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INTRODUCTION |
The formation of the mammalian central nervous system involves a
complex pattern of neuronal migration that results in the organization
of several neuronal populations into precise layers (1, 2). Over the
past five years, a great deal has been learned about the molecular
mechanisms that control cell migration in the developing brain from the
study of mutant mice. In particular, the identification of
reelin, the gene mutated in the classic ataxic mutant mouse
reeler, has uncovered a signaling pathway that is critical
for cell positioning (3). Detailed histological studies of
reeler mice revealed severe lamination defects in the cerebral cortex, cerebellum, hippocampus, and other laminated structures in the central nervous system (4). Remarkably, mutations in
Disabled 1 (Dab1),1 an
intracellular adapter protein (5-8), or both the very low density
lipoprotein receptor and apolipoprotein E receptor 2 (9) result in
identical defects in mice. The reelin gene encodes a large
protein secreted by several populations of neurons in the developing
brain, including Cajal-Retzius cells in the neocortex and granule cell
precursors in the cerebellum (3, 10). Biochemical studies have
demonstrated that Reelin serves as a ligand for lipoprotein receptors
(11, 12). Taken together, these findings suggest a model in which
Reelin binds to lipoprotein receptors present on target neurons that
express Dab1, initiating an intracellular signaling cascade. Recently,
reelin has been shown to be mutated in a rare form of
autosomal lissencephaly in humans (13), and alterations in the Reelin
pathway have also been proposed to be associated with other
neurological disorders (14, 15).
Although the exact mechanism involved in Reelin signaling is unclear,
it has been proposed that Dab1 acts as an intracellular adapter
molecule downstream of Reelin. Dab1 was first identified as a
Src-binding protein in a yeast two-hybrid screen (16). The amino
terminus of Dab1 contains a PTB (phosphotyrosine binding) domain that
is structurally similar to the PTB domains of Shc and Numb (17).
Although PTB domains were originally identified as protein interaction
domains that recognized tyrosyl-phosphorylated Asn-Pro-X-Tyr
(NPXY) motifs present on target proteins, they have recently
emerged as a more diverse family of protein modules that vary in their
recognition sequences (17). The Dab1 PTB domain interacts with
NPXY sequences within the cytoplasmic regions of several
membrane-bound proteins, including lipoprotein receptors (18) and
amyloid precursor protein family proteins (19, 20). The Dab1 PTB
domain has a preference for the unphosphorylated NPXY motif,
and phosphorylation of the tyrosine residue is inhibitory to the
interaction of Dab1 PTB domain with amyloid precursor protein (20). The
NPXY motif serves as an internalization signal for the low
density lipoprotein receptor (21), and the interaction with Dab1 has
been suggested to regulate the rate of receptor internalization
(22).
Dab1 accumulates in mice lacking Reelin or both very low density
lipoprotein receptor and apolipoprotein E receptor 2 (6), suggesting
that Dab1 is degraded as a consequence of Reelin signaling. In
wild-type mice, Dab1 is phosphorylated on tyrosines during embryogenesis (16). In contrast, tyrosyl phosphorylation of Dab1 is
greatly diminished in reeler mice, despite the elevated levels of the protein. However, stimulation of forebrain neurons from
reeler embryos with exogenous Reelin can result in a rapid and dramatic induction of Dab1 tyrosyl phosphorylation (23). This has
led to the hypothesis that tyrosyl phosphorylation of Dab1 is a
critical step in the Reelin-signaling pathway. Indeed, mice expressing
a mutant form of Dab1, in which a cluster of five tyrosine residues
located immediately downstream of the PTB domain were replaced with
phenylalanine, exhibit a reeler phenotype (24). Although
this study supported a role for Dab1 phosphorylation in neuronal
positioning, it did not identify the specific sites involved. Here, we
identify the sites of Reelin-induced tyrosyl phosphorylation on Dab1 in
neurons. This is a critical first step in understanding the mechanisms
that couple the molecular events triggered by Reelin to the cellular
processes that mediate neuronal positioning in the developing brain.
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EXPERIMENTAL PROCEDURES |
Construction of Expression Plasmids--
The GST-Dab1 fusion
construct and Dab1-hemagglutinin expression plasmid have been described
previously (19). GST fusions of the Dab1 PTB domain
(GST-Dab11-179), middle region
(GST-Dab1180-399), and the carboxyl-terminal region
(GST-Dab1400-555) were prepared by subcloning the
corresponding polymerase chain reaction products into pGEX4T3 (Amersham
Pharmacia Biotech). Site-directed mutagenesis was conducted using the
QuikChange site-directed mutagenesis kit (Stratagene). The mutagenesis
reactions were performed in the pGEX4T3 and pcDNA3 vectors
containing a full-length Dab1 clone according to the manufacturer's
protocol and confirmed by DNA sequencing.
