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Current Biology, Vol. 13, 18–26, January 8, 2003, 2003 Elsevier Science Ltd. All rights reserved.
Reelin Activates Src Family
Tyrosine Kinases in Neurons
Hans H. Bock* and Joachim Herz*
Department of Molecular Genetics
University of Texas Southwestern Medical Center
Dallas, Texas 75390
Summary
Background: Reelin is a large signaling molecule that
regulates the positioning of neurons in the mammalian
brain. Transmission of the Reelin signal to migrating
embryonic neurons requires binding to the very-low-
density lipoprotein receptor (VLDLR) and the apolipo-
protein E receptor-2 (apoER2). This induces tyrosine
phosphorylation of the adaptor protein Disabled-1
(Dab1), which interacts with a shared sequence motif in
the cytoplasmic tails of both receptors. However, the
kinases that mediate Dab1 tyrosine phosphorylation and
the intracellular pathways that are triggered by this event
remain unknown.
Results: We show that Reelin activates members of the
Src family of non-receptor tyrosine kinases (SFKs) and
that this activation is dependent on the Reelin receptors
apoER2 and VLDLR and the adaptor protein Dab1. Dab1
is tyrosine phosphorylated by SFKs, and the kinases
themselves can be further activated by phosphorylated
Dab1. Increased Dab1 protein expression in fyn-defi-
cient mice implies a response to impaired Reelin signal-
ing that is also observed in mice lacking Reelin or its
receptors. However, fyn deficiency alone does not com-
pound the neuronal positioning defect of vldlr- or
apoer2-deficient mice, and this finding suggests func-
tional compensation by other SFKs.
Conclusions: Our results show that Dab1 is a physio-
logical substrate as well as an activator of SFKs in neu-
rons. Based on genetic evidence gained from multiple
strains of mutant mice with defects in Reelin signaling,
we conclude that activation of SFKs is a normal part of
the cellular Reelin response.
Introduction
The proper positioning of neurons in the CNS depends
on a signaling pathway that involves the large secreted
signaling protein Reelin, at least two receptors for this
protein on the surface of migrating neurons, and the
cytosolic adaptor protein Disabled-1 (Dab1) [1, 2]. Two
members of the low-density lipoprotein (LDL) receptor
family of apolipoprotein E (ApoE) receptors, the very-
low-density lipoprotein receptor (VLDLR) and the ApoE
receptor-2 (apoER2), have been shown to function as
Reelin receptors. Dab1 interacts with a conserved Asn-
Pro-X-Tyr tetra amino acid motif (NPxY, where x repre-
sents any amino acid) in the cytoplasmic domain of both
receptors. Binding of Reelin to the extracellular domains
of VLDLR and apoER2 rapidly induces tyrosine phos-
*Correspondence: hans.bock@utsouthwestern.edu; joachim.herz@
utsouthwestern.edu
Metadata, citation and similar papers at core.ac.
PII S0960-9822(02)01403-3
phorylation of Dab1 [3, 4], which is essential for trans-
mission of the positional cue that is conveyed by Reelin
to the migrating cell.
The nature of the tyrosine kinase or kinases that medi-
ate the phosphorylation of Dab1 has not been ascer-
tained. We have previously proposed a model in which
a coreceptor, which could either be a transmembrane
tyrosine kinase or a membrane protein that can recruit
a non-receptor tyrosine kinase to the membrane, also
interacts with Reelin and is thereby brought into proxim-
ity to Dab1 [5]. Cadherin-related neuronal receptors
(CNRs) [6] and members of the integrin family of adhe-
sion proteins [7] have been suggested as such core-
ceptors, but further genetic and biochemical data are
required to establish the respective roles of these recep-
tors in this signaling pathway.
A role for Src family kinases (SFKs) in Reelin signaling
has been suggested by several lines of evidence. First,
the mammalian form of Dab1 was originally identified
as a Src-interacting protein in a yeast two-hybrid screen
by Howell and colleagues [8]. Second, mice that are
genetically deficient in the SFK Fyn show neuronal posi-
tioning defects in the hippocampal CA3 region and the
dentate gyrus [9, 10]. Third, Fyn has been reported to
bind to the cytoplasmic tails of the CNRs [11], and,
fourth, integrins, which have been shown to be present
in a complex with the Reelin receptors VLDLR and
apoER2 [7], have also been recognized as activators of
SFKs [12].
Here, we have investigated the role of SFKs in Reelin
signaling. Our results demonstrate that Reelin activates
SFKs through a mechanism that depends on the Reelin
receptors VLDLR and apoER2 and the adaptor protein
Dab1. Efficient tyrosine phosphorylation of Dab1 re-
quires SFK activity, and SFKs can undergo autoactiva-
tion in the presence of phosphorylated Dab1. Upregu-
lation of Dab1 expression in fyn-deficient neurons is
consistent with a response mechanism by which the cell
attempts to compensate for reduced kinase availability.
Taken together, these results suggest that SFKs func-
tion as intrinsic components of the Reelin signaling
pathway.
Results and Discussion
Activation of Src Family Kinases by Dab1
It has been shown that Src family kinases (SFKs) can
phosphorylate Dab1 in vitro and can interact with tyro-
sine-phosphorylated Dab1 through their SH2 domain [4,
8, 13]. We questioned whether Dab1 could activate SFKs
by established mechanisms, i.e., either by displacement
of the inhibitory phosphorylated tyrosine residue 529
from the SH2 domain or by an SH3 domain-mediated
interaction with proline-rich sequences [14]. To test this,
we transfected HEK-293 cells with expression plasmids
for c-Src (Figure 1A, lane 1) and Dab1 (lane 3) alone or
with both plasmids (lane 2). Simultaneous expression
of both proteins led not only to increased tyrosine phos-
Reelin Activates Src Family Tyrosine Kinases
19

