"Experimental Procedures").
Briefly, GST fusion proteins containing either the Dok amino-terminal
PH and PTB domains (GST-DokN) or the Dok PTB domain alone (GST-PTB)
were purified and captured on GSH beads. The beads were then incubated
with a soluble degenerate phosphopeptide library mixture to select for
specific peptides that would bind to the PTB domains. The peptide
library used had a sequence of
MAXXXNXXpYXAKKK. The amino acid at
position
3 to the phosphotyrosine was fixed (Asn), because PTB
domains prefer turn-forming sequences (9, 22). The phosphopeptide
mixtures that bound specifically to the Dok PTB domain were then
isolated and sequenced by Edman degradation. A comparison of these
sequences to those obtained using GST alone revealed that the Dok PTB
domain recognizes phosphopeptides with the unique motif of
Y/MXXNXLpY (Fig.
3). At position pY-1, Leu was exclusively
selected, indicating the importance of this residue. At position pY-6,
hydrophobic amino acids Tyr, Met, and Phe were strongly selected.
Similar preferences for hydrophobic residues at positions pY-5 to pY-8
have been reported for other PTB domains as well (8, 22).

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Fig. 3.
The Dok PTB domain binds phosphopeptides with
distinct sequences. The binding specificity of the Dok PTB domain
was examined using the phosphopeptide library
MAXXXNXXpYXAKK. The relative
preference of amino acids at each degenerate position (pY as position
0) was plotted. A value of 1 higher indicates a preferred amino acid.
Selections are shown at position -6 (A), position -5 (B), position -4 (C), position -3 (D), position -2 (E),
and position -1 (F).
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A region within the Dok PTB domain (amino acids 204-232) is 41 and
52% identical to those of IRS-1 and SNT-1, respectively. The latter
two PTB domains bind phosphopeptides with the consensus motif of
NPXpY (8-11). Importantly, two critical Arg residues (Arg207 and Arg222) that are known to mediate
phosphotyrosine recognition in the IRS-1 PTB domain are conserved in
the Dok PTB domain as well (Arg207 and Arg222).
To test whether Arg207 also mediates the interaction
between the Dok PTB domain and the phosphotyrosine moiety, we examined
the Dok-AA mutant in which Arg207 was replaced with Ala. As
predicted, this mutation eliminated the ability of the Dok PTB domain
to bind phosphopeptides (data not shown). Combined with the observation
that Dok-AA could no longer inhibit Src transformation, these data
support the model that Dok function depends on the PTB domain.
Furthermore, despite their similarities in the structural basis for
phosphotyrosine recognition, the Dok PTB domain recognizes distinct
sequences (NXLpY) compared with the IRS-1 PTB domain
(NPXpY). Therefore, the Dok PTB domain may associate
in vivo with a set of tyrosine-phosphorylated proteins
distinct from that bound by IRS-1.
The Dok PTB Domain Mediates Phosphotyrosine-dependent,
Homotypic Interactions of Dok--
We have shown that the PTB domain
recognizes specific tyrosine-phosphorylated sequences and is required
for the inhibitory activity of Dok. It is likely that Dok may exert its
inhibitory function by binding to specific phosphoproteins through the
PTB domain. To further investigate the phosphoproteins that might associate with the PTB domain and the mechanism by which the PTB domain
might regulate Dok function, the GST-DokN fusion protein containing
both PH and PTB domains was used to precipitate proteins from
Src-transformed cells. As shown in Fig.
4A, multiple
tyrosine-phosphorylated bands were specifically copurified with the Dok
amino-terminal domain, including major proteins at ~60 kDa, which is
similar to the molecular mass of Dok. We therefore speculated
that some of these proteins might be Dok family members. To test this
idea, GST-DokN fusion proteins were incubated with cell lysates from Src-transformed cells that also expressed various Dok mutants. Although
GST alone did not precipitate any Dok molecules, GST-DokN bound
specifically to HA-tagged, wild-type Dok and various mutant Dok
proteins (Fig. 4B). The carboxyl-terminal tail of Dok is not required for this interaction, because Dok277 appeared to bind GST-DokN
with the same efficiency as wild-type Dok. In addition, the GST-PTB
domain alone was able to associate with HA-tagged Dok277, indicating
that the PTB domain may mediate homotypic interactions of Dok (Fig.