Expression of GST Fusion Proteins--
Fusion proteins were
expressed in BL21 bacterial strains as described previously (19).
Isopropyl-1-thio-
-D-galactopyranoside-induced bacterial
pellets were lysed by sonicating in phosphate-buffered saline/Tween
containing 5 mM EDTA, 2 mM phenylmethylsulfonyl
fluoride, 40 µg/ml aprotinin, and 40 µg/ml leupeptin. The lysates
were incubated with glutathione-Sepharose (Amersham Pharmacia Biotech)
at 4 °C. Bound proteins were then used as substrates in Src kinase
reactions in vitro.
Kinase Reactions--
GST fusion proteins (~5 µg each)
immobilized on glutathione-Sepharose were phosphorylated by ~15 units
of purified active c-Src (Upstate Biotechnology) in 50 µl of kinase
reaction buffer (25 mM Tris-HCl, pH 7.3, 25 mM
MgCl2, 10 mM MnCl2, 0.5 mM EGTA, 0.05 mM sodium orthovanadate, 0.5 mM dithiothreitol, 100 µM ATP, and 25 µCi
of [
-32P]ATP). The kinase reactions were carried out
at 30 °C for 30 min. The reactions were stopped by the addition of
SDS sample loading buffer, and protein samples were boiled for 5 min.
Phosphoproteins were separated by SDS-PAGE, transferred to
nitrocellulose membranes, and visualized by autoradiography.
Phosphopeptide Mapping--
Phosphoproteins were excised from
nitrocellulose membranes and digested with trypsin essentially as
described (25). Briefly, membrane pieces were first incubated in 0.5%
polyvinylpyrrolidone (PVP-360; Sigma), 100 mM acetic acid
for 30 min at 37 °C. After extensive washing with H2O,
membranes were incubated for 2 h at 37 °C with 10 µg of
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin (Sigma) in 50 mM NH4HCO3.
This was followed by an overnight digestion with an additional 10 µg
of trypsin. Samples were lyophilized and dissolved in alkaline PAGE
sample buffer (125 mM Tris-HCl, pH 6.8, 6 M
urea, and bromphenol blue). The tryptic phosphopeptides were
resolved by electrophoresis on an alkaline 40% polyacrylamide gel, as
described (26). The samples were electrophoresed overnight at 7 mA
until the tracking dye had migrated to RF = 0.5. The
gel was dried, and phosphopeptides were detected by autoradiography.
Cell Culture and Immunoprecipitations--
HEK-293T cells (ATCC)
were transfected with pcDNA3.1-Dab1-hemagglutinin or mutant forms
of Dab1 and a constitutively active form of Src (pcDNA-Src527F)
using FuGene 6 transfection reagent (Roche Molecular Biochemicals).
24 h after transfection, cells were lysed in cell lysis buffer (25 mM Tris-HCl, 1% Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate, 5 mM NaF, 10 µg/ml each aprotinin and leupeptin). The
lysates were cleared by centrifugation and incubated with
anti-hemagglutinin antibodies (Covance). Immunoprecipitates were
resolved by SDS-PAGE and transferred to nitrocellulose for analysis by
Western blotting.
Reelin Purification--
Expression of Reelin in 293T cells has
been described previously (11). Briefly, Nephrigen (Celox Laboratories
Inc.) supernatant containing Reelin was clarified by centrifugation
for10 min at 5000 × g at 4 °C. A saturated ammonium
sulfate (4.1 M) solution was added to the Nephrigen
supernatant to bring it from 0 to 45% saturation. Ammonium sulfate was
gradually added to a slowly stirring solution over a period of 30 min
at 4 °C, and the solution was stirred for an additional 30 min to
allow proteins to precipitate completely. The precipitate was collected
by centrifugation for 30 min at 12,000 × g at 4 °C.
After air-drying, the pellet was redissolved in 10 mM HEPES
(pH 7.5) containing 10% glycerol and stored at
80 °C. To estimate
the concentration of Reelin, samples were separated on SDS-PAGE gels
and compared with known quantities of bovine serum albumin by staining
with Coomassie Brilliant Blue. This technique yielded a 50-fold
purification, and partially purified full-length Reelin represented
~6% of the total precipitated proteins.
Primary Culture and Reelin Treatment--
Treatment of neurons
with Reelin-enriched supernatants has been described previously (11).