Figure 1. Activation of SFKs by Coexpression with Dab1 in Transfected Cells

(A) HEK-293 cells were transfected with plasmids containing c-Src (lane 1), Dab1 (lane 3), or were cotransfected with c-Src and Dab1 (lane
2). Tyrosine-phosphorylated proteins (5 ␮g total protein/lane) were detected by immunoblotting with an ␣-phosphotyrosine antibody (4G10).
Erk served as a loading control.
(B) An analogous experiment was performed with c-Fyn instead of c-Src. Immunoblotting with an antibody directed against a conserved
phosphotyrosine residue in the activation domain of SFKs (␣-pY418) was used to monitor Dab1-dependent Fyn activation. Cdk5 served as a
loading control.

phorylation of Dab1, but also to a dramatic overall in-
crease in total cellular Src kinase activity. In contrast,
expression of recombinant c-Src alone only modestly
increased cellular phosphotyrosine levels (lane 1). In-
creased tyrosine kinase activity was also observed when
Dab1 and another Src family kinase, c-Fyn, were coex-
pressed in HEK-293 cells (Figure 1B, lane 3). This corre-
lated with an ⵑ4-fold increase in phosphorylation of a
tyrosine residue in the activation loop. This residue is
conserved in all SFK members (referred to as Y418, its
position in human Src) and stimulates SFK activity in the
phosphorylated state (reviewed in [14]). These results
suggest that Dab1 and SFKs can mutually activate each
other if the local concentration exceeds a critical thresh-
old. Physiologically, this could be achieved by ligand-
induced clustering at the cell surface, for instance, by
oligomerized Reelin [15].
Reelin-Induced Activation of SFKs in Primary
Cortical Neurons
Tyrosine phosphorylation of Dab1 has been shown to
represent a key step in Reelin signaling and is required
for the physiological function of Reelin during brain de-
velopment [1, 3, 13]. As it had previously been shown
that tyrosine-phosphorylated Dab1 interacts with the
Src SH2 domain [8], and coexpression of Dab1 and SFKs
in cultured cells greatly enhances overall tyrosine kinase
activity, we wanted to investigate a possible involve-
ment of SFKs in Reelin signaling in primary neurons. By
improving the sensitivity and specificity of the biochemi-
cal assay developed by Howell and collegues [3], which
detects Reelin-induced tyrosine phosphorylation of
Dab1 in cultured cortical neurons, we were able to moni-
tor Reelin signaling by direct immunoblotting of crude
cell lysates from wild-type embryonic neurons with an
anti-phosphotyrosine antibody without the need for
prior immunoprecipitation (see Figure S1 in the Supple-
mentary Material available with this article online). Using
this simplified assay, Reelin-induced tyrosine phosphor-
ylation of Dab1 was observed not only in cortical neu-
rons from mouse embryos, as had been previously dem-
onstrated [3], but also in neurons that had been prepared
from newborn (P0.5) pups (Figure S1); this finding sug-
gests that the Reelin signaling pathway remains func-
tional beyond the birth period of neurons during embry-
onic development. Reelin expression continues in the
adult brain [16], where it was recently shown to modulate
synaptic plasticity [17]. Taken together, these findings
suggest functions of this signaling pathway that go be-
yond the regulation of cortical lamination.
Treatment of cortical neurons with Reelin induced not
only tyrosine phosphorylation of Dab1 (Figure 2A, upper
panel) by ⵑ7-fold, but also the activation of SFKs by
increasing the level of tyrosine phosphorylation at the
activating Y418 residue by ⵑ4-fold. In contrast, no
change in phosphorylation levels was observed at the
inhibitory autophosphorylation site of Src (Y529 in hu-
man; Y534 in mouse Src). The phosphorylation state of
the main activating residue (Y397) of focal adhesion
kinase (FAK), another non-receptor tyrosine kinase that
mediates signaling by integrins and thereby regulates
cellular adhesion and migration [18], was determined as
a control and did not change upon Reelin stimulation.
Total cellular levels of c-Src, FAK, and the serine/threo-
nine kinases Cdk5 and Akt also remained unaffected.
Current Biology
20
Since the Y418 epitope is highly conserved between
the different SFKs, this antibody did not allow us to
distinguish which family members were specifically acti-
vated by the Reelin signal. Circumstantial evidence sug-
gested a particular role for the SFK Fyn in the Reelin
pathway. Fyn is expressed in regions of the developing
brain that are affected in the Reelin-deficient reeler mice
and enriched in embryonic growth cones [19], the pre-
sumptive Reelin-responsive site of migrating neurons
(our unpublished data), together with other SFKs [20].
Furthermore, Fyn has been reported to interact with the
cytoplasmic tails of putative Reelin coreceptors [6, 7].
Moreover, mutant mice lacking Fyn display structural
abnormalities in the hippocampus that resemble, but
are less severe than, those seen in reeler mice [9, 10].
Immunoprecipitation of Fyn and immunoblot analysis
with the nonselective anti-phosphotyrosine antibody
4G10 (Figure 2B, upper panel) and with the activation
loop-specific anti-pY418 antibody (middle panel) re-
vealed that Fyn was indeed tyrosine phosphorylated in
response to Reelin treatment (10-fold and 3-fold in-
crease, respectively). Fyn immunoprecipitation from
Reelin-treated and control samples was quantitative and
identical (lower panel).
Pharmacological Inhibition of Dab1 Tyrosine
Phosphorylation and Effect of Fyn Deficiency
on Reelin Signaling in Cortical Neurons
Our results so far had suggested that Dab1 can be both
a substrate and an activator of SFKs when both proteins
Figure 2. Reelin-Induced Activation of SFKs
in Primary Embryonic Cortical Neurons
(A) Immunoblot analysis of crude cell lysates
from neurons treated with control medium or
Reelin. Reelin-induced Dab1 tyrosine phos-
phorylation was detected with the 4G10 anti-
body (also see Figure S1). Activating phos-
phorylation of SFKs in response to Reelin was
revealed by using ␣-pY418. No change in the
phosphorylation state at the inhibitory tyro-
sine residue (Y529) in Src was observed.
Phosphorylation at the major activation site
(Y397) of another non-receptor tyrosine ki-
nase and Src substrate, focal adhesion ki-
nase (FAK), as well as total levels of c-Src,
FAK, and the serine/threonine kinases Cdk5
and Akt/PKB remained unchanged.
(B) Fyn is tyrosine phosphorylated in re-
sponse to Reelin in primary cortical neurons.
Fyn was immunoprecipitated from total cell
lysates of neurons treated with control me-
dium or with Reelin. Precipitated proteins
were immunoblotted with 4G10 (anti-phos-
photyrosine, upper panel). Increased overall
tyrosine phosphorylation of Fyn in Reelin-
treated neurons was paralleled by increased
phosphorylation in the activation loop (pY418).
Equal precipitation and loading was verified
by immunoblotting with ␣-Fyn (lower panel).
are overexpressed in cultured cells (Figure 1). Reelin
stimulation of cultured neurons resulted in phosphoryla-
tion of Dab1 and activation of SFKs (Figure 2). To investi-
gate whether Dab1 might also be a substrate for SFKs in
vivo, we used a panel of commercially available tyrosine
kinase inhibitors (Figure S2). The pyrazolopyrimidines
PP1 and PP2, two tyrosine kinase inhibitors with the
highest relative selectivity for SFKs [21], but not the
structurally related inactive compound PP3, blocked
Reelin-induced Dab1 tyrosine phosphorylation in a
dose-dependent manner. In contrast, Herbimycin A and
STI 571 (Gleevec), both inhibitors of c-Abl as well as
several other tyrosine kinases, were ineffective (Figure
S2). This result argues against a major role of c-Abl in
Dab1 tyrosine phosphorylation but does not exclude a
downstream function of c-Abl.
Because none of the commercially available inhibitors
including PP1 and PP2 are completely selective for any
specific tyrosine kinase, these results are consistent
with, but do not in themselves prove, a direct role of SFKs
in Reelin-mediated signaling and Dab1 phosphorylation
in vivo.
We thus sought to obtain genetic evidence to support
a role of SFKs in Reelin-induced signaling in vivo. Mice
that lack only one of several SFKs show none of the
gross cortical lamination defects that are characteristic
for mutants with defects in the Reelin signaling pathway
[22]. An exception are fyn-deficient mice that show a
subtle malformation of the dentate gyrus [9, 10]. We
therefore focused our attention on the fyn mutants and
Reelin Activates Src Family Tyrosine Kinases
21