4C).

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Fig. 4.
The Dok PTB domain mediates oligomerization
of Dok proteins. A, the amino-terminal domain of Dok
binds to phosphoproteins at ~60 kDa. The Dok amino-terminal domain
fusion protein (GST-DokN) or GST alone was incubated with
lysates from Src527F-expressing NIH3T3 cells. The associated proteins
were then detected by anti-phosphotyrosine antibodies and enhanced
chemiluminescence. B, the amino-terminal domain of Dok
interacts with different Dok mutant proteins. The GST-DokN fusion
protein was incubated with lysates from cells expressing various
HA-tagged Dok mutants. As a negative control, GST proteins were
incubated with cell lysates from HA-Dok- and Src527-coexpressing cells.
Proteins that bound to GST or GST-DokN were visualized using an anti-HA
monoclonal antibody and enhanced chemiluminescence. *, cross-reactive
bands. C, the Dok PTB domain mediates homotypic interaction
through Tyr146 of Dok. GST fusion proteins of DokN
(GST-DokN), Dok PTB (GST-PTB), and Dok PTB mutant
(GST-PTB AA) were incubated with lysates from cells
coexpressing Src527F and HA-tagged Dok277 or Dok313 with and
Tyr146 to Phe mutation (Dok313Y146F). Proteins
that bound to GST fusion proteins were visualized using an anti-HA
monoclonal antibody and enhanced chemiluminescence. D, the
Dok PTB domain binds directly to the phosphorylated Tyr146
site. GST-PTB fusion proteins were incubated with purified His-tagged
DokN (right) or c-Src-phosphorylated, His-tagged DokN
(His-DokN) and DokN-Y146F (His-DokNY146F;
left). Proteins that bound to the GST PTB domain were
visualized using anti-His-tag or anti-phosphotyrosine antibodies and
enhanced chemiluminescence.
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On the basis of our combinatorial peptide library mapping of the
consensus substrate motif (Y/MXXNXLpY) of the Dok
PTB domain, sequences surrounding residue Tyr146
(LEMLENSLYS) on Dok constitute a perfect binding site for the Dok PTB
domain when this tyrosine is phosphorylated. Tyr146,
located between the Dok PH and PTB domains, is also a potential tyrosine phosphorylation site for the Src family PTKs (24). We
hypothesized that Tyr146 might be necessary for the
homotypic interactions mediated by the Dok PTB domain. Consistent with
our hypothesis, the HA-tagged Dok mutant with its Tyr146
mutated to Phe (Dok313-Y146F) failed to copurify with the GST-PTB domain, even though the GST-PTB domain fusion proteins were able to
pull down the Dok amino-terminal region (Fig. 4C). These
data strongly indicate a PTB domain-mediated, direct interaction
between Dok molecules.
PTB domains have been shown to mediate both
phosphotyrosinedependent and -independent interactions. To
confirm that Dok-Dok interactions through the PTB domain were direct
and tyrosine phosphorylation-dependent, we generated the
His-tagged Dok amino-terminal domain (His-DokN) and His-DokN with the
Y146F mutation (His-DokN-Y146F) in E. coli. These fusion
proteins were in vitro-phosphorylated using recombinant c-Src and incubated with GST-Dok PTB. As shown in Fig. 4D,
only His-DokN was able to bind GST-Dok PTB, although His-DokN and
His-DokN-Y146F were equally phosphorylated by c-Src. In addition, the
GST-PTB domain failed to bind His-DokN in the absence of c-Src. These data demonstrate that the Dok PTB domain mediates
phosphotyrosine-dependent homotypic interactions through
residue Tyr146.
Tyr146 Is Important for Regulating Dok
Activity--
We showed that Dok363 was the shortest mutant to still
retain the inhibitory activity (Fig. 1). To determine the role of
Tyr146 on Dok function, Dok363 with the Tyr146
to Phe mutation (Dok363-Y146F) was generated and compared with Dok363
for its effect on Src-induced transformation in NIH3T3 cells. Notably,
the Y146F mutation significantly reduced the inhibitory activity of
Dok363 (Fig. 5). Therefore, mutations (in
either the PTB domain or Tyr146) that prevent Dok
oligomerization also abrogated its inhibitory activity. These results
indicate that the homotypic interaction through Tyr146 and
the Dok PTB domain is necessary for Dok function.