Briefly, brains of E16 reeler embryos were removed, and neurons were
isolated by trituration in Hanks' buffered salt solution. Neurons were
separated into aliquots in several microcentrifuge tubes and then
resuspended in either Dulbecco's modified Eagle's medium (DMEM)
or DMEM containing 1 ng/µl Reelin. After incubation at 37 °C for
15 min, neurons were collected by centrifugation and either frozen at
80 °C or lysed immediately in cell lysis buffer. The lysates were
pre-cleared by centrifugation at 14,000 rpm for 15 min, and the
supernatant was used for anti-Dab1 (goat anti-Dab1 PTB, ExAlpha
Biologics) immunoprecipitations.
Phosphopeptide Antibodies and Immunoblotting--
Synthesis of
peptides and generation of phosphopeptide antibodies were carried out
by Alpha Diagnostic International (San Antonio, TX). Rabbits were
immunized with KLH-coupled phosphopeptides PY185,
CEQAVY(PO4)QTILEED; PY198/200,
CEDPVY(PO4)QY(PO4)IVFEAG; PY220,
CETEENIVY(PO4)QVPTSQK; and PY232,
CKKEGVY(PO4)DVPKSQP. Phosphopeptide PY185 and an
unphosphorylated form of this peptide were conjugated to bovine serum
albumin (BSA) using Imject maleimide-activated BSA (Pierce).
Unphosphorylated GST-Dab1, Src-phosphorylated GST-Dab1, and GST-Dab1
mutant proteins were used in immunoblotting assays to characterize the antisera.
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RESULTS |
Dab1 Is Phosphorylated by Src in the Middle
Region--
Previous studies demonstrated that Dab1 binds to members
of the Src family of kinases and that Dab1 can be phosphorylated by Src
in transfected cells (16, 24). To determine if Src phosphorylates Dab1
directly, we used purified active Src (Src527F) to
phosphorylate GST fusion proteins containing Dab1. Indeed, as shown in
Fig. 1A, Src efficiently
phosphorylated GST-Dab1, whereas GST alone was not phosphorylated. As a
first step toward identifying the in vitro sites of
phosphorylation, we used GST fusion proteins containing three distinct
Dab1 regions as substrates. As illustrated in Fig. 1B, these
domains comprised the PTB domain (GST-Dab11-179),
the middle region (GST-Dab1180-399), and the carboxyl
terminus (GST-Dab1400-555) of Dab1. Interestingly, only
the middle region of Dab1 was phosphorylated by Src (Fig.
1A). This region contains six tyrosines, five of which were
previously suggested as sites of Dab1 phosphorylation in transient
transfection assays.

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Fig. 1.
Region of Src phosphorylation on Dab1.
A, GST alone or GST fusion proteins containing either
full-length Dab1, PTB domain (GST-Dab11-179), Mid region
(GST-Dab1180-399), or carboxyl-terminal region
(CT; GST-Dab1400-555) were phosphorylated with
Src in the presence of [ -32P]ATP. The proteins were
resolved by SDS-PAGE, transferred to nitrocellulose, and detected by
autoradiography. B, a schematic showing the region of
phosphorylation in relation to the remaining molecule. A partial amino
acid sequence of this region containing the cluster of tyrosines is
shown. All six tyrosine residues (Tyr185,
Tyr198, Tyr200, Tyr220,
Tyr232, and Tyr300) present within the middle
region are indicated.
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Mapping of the in Vitro Phosphorylation Sites--
A combination
of peptide mapping and mutagenesis approaches was used to identify the
specific sites of phosphorylation by Src in vitro.
Phosphopeptides generated by tryptic digestion of in vitro
phosphorylated Dab1 were readily separated by one-dimensional 40%
alkaline-PAGE. As shown in Fig.
2A, complete digestion of phosphorylated wild-type GST-Dab1 consistently yielded four
phosphopeptides. These phosphopeptides were designated numbers 1-4
starting from the most slowly migrating peptide. Occasionally, an
additional phosphopeptide (indicated by an asterisk, Fig.
2A) was also observed to migrate slightly faster than
phosphopeptide 3. Tyrosine 300 is not present in any of these
phosphopeptides as it lies in a 16-kDa fragment of Dab1 that is never
found to be phosphorylated in vitro.

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Fig. 2.