Figure 3. Tyrosine Phosphorylation of Dab1 by Reelin Is Not Impaired in Fyn-Deficient Neurons

(A) Total cellular proteins from cortical embryonic neurons containing two (lane 5), one (lanes 2 and 3), or no (lanes 1 and 4) functional fyn
alleles were separated by SDS gelelectrophoresis and were analyzed by immunoblotting with the indicated antibodies. The relative amount
of Fyn immunoreactivity in the samples correlates with the number of intact fyn alleles. Total cellular amounts of VLDLR, apoER2, FAK, and
Cdk5 remain unchanged irrespective of the genotype, whereas Dab1 protein levels are significantly increased in fyn-deficient neurons.
(B) Cortical neurons from fyn-deficient (lanes 1 and 2) and wild-type (lanes 3 and 4) embryos were incubated with control medium (lanes 1
and 3) or Reelin (lanes 2 and 4). Endogenously produced Reelin in the control samples causes the low background phosphorylation of Dab1.
Reelin-induced Dab1 tyrosine phosphorylation occurs normally in the absence of Fyn (lane 2), but phosphorylation of the activation domain
of neuronal SFKs (␣-pY418) is greatly reduced. Expression of endogenous Src is slightly reduced in Fyn-deficient neurons (lanes 1 and 2),
while Yes remains unchanged. Cdk5 served as a loading control.