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Fig. 5.
Tyr146 is important for the
inhibitory activity of Dok. Parental NIH3T3 fibroblast cells and
those that expressed HA-tagged Dok363 or Dok363-Y146F were infected
with low-titer Src527F viruses. Focus formation was then assayed to
compare the effect of various Dok mutants on Src transformation in
these cells. Error bars indicate S.E. from three independent
experiments.
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Dok Interacts with the Ras Pathway--
Src is known to activate
the Ras pathway, and Dok is a rasGAP-binding protein. We therefore
reasoned that Dok might inhibit Src transformation by recruiting
rasGAP, thereby regulating Ras activity in the cell. V12Ras (activated
Ras) is locked in the GTP-bound state and therefore should be
unaffected by rasGAP activity (25). We first tested how Dok might
affect V12Ras-mediated transformation in NIH3T3 cells. Consistent with
its rasGAP binding ability, Dok expression did not affect focus
formation induced by activated V12-K-Ras (Fig.
6A), suggesting that Dok
likely acts upstream of Ras to block Ras activation.

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Fig. 6.
Dok binds rasGAP and interacts with the Ras
signaling pathway. A, Dok does not inhibit
V12-K-Ras-induced transformation. Focus-forming abilities of NIH3T3
cells expressing V12-K-Ras alone or with Dok were compared.
B, mutant Dok proteins are impaired in their abilities to
bind endogenous rasGAP. Lysates from cells that coexpressed Src527F and
various HA-tagged Dok constructs were immunoprecipitated with anti-HA
antibodies and Western blotted with anti-rasGAP antibodies. Whole-cell
lysates were also probed with anti-HA monoclonal antibodies.
C, Dok inhibits Ras GTP loading in Src-transformed cells.
Active Ras (GTP-bound) was affinity-precipitated with Raf-1 RBD
agarose conjugate from NIH3T3 cell lysates that expressed Src527 alone
or with Dok and Western blotted using anti-Ras monoclonal antibodies.
Ctr, control.
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We postulated that Dok might function by increasing the local
concentration of rasGAP. To test this hypothesis, anti-HA
immunoprecipitates from Src-transformed cells that also expressed
full-length HA-tagged Dok, Dok363, Dok336, Dok277, or Dok-AA were
probed with anti-rasGAP antibodies (Fig. 6B). Full-length
Dok and Dok363 were able to associate with rasGAP; however, Dok336,
Dok277, and the PTB domain mutant Dok-AA were impaired in their
abilities to bind rasGAP. The inability to bind rasGAP may explain the
failure of Dok336, Dok277, and Dok-AA to inhibit Src transformation
(Fig. 1B). These results further imply that the PTB domain
together with the carboxyl-terminal region of Dok may function in
clustering and recruiting rasGAP to the site of action. We further
speculated that the recruitment of rasGap by Dok may lead to inhibition
of Ras GTP loading. Consistent with this idea, the amount of Ras GTP
(active Ras) was found to be significantly reduced in Src-transformed
cells coexpressing Dok compared with Src-transformed cells (Fig.
6C).
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DISCUSSION |
We have shown that expression of Dok can block c-Src-induced
transformation in NIH3T3 fibroblasts, indicating that Dok may negatively regulate signal pathways that are activated by PTKs. It is
possible that Dok functions to recruit negative regulators of PTK
cascades. For example, Csk family kinases are known to down-regulate
c-Src activity by phosphorylating Tyr527 on c-Src (26, 27).
Furthermore, Dok family proteins have been reported to associate
directly with Csk (18, 28, 29). These data suggest that Dok may
attenuate Src signaling by regulating Csk. However, this model was
ruled out because Dok also inhibits transformation by activated Src (527F).