One-dimensional tryptic phosphopeptide map of
Dab1 phosphorylated by Src. A, GST fusion proteins of
either wild-type (Wt) Dab1 or Dab1 mutants containing
phenylalanine substitutions in place of tyrosines 185 (185F), 198 (198F), 220 (220F), 232 (232F), or both 198 and 200 (198F/200F) were
phosphorylated using Src. The phosphorylated proteins were separated by
SDS-PAGE, transferred to nitrocellulose, and located by
autoradiography. The proteins were trypsinized off the membrane, and
the tryptic fragments were resolved by alkaline 40% polyacrylamide gel
electrophoresis. The phosphopeptides were visualized by
autoradiography. B, a schematic showing the identity of the
tryptic phosphopeptides, including the sites of phosphorylation within
each peptide.
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To identify the specific sites of phosphorylation within Dab1, we
generated GST-Dab1 fusion proteins containing single phenylalanine substitutions at tyrosines 185 (185F), 198 (198F), 200 (200F), 220 (220F), and
232 (232F) and a GST-Dab1 fusion with double substitutions at residues 198 and 200 (198F/200F). The mutant GST-Dab1
fusion proteins were phosphorylated by Src in vitro,
tryptic phosphopeptide maps were generated, and the results are shown
in Fig. 2. Phenylalanine substitution at residues 220 and 232 resulted
in the loss of phosphopeptides 2 and 4, respectively. Therefore, Src
phosphorylated Tyr220 and Tyr232 of Dab1
in vitro. However, given the low intensity of the band corresponding to peptide 4, Tyr232 was considered to be a
minor site of in vitro phosphorylation. The remaining three
potential sites of phosphorylation, Tyr185,
Tyr198, and Tyr200, are located within a single
tryptic peptide fragment, which complicates analysis. Single
substitution of either Tyr185 or Tyr200 did not
result in the loss of any of the four major peptides, indicating that
these two tyrosines were not major phosphorylation sites. In contrast,
substitution of Tyr198 with phenylalanine resulted in a
dramatic loss of phosphopeptide 1 and phosphopeptide 3 and the
appearance of a novel minor peptide (indicated by an arrow,
Fig. 2A). Therefore, phosphopeptide 1 probably represented a
tryptic fragment that was almost exclusively phosphorylated on
Tyr198, whereas phosphopeptide 3 likely represented a
doubly phosphorylated form of the same tryptic fragment containing
Tyr198(P) and either Tyr185(P) or
Tyr200(P). The novel minor phosphopeptide in tryptic digest
of GST-Dab1198F probably appeared as a result of a single
phosphate present on the same peptide at either Tyr185 or
Tyr200. Interestingly, tryptic digest of wild-type GST-Dab1
contained an additional phosphopeptide (indicated by an
asterisk, Fig. 2A) with a slightly faster
migration rate than phosphopeptide 3. It is possible that this band
represented a variant form of phosphopeptide 3 with a migration rate
that would depend on which of the other tyrosine residues was
phosphorylated within the single tryptic fragment. The exact identity
of this phosphopeptide was difficult to establish, because this band
was absent in digests of all mutant forms of GST-Dab1 fusion proteins.
Consistent with the possibility that phosphopeptide 3 resulted from
phosphorylation of Tyr198 and phosphorylation of either
Tyr185 or Tyr200, both phosphopeptides 1 and 3 were absent from tryptic digests of the GST-Dab1 fusion protein
containing phenylalanine substitutions at both residues 198 and 200. However, since phosphopeptide 3 persisted in the absence of
Tyr200, it is likely that Tyr185 was also
phosphorylated by Src in vitro. Therefore, the most straightforward interpretation of our results is that phosphopeptide 3 resulted from phosphorylation of Tyr198 and either
Tyr185 or Tyr200. Although it was difficult to
establish the relative contributions of Tyr185(P) and
Tyr200(P) toward the composition of phosphopeptide 3, our
results suggest that Tyr198 and Tyr220 were
major in vitro phosphorylation sites, and
Tyr185, Tyr200, and Tyr232 were
minor Src phosphorylation sites.