investigated whether the loss of Fyn had any measurable
effect on the known components of the Reelin signaling
pathway.
Figure 3A shows an immunoblot analysis of neocorti-
cal brain extracts from wild-type, fyn⫹/⫺, and fyn⫺/⫺ em-
bryos. Dab1 expression was increased ⵑ6-fold in homo-
zygous fyn-deficient mice (lanes 1 and 4) and ⵑ2-fold
in heterozygotes (lanes 2 and 3) compared to wild-type
animals (lane 5). Intriguingly, there was also a small,
but reproducible (by ⵑ2-fold), increase in the smaller
unglycoslylated form of one of the Reelin receptors, the
VLDLR, although total protein levels were unchanged.
Expression of the second Reelin receptor, apoER2, was
unaffected, and total levels of FAK and Cdk5, which is
also involved in the regulation of neuronal migration,
were also unaffected. Increase of Dab1 expression also
occurs in mice lacking Reelin or its receptors [1, 5] and
likely reflects an attempt of the cell to adjust the “gain”
of the expected Reelin signal input.
We next isolated primary cortical neurons from wild-
type (Figure 3B, lanes 3 and 4) and fyn-deficient (lanes
1 and 2) mice and tested the ability of Reelin to induce
tyrosine phosphorylation of Dab1 as well as phosphory-
lation of other SFKs in the activation loop when Fyn was
absent. As in the brain extracts, Dab1 expression was
increased, but Reelin-induced tyrosine phosphorylation
of Dab1 occurred normally in fyn-deficient cortical neu-
rons (lane 2); this finding is consistent with the absence
of the typical neuroanatomical malformations that occur
in mice in which the Reelin signaling pathway is com-
pletely disrupted [1, 23]. However, Reelin-induced phos-
phorylation at the activating tyrosine residue of SFKs
was reduced by almost 5-fold in Fyn-deficient neurons
(compare lanes 2 and 4). Yet the relative, but not the
absolute, increase in Y418 phosphorylation upon Reelin
stimulation (lanes 1 and 2, ⵑ7-fold increase) was at least
as robust as that seen in wild-type neurons (lanes 3 and
4, ⵑ3-fold increase). These findings are also consistent
with a dominant role of Fyn in Reelin signaling, because
the phosphorylation site-specific antibody (␣-pY 418)
does not distinguish between different SFK members.
Thus, the overall reduced levels of cellular Y418-phos-
phorylated SFKs upon Reelin stimulation appear to re-
flect the contribution of Fyn to this signaling pathway.
Compensation for the absence of Fyn apparently occurs
through an increase in Dab1 levels, but does not involve
upregulation of the related SFKs Yes or Src. In fact,
expression of the latter was even reduced by ⵑ50% in
fyn knockout neurons (Figure 3B, lanes 1 and 2).
Reelin-Mediated Activation of SFKs
Requires Dab1
The increased expression of Dab1 in the brains of fyn
knockout mice and in fyn-deficient neurons suggests a
functional role of Fyn in Reelin signaling and further
supports a physiological role for SFKs in this signaling
Current Biology
22
Figure 4. Activation of SFKs by Reelin Is Blocked in dab1-Deficient
Cortical Neurons
(A) Total levels of Fyn and Src are unaffected by dab1 genotype in
E15.5 embryonic brains containing two (lanes 1 and 6), one (lanes
2 and 3), or no (lanes 4 and 5) functional dab1 alleles. Cdk5 served
as a loading control.
(B) Reelin-induced phosphorylation of neuronal SFK activation do-
mains (␣-pY418) is normal in dab1⫹/⫹ (lanes 1 and 2), but absent in
dab1⫺/⫺, neurons (lanes 3 and 4).
pathway. To obtain additional genetic evidence for a
functional relationship between components of the Ree-
lin signaling pathway and SFKs, we investigated the
effect of dab1 inactivation on Fyn expression and SFK
activation. Neither Fyn nor Src protein levels were al-
tered in the brains of dab1 knockout mice (Figure 4A,
lanes 4 and 5). Dab1 protein was reduced by approxi-
mately 50% in dab1⫹/⫺ brains (lanes 2 and 3) compared
to wild-type (lanes 1 and 6).
As expected, the Reelin-induced, tyrosine-phosphor-
ylated protein band at ⵑ80 kDa, which corresponds to
Dab1 (also see Figure S1), was absent from lysates of
dab1⫺/⫺ neurons (Figure 4B, lane 4). SFK activation by
Reelin was monitored by using the ␣-pY418 antibody
and was normal in wild-type neurons (lanes 1 and 2,
ⵑ7-fold increase), but it was completely abolished in
dab1⫺/⫺ neurons (lane 4, no change). However, the basal
levels of Y418 phosphorylation were identical in the
dab1⫹/⫹ and dab1⫺/⫺ neurons, indicating that dab1 defi-
ciency abrogates Reelin responsiveness, but has no
other discernible impact on the steady state of SFK
activation in embryonic neurons.
Activation of SFKs by Reelin Requires the Reelin
Receptors VLDLR and apoER2
Tyrosine phosphorylation and thus activation of Dab1
in response to Reelin requires the binding of Reelin to
its receptors VLDLR and apoER2 [24, 25]. To confirm
that Reelin binding to its receptors is required for the
Dab1-dependent activation of SFKs, we exposed wild-
type neurons to Reelin in the absence (Figure 5A, lanes
1–3) or presence (lane 4) of GST-RAP, a fusion protein
between glutathione-S-transferase (GST) and the recep-
tor-associated protein (RAP). The latter is a universal
inhibitor of ligand binding to LDL receptor family mem-
bers [26] including the VLDLR and apoER2. We had
previously shown that GST-RAP antagonizes Reelin
binding to both receptors and greatly reduces Reelin-
dependent Dab1 phosphorylation [24].
Consistent with our previous findings, GST-RAP re-
duced Dab1 phosphorylation (Figure 5A). The low resid-
ual phosphorylation of Dab1 by Reelin in the presence
of GST-RAP is explained by incomplete inhibition due
to the ⵑ20-fold higher binding affinity of Reelin to the
receptors [24]. Nevertheless, the Reelin-dependent in-
crease of SFK phosphorylation (compare lanes 1 and 4,
no change) was completely abrogated. GST alone
served as a negative control.
Dab1 phosphorylation and SFK activation were also
dependent upon the presence of functional Reelin re-
ceptors on the neurons. Cells lacking both vldlr and
apoer2 were unable to induce Dab1 or SFK tyrosine
phosphorylation in response to Reelin (Figure 5B, lane
2), while cells lacking vldlr but expressing functional
apoER2 activated Dab1 and SFKs almost normally (lane
4, ⵑ3-fold increase). Total Src or Fyn levels were unal-
tered in neurons from double-deficient mice, whereas
Dab1 protein expression was increased by approxi-
mately 10-fold (lanes 1 and 2).
Taken together, these data show that SFKs function
downstream of the Reelin receptor-Dab1 signaling com-
plex. However, pharmacological inhibition of SFKs also
interferes with Dab1 tyrosine phosphorylation, sug-
gesting that SFKs also participate in Dab1 tyrosine
phosphorylation. These findings are consistent with a
model in which Reelin-induced aggregation of the re-
ceptors and Dab1 at the plasma membrane generates
a “supercritical density” that results in mutual activation
of Dab1 and its binding partner and effector, i.e., SFKs.
It is possible, although not absolutely required, that a
coreceptor (e.g., CNRs or integrins) is recruited into the
complex by Reelin and serves as a primer to facilitate the
initial reaction (Figure 6). This model would be consistent
with the results shown in Figure 1 in which overexpres-
sion of Dab1 and an SFK, but not of either protein alone,
enhanced Dab1 tyrosine kinase phosphorylation as well
as total cellular tyrosine kinase activity.
Combined Genetic Deficiency of fyn
and Reelin Receptors
Extensive redundancy between Src kinase family mem-
bers impedes the dissection of their relative functional
importance in various physiological processes. Triple
mutant mice that completely lack the major neuronally
expressed SFKs Src, Yes, and Fyn [27] are embryonic
lethal around E9.5, too early to be useful to study the
effect of SFKs on brain development. In order to test
whether it might be possible to obtain genetic evidence
for a potential specific role of Fyn in Reelin signaling by
Reelin Activates Src Family Tyrosine Kinases
23