Alternatively, Dok may exert its inhibitory effects via the
GTPase-activating protein rasGAP. Several lines of evidence support this hypothesis. First, Dok is a rasGAP-binding protein. Association of
Dok with rasGAP can be readily detected during activation of PTKs such
as Src, Abl, and the Eph receptor kinase (1-5). Among the seven
potential tyrosine phosphorylation sites of Dok, five are predicted
docking sites for the Src homology 2 domains of rasGAP (4, 5). The
presence of multiple rasGAP binding sites on Dok suggests that Dok may
provide the molecular platform necessary for high local rasGAP
activity. In addition, the Ras pathway is activated by Src and required
for Src transformation of fibroblast cells (30, 31). Dok may block Src
transforming activity by interfering with Ras GTP loading and
mitogen-activated protein kinase activation. Consistent with this
model, we showed that Dok reduced Ras GTP loading and did not affect
cellular transformation triggered by V12Ras, which is resistant to
rasGAP activity. Furthermore, in correlation with their inability to
inhibit Src-mediated transformation, Dok336, Dok277, and Dok-AA mutants
failed to associate with rasGAP in Src527F-transformed cells. These
observations suggest that one major function of Dok is to cluster
rasGAP and thereby negatively regulate the Ras signal pathways.
Increased PTK activity can result in hyperphosphorylation of Dok. In
turn, more negative regulators such as rasGAP are recruited to the site
of PTK activation to prevent Ras activation. Consistent with this
model, it was demonstrated that Dok inhibits Ras activity in 293 cells
(32). However, this model may not be universal, because Dok could
inhibit rather than enhance rasGAP activity in some cells (33). One
potential target of Dok is the mitogen-activated protein kinase
pathway. We have shown recently that mitogen-activated protein kinase
activity is up-regulated in B lymphocytes from Dok
/
mice (34). In
addition, Dok is required to mediate the inhibitory effect of Fc
RIIB
on Erk activation (35). Furthermore, recent evidence has indicated that
the Dok homologues Dok-R and Dok-L inhibit mitogen-activated protein
kinase activation induced by the epidermal growth factor receptor and
v-Abl (17, 36).
Our results have indicated that there are at least two independent
functional domains in Dok, the carboxyl-terminal tail and the
amino-terminal PH and PTB domains. We have shown that the carboxyl-terminal tail of Dok is necessary for Dok in vivo
activity. Dok relies on its carboxyl-terminal tail to recruit Src
homology 2-containing molecules such rasGAP and Nck (5, 12). Deletion of residues 278-481, which encompass the cluster of potential tyrosine
phosphorylation sites, was found to impair the inhibitory ability of
Dok. Deletional analysis has located a minimum region (residues
336-363) on Dok that is essential for its function. Interestingly, the
sequence DPIY361DEPE within this region is conserved among
the Dok family proteins. Furthermore, Tyr361 is also the
major docking site for Nck and rasGAP (4, 13). A recent study has
demonstrated that Tyr361 plays a central role in
Dok-mediated cell migration on insulin stimulation (13). It is
therefore possible that Tyr361 is important for the
inhibitory activity of Dok.
How does the amino-terminal domain of Dok modulate Dok activities? The
PTB and PH domains of Dok-R were shown to be necessary for Dok-R
phosphorylation by the epidermal growth factor receptor (36).
Therefore, the amino-terminal PH and PTB domains may be important for
efficient phosphorylation by protein-tyrosine kinases. In addition,
phosphorylated Dok-AA proteins had significantly decreased binding with
endogenous rasGAP, suggesting that an intact PTB domain may be required
to phosphorylate the rasGAP binding sites on Dok or to recruit Dok to
where rasGAP is localized.
Importantly, the amino-terminal region of Dok may be needed for
oligomerization of Dok and may recruit other signaling proteins. Our
data on the Dok PTB domain and Tyr146 support this model.
We have shown that the Dok PTB domain is capable of binding to
phosphotyrosine-containing sequences. Such binding is required for Dok
function, because the Dok PTB domain mutant that failed to bind
phosphopeptides also lost its ability to inhibit Src-mediated
transformation. Furthermore, we have demonstrated that the PTB domain
also mediates Dok oligomerization by binding to the phosphorylated
Tyr146 site (located between the PH and PTB domains). The
Dok homotypic interaction may cluster Dok molecules at sites of PTK
activation. Consistent with the importance of oligomerization, the
Y146F mutation significantly decreased Dok inhibitory activity.
Alternatively, the Dok PTB domain may bind negative regulators such as
phosphatase SHIP1 (23). Therefore, the Dok amino-terminal domain
may not only facilitate tyrosine phosphorylation of Dok but also
cluster Dok and its associated proteins at the location for negative signaling.