Characterization of Phosphopeptide-specific Dab1 Antibodies--
A
combination of metabolic radiolabeling of cells with orthophosphate and
tryptic phosphopeptide mapping has been the traditional approach to
identify in vivo sites of protein phosphorylation. Due to
inefficient radiolabeling of Dab1 in neurons and the presence of
multiple tyrosines (Tyr185, Tyr198, and
Tyr200) within a single tryptic fragment, it was difficult
to employ this technique to identify the in vivo sites of
Dab1 phosphorylation. Therefore, we chose to prepare
phosphopeptide-specific antibodies to confirm the in vitro
phosphorylation sites and to investigate in vivo
phosphorylation of Dab1. Peptides containing single phosphotyrosines Tyr185(P) (PY185), Tyr220(P) (PY220), and
Tyr232(P) (PY232) were used to immunize rabbits. Given the
close proximity of Tyr198 and Tyr200, we used a
synthetic peptide containing both tyrosines in the phosphorylated form
(PY198/200) for immunization. The efficacy of the each antibody was
determined by Western blot assays in which the antisera were tested
against unphosphorylated GST-Dab1 and GST-Dab1 phosphorylated with Src
in vitro. As shown in Fig. 3A, the antisera raised
against PY198/200, PY220, and PY232 recognized only phosphorylated
GST-Dab1, with no significant cross-reactivity with unphosphorylated
GST-Dab1. Western blotting using anti-Dab1 antibodies confirmed that
equivalent levels of unphosphorylated and phosphorylated Dab1 were
present. Antisera from three different rabbits immunized with the PY185
peptide failed to show any specific reactivity toward phosphorylated
GST-Dab1. This lack of reactivity was not due to a low titer, because
the antisera strongly recognized bovine serum albumin-conjugated PY185
phosphopeptide in a Western blot assay without any cross-reactivity
toward an unphosphorylated form of the peptide. Therefore, since the
antisera recognized a synthetic phosphopeptide but did not recognize
Src-phosphorylated GST-Dab1, it can reasonably be concluded that
Tyr185 was not phosphorylated by Src in vitro.
This finding was consistent with our phosphopeptide mapping results,
which indicated that Tyr185 was a minor site of in
vitro phosphorylation.

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Fig. 3.
Characterization of the
phosphopeptide-specific antibodies. A, GST-Dab1 was
either left untreated (U) or phosphorylated (P)
by Src. The proteins were separated by SDS-PAGE and transferred to
nitrocellulose. The membranes were subjected to Western blotting using
anti-PY185, anti-PY198/200, anti-PY220, or anti-PY232 antibodies. The
membranes were stripped and reprobed with anti-Dab1 antibodies to show
equal loading of the proteins. The anti-PY185 antibody was also tested
in a Western blot using bovine serum albumin conjugates of the
unphosphorylated (Y185) and the phosphorylated
(PY185) peptides corresponding to this phosphorylation site
(right panel). B, GST fusions of Dab1 containing
phenylalanine substitutions at residues 198 (198F), 200 (200F), 198 and 200 (198F/200F), 220 (220F), or 232 (232F) were either phosphorylated
by Src (P) or left untreated (U). Immunoblotting
was then carried out using either anti-PY198/200 (upper
panel), anti-PY220 (middle panel), or anti-PY232
(lower panel) antibodies. The membranes were then stripped
and reprobed with anti-Dab1 antibodies to confirm equal protein
loading. Wt, wild type.
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Although the data in Fig. 3A show that the
phosphopeptide-specific antibodies specifically recognized
phosphorylated Dab1, the utility of these antibodies requires that they
also exhibit sequence specificity. To determine sequence specificity,
the antibodies were tested in Western blots against unphosphorylated
and Src-phosphorylated wild-type GST-Dab1 or GST-Dab1 fusion proteins
containing single phenylalanine substitutions at Tyr198,
Tyr200, Tyr220, and Tyr232 or
double mutations of Tyr198 and Tyr200. As shown
in Fig. 3B, the antibodies raised against PY198/200, PY220,
and PY232 exhibited strong sequence specificity. The anti-PY220 and
anti-PY232 antibodies did not recognize phosphorylated GST-Dab1 containing single phenylalanine substitutions in place of the corresponding tyrosines. The specificity of anti-PY198/200 was determined by testing it on GST-Dab1 fusion proteins that contained either single phenylalanine substitutions in place of
Tyr198 and Tyr200 or double substitution of
both Tyr198 and Tyr200. The antibodies did not
recognize Src-phosphorylated GST-Dab1 when Tyr198 alone or
both Tyr198 and Tyr200 were substituted with
phenylalanine. In contrast, the antisera contained partial reactivity
toward Dab1 containing a single mutation of Tyr200,
indicating that Tyr198(P) is the major epitope for this
antibody but both Tyr198(P) and Tyr200(P) are
required for optimal recognition of phosphorylated Dab1.
Phosphorylation of Dab1 in Transfected 293T Cells--
Dab1 is
efficiently phosphorylated by Src in transfected cells. Mapping studies
using mass spectrometry to identify tryptic phosphopeptides showed that
Tyr198 and Tyr232 were phosphorylated by Src
(24). However, tyrosyl phosphorylation of Dab1 remained undiminished
when these two residues were mutated to phenylalanine, suggesting that
Dab1 was phosphorylated on other tyrosine residues as well.