Figure 5. Reelin-Mediated Activation of SFKs Requires Binding to ApoE Receptors

(A) Cortical neurons were incubated in the absence (lane 1) or presence of Reelin (lanes 2–4) and in the absence (lanes 1–3) or presence (lane
4) of receptor-associated protein fused to glutathione-S-transferase (GST-RAP), a universal inhibitor of ligand binding to LDL receptor family
members. GST protein was used as a control (lane 3). Reelin-induced tyrosine phosphorylation of Dab1 (upper panel) and SFKs (␣-pY418) is
greatly reduced by the presence of RAP (lane 4).
(B) Reelin-mediated SFK activation is impaired in neurons from vldlr and apoer2 knockout mice. Cortical neurons were isolated from apoer2/
vldlr double knockout (lanes 1 and 2) or vldlr-deficient mice (lanes 3 and 4). Treatment with Reelin (lanes 2 and 4) was compared to control
medium (lanes 1 and 3) and induced Dab1 and SFK phosphorylation only in vldlr⫺/⫺;apoer2⫹/⫹ (lane 4), but not in neurons lacking both Reelin
receptors (lane 2). Total Dab1 protein levels were greatly increased in double-deficient neurons (lanes 1 and 2), whereas total levels of Src
and Fyn were not affected by the genotype of the neurons. Cdk5 served as a loading control. Neurons were obtained from individual littermate
embryos.

epistasis, we crossed vldlr⫺/⫺ or apoer2⫺/⫺ mice with
fyn⫺/⫺ animals. Mice lacking only one of these receptors
display characteristic hypomorphic and spatially re-
stricted phenotypes [5]. A combined deficiency in one
of the receptors and a tyrosine kinase involved in Dab1
phosphorylation might conceivably compound the ob-
served phenotype overall or in specific regions of the
brain. However, mice double mutant for either fyn and
apoer2 or for fyn and vldlr were viable and did not exhibit
any gross neurological phenotype or neuroanatomical
abnormalities beyond those already present in the re-
spective single-knockout mice (Figure S3). This finding
underscores the high degree of functional redundancy
among SFKs and the remarkable degree to which neu-
rons can compensate for the loss of some of the molecu-
lar components that relay and interpret the Reelin signal
within the cell.
The absence of a compounded phenotype in the dou-
ble mutants may reflect an ability of the cell to adjust
the responsiveness of the cellular processes that are