Phenylalanine substitution of all five tyrosines (Tyr185,
Tyr198, Tyr200, Tyr220, and
Tyr232) present immediately downstream of the PTB domain
abrogates Src phosphorylation in transfected cells (24). To identify
specific sites of tyrosyl phosphorylation, we transfected 293T cells
with Src and with either wild-type Dab1 or Dab1 carrying single
phenylalanine substitutions at Tyr198, Tyr200,
Tyr220, Tyr232 or a double substitution of
Tyr198 and Tyr200. Western blots using the
phosphopeptide-specific antibodies were then carried out to determine
the phosphorylation status of each of the mapped tyrosine residues. As
shown in Fig. 4A,
Tyr220 and Tyr232 were clearly phosphorylated
by Src. The antibody raised against the doubly phosphorylated PY198/200
peptide did not recognize Dab1 if Tyr198 alone or both
Tyr198 and Tyr200 were mutated to
phenylalanine. In contrast, mutation of Tyr200 did not have
any effect on the ability of this antibody to recognize phosphorylated
Dab1, implying that Tyr200 was not phosphorylated. To
determine if Tyr185 was phosphorylated by Src, Dab1 was
expressed either with Src or without Src in 293T cells, and
anti-Dab1 immunoprecipitates were immunoblotted with anti-PY185. As
shown in Fig. 4B, Tyr185 was not phosphorylated,
even though Dab1 was tyrosyl-phosphorylated in the presence of Src.
Therefore, these results show that Dab1 is phosphorylated by Src on
Tyr198, Tyr220, and Tyr232 in 293T
cells.

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Fig. 4.
Dab1 tyrosyl phosphorylation sites in 293T
cells. A, wild-type (Wt) Dab1 or mutant
forms of Dab1 carrying phenylalanine substitutions at residues 198 (198F), 200 (200F), 220 (220F), 232 (232F), or both 198 and 200 (198F/200F) were
expressed 293T cells along with active form of Src. Dab1
immunoprecipitates were immunoblotted with various phosphopeptide
antibodies and anti-Dab1 antibody. B, Dab1 expressed in 293T
cells alone ( ) or with Src (+) was immunoprecipitated (IP)
and probed with either anti-PY185 or anti-phosphotyrosine
(Anti-PTyr) antibodies.
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Identification of Sites of Reelin-induced
Phosphorylation--
Dab1 is tyrosyl-phosphorylated in a
Reelin-dependent manner, and Reelin-induced phosphorylation
of Dab1 in embryonic neurons is an important biochemical assay for
Reelin function. As shown in Fig.
5A, tyrosyl phosphorylation of
Dab1 increased dramatically when neurons from reeler embryos
were treated with Reelin. We used the phosphopeptide antibodies to
identify the sites of Reelin-induced tyrosyl phosphorylation on Dab1 to
detect Dab1 in cultured neurons. Neurons isolated from embryonic brains
of reeler mice were either left untreated or treated with
Reelin. Anti-PY185, anti-PY198/200, anti-PY220, and anti-PY232 were
used in Western blots to detect phosphorylated Dab1 in the anti-Dab1
immunoprecipitates. As shown in Fig. 5, only the anti-PY198/200 and
anti-PY220 recognized Dab1 in Reelin-treated neurons, indicating that
Tyr220 and either Tyr198 alone or both
Tyr198 and Tyr200 were phosphorylated in
neurons stimulated with Reelin. In three independent experiments,
anti-PY185 and anti-PY232 did not react against Dab1 from
Reelin-treated neurons, suggesting that these two tyrosines were not
phosphorylated to any significant extent in response to Reelin
treatment. Tyrosyl phosphorylation of Dab1 was very faint in the
absence of Reelin, and only the anti-PY220 antisera reacted against
Dab1 from unstimulated neurons. Therefore, although it is possible that
Dab1 is basally phosphorylated on other tyrosines, our results suggest
that Tyr220 is the major site of phosphorylation in absence
of Reelin.

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Fig. 5.
Reelin-induced sites of Dab1
phosphorylation. A, neurons from E16 reeler
embryonic brains were incubated with either Dulbecco's modified
Eagle's medium (DMEM) alone or Reelin-enriched DMEM. The neurons were
then lysed and immunoprecipitated with anti-Dab1 antibodies. The
immunoprecipitates were separated by SDS-PAGE, and immunoblots were
carried out using anti-phosphotyrosine (anti-PTYR),
anti-PY185, anti-PY198/200, anti-PY220, anti-PY232, or anti-Dab1
antibodies. B, a schematic indicating the positions of the
tyrosines phosphorylated on Dab1 in neurons treated with Reelin.
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DISCUSSION |
Dab1 plays a critical role in controlling neuronal positioning in
the developing brain. It functions as an intracellular adapter protein
in the Reelin-signaling pathway, although the mechanism by which it
mediates downstream signaling is unknown. The most important clue to
Dab1 function is that it accumulates in a hypophosphorylated form in
reeler mice, and binding of Reelin to lipoprotein receptors triggers tyrosyl phosphorylation of Dab1 in neurons (11, 12). The
phosphorylation sites were suspected to be contained within a cluster
of five tyrosines located immediately downstream of the Dab1 PTB
domain. Indeed, Howell et al. (24) show that a mutant mouse
expressing Dab1 with phenylalanine substitutions in place of these five
tyrosines exhibited the same phenotype as reeler mice. This
provided genetic evidence to support the critical role of Dab1 tyrosyl
phosphorylation in the Reelin-signaling pathway. The biochemical
consequences of Dab1 tyrosyl phosphorylation are not known, but it is
possible that the phosphorylated tyrosines serve to recruit signaling
molecules that bind to phosphorylated Dab1 via their SH2 domains.
Alternatively, tyrosyl phosphorylation could affect the stability of
Dab1 or its interaction with lipoprotein receptors. Therefore,
identification of the Reelin-induced phosphorylation sites on Dab1 is
critical to identifying downstream signaling components of this pathway.
Dab1 was originally identified in a yeast two-hybrid screen as a
Src-binding protein, and it was subsequently shown to be phosphorylated
in an Src-dependent manner in cell lines (16). Given the
likely role of Src in Dab1 phosphorylation, we used recombinant active
Src to map the in vitro sites of Dab1 tyrosine phosphorylation. Using GST fusions of three distinct Dab1 domains as
substrates for Src in vitro, we determined that Src
phosphorylates several tyrosines that are clustered in the middle
region of Dab1 immediately downstream of the PTB domain. Using a
combination of site-directed mutagenesis and tryptic phosphopeptide
mapping, we showed that the major sites of Src-catalyzed in
vitro phosphorylation are Tyr198 and
Tyr220, and the minor sites of phosphorylation are
Tyr185, Tyr200, and Tyr232. Using
antibodies raised against phosphopeptides corresponding to these sites
of phosphorylation, we confirmed that Tyr198,
Tyr200, Tyr220, and Tyr232 are
in vitro Src phosphorylation sites. In transfected 293T
cells, Src phosphorylated Dab1 on Tyr198,
Tyr220, and Tyr232. Recently, a mass
spectrometric approach was used to identify Tyr198 and
Tyr232 as sites of Src-catalyzed phosphorylation on Dab1 in
transiently transfected 293T cells (24). However, since Dab1 mutants
with single phenylalanine substitutions of these two residues did not significantly reduce Dab1 tyrosyl phosphorylation, it was concluded that additional sites must be phosphorylated that are not detected by
mass spectrometry. Our finding that Tyr220 is also
phosphorylated is consistent with this conclusion. Since the antibodies
raised against a synthetic peptide corresponding to the
Tyr185(P) phosphorylation site failed to react with
phosphorylated Dab1, Tyr185 is unlikely to be a major
phosphorylation site.
To identify the sites phosphorylated in response to Reelin, we treated
reeler embryonic neurons with Reelin and used phosphopeptide antibodies raised against these putative sites in Western blots. The
results of these experiments clearly indicate that Tyr198
and Tyr220 are phosphorylated in response to Reelin
treatment. We were unable to determine whether Dab1 is phosphorylated
on tyrosine 198 alone or both tyrosines 198 and 200, because the
antibody against the doubly phosphorylated PY198/200 phosphopeptide
retains reactivity in the absence of Tyr200(P). However,
Tyr200 was not a major Src phosphorylation site in
vitro or in transfected cells. Therefore, it is unlikely that this
tyrosine is a major site of Reelin-induced phosphorylation. Antibodies
against PY185 and PY232 did not recognize Dab1 in either Reelin-treated
or untreated cells, suggesting that these tyrosines are not
phosphorylated to any significant extent in response to Reelin
stimulation. Despite the elevated levels of Dab1, tyrosyl
phosphorylation of Dab1 is significantly diminished in
reeler mice. This makes it very difficult to identify
tyrosines that are phosphorylated in the absence of Reelin. However, we
were able to detect a very low level of phosphorylation of
Tyr220 in untreated neurons. Therefore, our results suggest
that a population of Dab1 may be constitutively phosphorylated on this
tyrosine in the absence of Reelin.