Figure 6. Hypothetical Model for SFK Activa-

tion by Reelin
Oligomerized Reelin induces clustering of the
structurally closely related VLDLR and
apoER2. This results in a high local density
of Dab1 at the cytoplasmic leaflet. Dab1 may
activate SFKs directly, or coreceptors (e.g.,
CNRs or integrins) may serve as primers to
initiate the reaction. Tyrosine-phosphory-
lated Dab1 can recruit additional SFKs
through SH2 domain/phosphotyrosine inter-
actions. This would result in rapid and local-
ized amplification of Reelin-induced tyrosine
kinase activity. Blue ovals indicate alterna-
tively spliced cysteine-rich repeats in the
Reelin binding domain of apoER2.
Current Biology
24
targeted by the Reelin-activated SFKs. Thus, the relative
change of SFK activity, rather than the absolute amount,
may be critical to elicit the intended physiological effect.
This could include remodeling of the cytoskeleton dur-
ing the period of neuronal migration [28].
Experimental constraints allow us only to reasonably
estimate the relative quantitative change of SFK activa-
tion in whole-cell extracts. Thus, we can draw no conclu-
sions about the absolute change of SFK activity at spe-
cific subcellular locations in response to Reelin. The
finding that the levels of phosphorylated Dab1 are not
affected by the reduced SFK activation in fyn⫺/⫺ neurons
(Figure 3B) can be explained by the increased availability
of unphosphorylated Dab1 substrate. Together, these
mechanisms could account for this remarkable degree
of functional compensation.
It is likely that SFK activation by Reelin reflects only
one branch of a complex cytoplasmic signaling pathway
that may not manifest itself primarily in the regulation
of neuronal positioning but, for instance, in the modula-
tion of synaptic plasticity. Our findings that Reelin en-
hances synaptic plasticity, and that mice genetically
deficient in Reelin receptors display LTP defects [17]
that closely resemble those seen in fyn-deficient mice
[9], are consistent with such a hypothesis.
Conclusions
We have shown that tyrosine-phosphorylated Dab1 can
activate SFKs and that Reelin induces SFK activation
in embryonic cortical neurons. This requires the Reelin
receptors apoER2 and VLDLR as well as the cytoplasmic
adaptor protein Dab1. Results with pharmacological in-
hibitors of non-receptor tyrosine kinases are consistent
with a functionally redundant role of SFKs in Reelin-
induced Dab1 tyrosine phosphorylation. Increased
Dab1 protein expression in neurons from fyn⫺/⫺ embryos
suggests a prominent role of Fyn in Reelin signaling.
Reelin-induced activation of Fyn and related SFKs in
neurons further implies a functional role for this family
of tyrosine kinases in brain development or neuronal
function in the mature brain. Taken together with the
complementary findings by Arnaud et al. [29], the pres-
ent findings have thus revealed another part of the path-
way by which Reelin shapes the developing nervous
system and regulates neuronal cell function.
Experimental Procedures
Reagents
The tyrosine kinase inhibitors radicicol, herbimycin A, PP1, and PP2
were purchased from Biomol; the inactive PP2 analog PP3 was
purchased from Sigma.
Antibodies
The rabbit polyclonal antisera raised against the 13 carboxyl-termi-
nal amino acids of murine Dab1, VLDLR, and ApoER2 have been
described elsewhere [5, 24]. The monoclonal anti-phosphotyrosine
antibody 4G10, the Src monoclonal antibody GD11, and a Fyn rabbit
polyclonal antibody were purchased from Upstate Biotechnology.
Phosphorylation site-specific antibodies against p-Src (Y418), p-Src
(Y529), and p-FAK (Y397) were obtained from Biosource. Antibodies
against Erk and AKT were purchased from Cell Signaling Technol-
ogy; a mouse monoclonal antibody against Fyn and the Cdk5 antibody
were from Santa Cruz Biotechnology. The mouse monoclonal anti-
Reelin antibody (G10) was a generous gift from Andre Goffinet [30].
Animals
The vldlr⫺/⫺;apoer2⫺/⫺ mutant mice [5] were maintained on a mixed
C57BL/6J ⫻ Sv129Ev strain background. The fyn⫺/⫺ mice [31] were
purchased from the Jackson Laboratory, and the dab1⫺/⫺ mutant
mice were a kind gift from Jon Cooper and Brian Howell and have
been described elsewhere [32]. All animals were maintained in ac-
cordance with National Institutes of Health and institutional animal
care guidelines. Wild-type mice were maintained on the mixed
C57BL/6J ⫻ Sv129Ev strain background. For neuronal cultures,
male and female mice were mated, and, if a vaginal plug was ob-
served the next morning, the developmental stage was designated
E0.5.
Production of Recombinant Proteins
Recombinant Reelin was produced as described in the Supplemen-
tary Material, and receptor-associated protein (RAP) was prepared
as described previously [26].
Cell Culture and Transfection
HEK-293 cells (CRL-1573, ATCC) were grown in DMEM supple-
mented with 10% fetal calf serum and were transfected by calcium
phosphate precipitation (MBS kit) (Stratagene) with 1 ␮g pcDNA3.1
zeo(⫹) mDab555 [33] and 1 ␮g pCMV5-cSrc (murine neuronal) or
with pRc/CMV-cFyn (human, T: obtained from Gottfried Baier, Inns-
bruck) per 60-mm dish. Transfections with the respective empty
vectors served as controls. After 36–48 hr, cells were harvested by
aspirating the medium, rinsing with phosphate-buffered saline, and
adding 500 ␮l lysis buffer (20 mM Tris-buffered saline [pH 7.5], 2
mM EDTA, 1% Triton X-100 supplemented with 1 tablet protease
inhibitor cocktail [Roche Molecular Biochemicals]/10 ml, and phos-