The finding that Reelin triggers phosphorylation of Tyr198
and Tyr220 has important implications. Although the
immediate biochemical consequences of Reelin-induced Dab1 tyrosyl
phosphorylation are unknown, tyrosyl-phosphorylated Dab1 is likely to
be involved in protein-protein interactions. Dab1 has been shown to
interact with SH2 domains of various signaling proteins in
vitro (16, 24), but the in vivo interaction of Dab1
with any of these proteins has not been demonstrated. Our results
suggest that physiologically relevant interactions of Dab1 with
SH2-containing proteins would be mediated by Tyr198(P) and
Tyr220(P). The amino acid sequence surrounding
Tyr198 is very similar to the juxtamembrane region of
platelet-derived growth factor
-receptor that binds to the SH2
domains of Src family kinases (27). It is conceivable that
phosphorylation of this tyrosine would serve to recruit Src family
kinases. These kinases may then phosphorylate other proteins that may
be associated with Dab1. Alternatively, such kinases may also play a
role in amplifying the Reelin signal by increasing Dab1 phosphorylation in a positive feedback loop. The amino acid sequence downstream of
Tyr220 represents a minimal consensus sequence,
YXXP, for interaction with SH2 domains of various signaling
proteins, such as Abl, phospholipase C
, Crk, and Nck (28). Thus,
Reelin-induced phosphorylation of Tyr198 and
Tyr220 could result in formation of a Dab1-associated
signaling complex that would mediate the downstream effects of Reelin.
The kinases responsible for in vivo tyrosyl phosphorylation
of Dab1 are not known. It is interesting that the Src-catalyzed in vitro phosphorylation sites are also phosphorylated in
neurons stimulated with Reelin. The amino acid sequences upstream of
the two major sites of phosphorylation, Tyr198 and
Tyr220, share a marked sequence similarity. A hydrophobic
residue at the
1 position and acidic residues at positions
3,
4,
6, and
8 relative to the tyrosines are characteristic of
phosphorylation sites on substrates of the Src family of kinases (29).
Indeed, we have been able to phosphorylate Dab1 in vitro
using various tyrosine kinases, including Src and Fyn, and to a lesser
extent, Abl. Several Src family tyrosine kinases are widely expressed in the brain during embryogenesis (30). Although Fyn has been suggested
to play a role in synaptic plasticity (31), no overt brain phenotype
has been associated with src or fyn mutations. Furthermore, tyrosyl phosphorylation of Dab1 is undiminished in mice
lacking several Src family kinases (16). However, functional redundancy
within this family of kinases has made it very difficult to elucidate
the roles of individual kinases by genetic approaches, and it is
possible that Dab1 is phosphorylated by more than one Src family
kinase. Abl, a close relative of the Src family, has also been
implicated in the Reelin-Dab1 pathway. In Drosophila, Dab
and Abl are involved in a common pathway that controls the development
of axonal tracts (32). Although mice deficient in Abl or Arg, an Abl
family member, do not exhibit any phenotypic similarity to
reeler mice, mice deficient in both kinases suffer from
serious neural tube defects, and embryos fail to survive past embryonic
day 9 (33). Therefore, it is possible that Abl or Arg are required for
phosphorylation of Dab1, but functional redundancy within this family
presents some difficulties in determining their role in Dab1
phosphorylation. We cannot preclude the possibility that more than one
tyrosine kinase is involved in complete activation of the Reelin-Dab1
pathway. For example, one kinase may be responsible for the
constitutive phosphorylation of Dab1, whereas another kinase may
increase Dab1 tyrosyl phosphorylation in response to Reelin.
Identification of the sites of Reelin-induced Dab1 tyrosyl
phosphorylation is an important step toward understanding the
biochemical function of Dab1 in the Reelin-signaling pathway. Synthetic
peptides containing Tyr198 and Tyr220 can be
used in biochemical assays to identify and purify tyrosine kinases
involved in Dab1 phosphorylation. Furthermore, the availability of
antibodies specific for these phosphorylation sites makes it possible
to identify other upstream signaling pathways that may cross-talk with
the Reelin pathway. Tyrosine phosphorylation of Dab1 is an obligatory
step in Reelin-Dab1 signaling, and Reelin-induced phosphorylation
served as an important assay for identifying lipoprotein receptors as
key components of the Reelin-signaling pathway. The availability of
phosphorylation site-specific antibodies may not only allow us to
identify additional components of this pathway, but they may also help
identify other signaling pathways that cross-talk with the Reelin-Dab1 pathway.