phatase inhibitor cocktails I and II [Sigma] at the recommended
dilutions) per 60-mm dish. Lysates were cleared by centrifugation
at 20,000 ⫻ g, adjusted for protein content, mixed with 1 volume
2⫻ gel-loading buffer (2% sodium dodecyl sulfate [SDS], 10% glyc-
erol, 0.25 M Tris-HCl [pH 6.8], 0.02% bromophenol blue, 5%
␤-mercaptoethanol), boiled for 5 min, and stored at ⫺20⬚C or used
directly for immunoblot analysis.
Neuronal Cultures
Primary cortical neurons were prepared essentially as described
previously [3] (also see the Supplementary Material).
Reelin Stimulation of Cortical Embryonic Neurons
and Treatment with Inhibitors
Reelin-containing or control concentrated media were diluted corre-
sponding to a final estimated Reelin concentration of approximately
5 nM and were incubated at 37⬚C for 25 min if not indicated other-
wise. Treatment was terminated by aspirating the medium, rinsing
the cells with PBS containing 50 mM NaF, 25 mM ␤-glycerophos-
phate, and 2 mM sodium orthovanadate (Sigma), and adding ice-
cold lysis buffer (PBS [pH 7.4], 2 mM EGTA, 5 mM EDTA, 1% [v/v]
Triton X-100, 0.5% [w/v] SDS, 0.25% [w/v] sodium deoxycholate
and protease and phosphatase inhibitor cocktails as described
above; 16 ␮l/cm2 surface area). Lysates were adjusted for protein
content, mixed with twice-concentrated gel-loading buffer, and
used for immunoblot analysis. Stocks of the phosphatase inhibitors
were prepared in dimethyl sulphoxide (DMSO; Sigma) (10 mM; herbi-
mycin A was dissolved at 1 mM). Neurons were pretreated for 60
min (herbimycin A: 18 hr) at the indicated final inhibitor concentra-
tions before adding the Mock- or Reelin-containing media.
Immunoprecipitation
For immunoprecipitation of Dab1 or Fyn protein, neurons plated on
100 mm-dishes were harvested in PBS (pH 7.4) (1% Triton X-100,
0.1% SDS and 0.1% deoxycholate, 2 mM EDTA) containing protease
and phosphatase inhibitors (1 ml per dish). Lysates were cleared
by centrifugation and were incubated with 10 ␮l/ml rabbit nonim-
mune or anti-Dab1 serum, or 4 ␮g/ml anti-Fyn monoclonal antibody
on ice. Immune complexes were precipitated by adding 50 ␮l protein
A (Sigma)- or protein G (Amersham)-sepharose slurry (correspond-
ing to 25 ␮l packed beads, washed three times with lysis buffer)
per milliliter of lysate. The IP supernatant was collected, and the
beads were washed once with 500 ␮l lysis buffer, twice with PBS,
Reelin Activates Src Family Tyrosine Kinases
25
and were resuspended and boiled in 30 ␮l twice-concentrated gel
loading buffer. Aliquots of the lysate, the IP supernatants, and the
extracted beads were analyzed by immunoblotting (see the Supple-
mentary Material).
Protein Determination and Quantitation
Protein concentrations of cellular lysates were determined with a
detergent-compatible protein assay based on the Lowry method
(Bio-Rad). Relative intensities of protein bands on immunoblots were
determined by using NIH image 1.62 software.
Histology
Three- to five-week-old animals were perfused transcardially with
warm phosphate-buffered saline (PBS), followed by 4% paraformal-
dehyde. Sagittal paraffin sections in the region of the cerebellar
peduncle (7 ␮m) were stained with hematoxylin and eosin (H&E).
Supplementary Material
Supplementary Material including data relevant to assay proce-
dures, pharmacological inhibition of Reelin-activated tyrosine ki-
nase, and histology of mutant mouse brains is available at http://
images.cellpress.com/supmat/supmatin.htm.
Acknowledgments
We are indebted to Wen-Ling Niu, Jenny Hayes, Lisa Beatty, and
LaMetria Blair for invaluable technical support, to Jim Richardson,
Jeff Stark and John Shelton for help with the histological analysis,
to Tom Curran and Eckardt Förster for Reelin expression vectors
and for sharing the Reelin-expressing 293 cell line, Gottfried Baier
for the Fyn expression plasmid, and to Axel Ullrich and Petra May
for critical suggestions. This work was supported by grants from the
National Institutes of Health (HL20948, NS43408, and HL63762), the
Alzheimer Association, and the Perot Family Foundation. J.H. is
the recipient of a Wolfgang-Paul Award by the Humboldt Foundation
and a Visiting Professor at the Center for Molecular Biology at the
University of Heidelberg. H.B. is supported by a fellowship from the
Deutsche Forschungsgemeinschaft.
Received: August 14, 2002
Revised: September 26, 2002
Accepted: October 18, 2002
Published: January 8, 2003
References
1. Rice, D.S., and Curran, T. (2001). Role of the reelin signaling
pathway in central nervous system development. Annu. Rev.
Neurosci. 24, 1005–1039.
2. Herz, J. (2001). The LDL receptor gene family: (un)expected
signal transducers in the brain. Neuron 29, 571–581.
3. Howell, B.W., Herrick, T.M., and Cooper, J.A. (1999). Reelin-
induced tryosine phosphorylation of disabled 1 during neuronal
positioning. Genes Dev. 13, 643–648.
4. Keshvara, L., Benhayon, D., Magdaleno, S., and Curran, T.
(2001). Identification of reelin-induced sites of tyrosyl phosphor-
ylation on disabled 1. J. Biol. Chem. 276, 16008–16014.
5. Trommsdorff, M., Gotthardt, M., Hiesberger, T., Shelton, J.,
Stockinger, W., Nimpf, J., Hammer, R.E., Richardson, J.A., and
Herz, J. (1999). Reeler/Disabled-like disruption of neuronal mi-
gration in knockout mice lacking the VLDL receptor and ApoE
receptor 2. Cell 97, 689–701.
6. Senzaki, K., Ogawa, M., and Yagi, T. (1999). Proteins of the CNR
family are multiple receptors for Reelin. Cell 99, 635–647.
7. Dulabon, L., Olson, E.C., Taglienti, M.G., Eisenhuth, S., McGrath,
B., Walsh, C.A., Kreidberg, J.A., and Anton, E.S. (2000). Reelin
binds ␣3␤1 integrin and inhibits neuronal migration. Neuron 27,
33–44.
8. Howell, B.W., Gertler, F.B., and Cooper, J.A. (1997). Mouse dis-
abled (mDab1): a Src binding protein implicated in neuronal
development. EMBO J. 16, 121–132.
9. Grant, S.G., O’Dell, T.J., Karl, K.A., Stein, P.L., Soriano, P., and
Kandel, E.R. (1992). Impaired long-term potentiation, spatial
learning, and hippocampal development in fyn mutant mice.
Science 258, 1903–1910.
10. Yagi, T., Aizawa, S., Tokunaga, T., Shigetani, Y., Takeda, N.,
and Ikawa, Y. (1993). A role for Fyn tyrosine kinase in the suckling
behaviour of neonatal mice. Nature 366, 742–745.
11. Kohmura, N., Senzaki, K., Hamada, S., Kai, N., Yasuda, R., Wata-
nabe, M., Ishii, H., Yasuda, M., Mishina, M., and Yagi, T. (1998).
Diversity revealed by a novel family of cadherins expressed in
neurons at a synaptic complex. Neuron 20, 1137–1151.
12. Schlaepfer, D.D., and Hunter, T. (1998). Integrin signalling and
tyrosine phosphorylation: just the FAKs? Trends Cell Biol. 8,
151–157.
13. Howell, B.W., Herrick, T.M., Hildebrand, J.D., Zhang, Y., and
Cooper, J.A. (2000). Dab1 tyrosine phosphorylation sites relay
positional signals during mouse brain development. Curr. Biol.
10, 877–885.
14. Brown, M.T., and Cooper, J.A. (1996). Regulation, substrates
and functions of src. Biochim. Biophys. Acta 1287, 121–149.
15. Utsunomiya-Tate, N., Kubo, K., Tate, S., Kainosho, M., Kata-
yama, E., Nakajima, K., and Mikoshiba, K. (2000). Reelin mole-
cules assemble together to form a large protein complex, which
is inhibited by the function-blocking CR-50 antibody. Proc. Natl.
Acad. Sci. USA 97, 9729–9734.
16. Pesold, C., Impagnatiello, F., Pisu, M.G., Uzunov, D.P., Costa, E.,
Guidotti, A., and Caruncho, H.J. (1998). Reelin is preferentially
expressed in neurons synthesizing gamma-aminobutyric acid
in cortex and hippocampus of adult rats. Proc. Natl. Acad. Sci.
USA 95, 3221–3226.
17. Weeber, E.J., Beffert, U., Jones, C., Christian, J.M., Förster, E.,
Sweatt, J.D., and Herz, J. (2002). Reelin and ApoE receptors
cooperate to enhance hippocampal synaptic plasticity and
learning. J. Biol. Chem. 277, 39944–39952.
18. Ilic, D., Damsky, C.H., and Yamamoto, T. (1997). Focal adhesion
kinase: at the crossroads of signal transduction. J. Cell Sci. 110,
401–407.
19. Bixby, J.L., and Jhabvala, P. (1993). Tyrosine phosphorylation
in early embryonic growth cones. J. Neurosci. 13, 3421–3432.
20. Helmke, S., and Pfenninger, K.H. (1995). Growth cone enrich-
ment and cytoskeletal association of non-receptor tyrosine ki-
nases. Cell Motil. Cytoskeleton 30, 194–207.
21. Hanke, J.H., Gardner, J.P., Dow, R.L., Changelian, P.S., Bris-
sette, W.H., Weringer, E.J., Pollok, B.A., and Connelly, P.A.
(1996). Discovery of a novel, potent, and Src family-selective
tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T
cell activation. J. Biol. Chem. 271, 695–701.
22. Stein, P.L., Vogel, H., and Soriano, P. (1994). Combined deficien-
cies of Src, Fyn, and Yes tyrosine kinases in mutant mice. Genes
Dev. 8, 1999–2007.
23. Herz, J., and Bock, H.H. (2002). Lipoprotein receptors in the
nervous system. Annu. Rev. Biochem. 71, 405–434.
24. Hiesberger, T., Trommsdorff, M., Howell, B.W., Goffinet, A.,
Mumby, M.C., Cooper, J.A., and Herz, J. (1999). Direct binding
of Reelin to VLDL receptor and ApoE receptor 2 induces tyrosine
phosphorylation of disabled-1 and modulates tau phosphoryla-
tion. Neuron 24, 481–489.
25. D’Arcangelo, G., Homayouni, R., Keshvara, L., Rice, D., Sheldon,
M., and Curran, T. (1999). Reelin is a ligand for lipoprotein recep-
tors. Neuron 24, 471–479.
26. Herz, J., Goldstein, J.L., Strickland, D.K., Ho, Y.K., and Brown,
M.S. (1991). 39-kDa protein modulates binding of ligands to low
density lipoprotein receptor-related protein/alpha 2-macro-
globulin receptor. J. Biol. Chem. 266, 21232–21238.
27. Klinghoffer, R.A., Sachsenmaier, C., Cooper, J.A., and Soriano,
P. (1999). Src family kinases are required for integrin but not
PDGFR signal transduction. EMBO J. 18, 2459–2471.
28. Thomas, S.M., Soriano, P., and Imamoto, A. (1995). Specific and
redundant roles of Src and Fyn in organizing the cytoskeleton.
Nature 376, 267–271.
29. Arnaud, L., Ballif, B.A., Förster, E., and Cooper, J.A. (2003). Fyn
tyrosine kinase is a critical regulator of Disabled-1 during brain
development. Curr. Biol. 13, 9–17.
30. de Bergeyck, V., Naerhuyzen, B., Goffinet, A.M., and Lambert
de Rouvroit, C. (1998). A panel of monoclonal antibodies against
Current Biology
26

reelin, the extracellular matrix protein defective in reeler mutant

mice. J. Neurosci. Methods 82, 17–24.
31. Stein, P.L., Lee, H.M., Rich, S., and Soriano, P. (1992). pp59fyn
mutant mice display differential signaling in thymocytes and
peripheral T cells. Cell 70, 741–750.
32. Howell, B.W., Hawkes, R., Soriano, P., and Cooper, J.A. (1997).
Neuronal position in the developing brain is regulated by mouse
disabled-1. Nature 389, 733–737.
33. Trommsdorff, M., Borg, J.P., Margolis, B., and Herz, J. (1998).
Interaction of cytosolic adaptor proteins with neuronal apolipo-
protein E receptors and the amyloid precursor protein. J. Biol.
Chem. 273, 33556–33560.

Note Added in Proof

The data referred to throughout as “unpublished data” are now in

press: Beffert, U., Morfini, G., Bock, H.H., Reyna, H., Brady, S.T., and
Herz, J. (2002). Reelin-mediated signaling locally regulates protein
kinase B/Akt and glycogen synthase kinase 3␤. J. Biol. Chem. 277,
49958–49964.