Current Biology 20, 783–791, May 11, 2010 ª2010 Elsevier Ltd All rights reservedDOI 10.1016/j.cub.2010.03.048
c-Abl, Lamellipodin, and Ena/VASP
Proteins Cooperate in Dorsal Ruffling
of Fibroblasts and Axonal Morphogenesis
Magdalene Michael,1Anne Vehlow,1Christel Navarro,1
and Matthias Krause1,*
1Randall Division of Cell and Molecular Biophysics,
King’s College London, New Hunt’s House, Guy’s Campus,
London SE1 1UL, UK
Background: Tight regulation of cell motility is essential for
many physiological processes, such as formation of a func-
tional nervous system and wound healing. Drosophila Abl
negatively regulates the actin cytoskeleton effector protein
Ena during neuronal development in flies, and it has been
postulated that this may occur through an unknown interme-
diary. Lamellipodin (Lpd) regulates cell motility and recruits
Ena/VASP proteins (Ena, Mena, VASP, EVL) to the leading
edge of cells. However, the regulation of this recruitment has
Results: Here we show that Lpd is a substrate of Abl kinases
and binds to the Abl SH2 domain. Phosphorylation of Lpd
positively regulates the interaction between Lpd and Ena/
VASP proteins. Consistently, efficient recruitment of Mena
and EVL to Lpd at the leading edge requires Abl kinases.
Furthermore, transient Lpd phosphorylation by Abl kinases
correlates with an increase in Lpd-Mena coprecipitation. Lpd
is also transiently phosphorylated by Abl kinases upon
platelet-derived growth factor (PDGF) stimulation, regulates
PDGF-induced dorsal ruffling of fibroblasts and axonal
morphogenesis, and cooperates with c-Abl in an Ena/VASP-
Conclusions: Our findings suggest that Abl kinases positively
regulate Lpd-Ena/VASP interaction, Ena/VASP recruitment to
Lpd at the leading edge, and Lpd-Ena/VASP function in axonal
morphogenesis and in PDGF-induced dorsal ruffling. Our data
do not support the suggested negative regulatory role of Abl
for Ena. Instead, we propose that Lpd is the hitherto unknown
intermediary between Abl and Ena/VASP proteins.
Drosophila ena was originally identified as a suppressor of
lethality induced by mutations in d-abl, and it was postulated
that abl and ena negatively regulate each other [1, 2]. Both
the Abl tyrosine kinase family (D-Abl, vertebrate c-Abl and
Arg) and the Ena/VASP family (Ena, vertebrate Mena, VASP,
and EVL) act downstream of the netrin-1 axon guidance
receptor DCC andregulate cellmotility[3–6].Ablkinasesregu-
late platelet-derived growth factor (PDGF)-induced dorsal
ruffling of fibroblasts, but it is not known whether Lamellipodin
(Lpd) or Ena/VASP proteins function in this pathway .
Ena/VASP proteins play a crucial role in cell motility by
antagonizing actin filament capping. This alters the geometry
of the actin network toward longer, less-branched filaments,
thereby changing the speed and persistence of lamellipodia
[3, 5, 8]. Ena/VASP proteins are recruited to the leading edge
through interactions between their EVH1 domain and FP4
motifs within Lpd, a member of the MRL family of Ras effector
proteins, which includes C. elegans MIG-10, vertebrate RIAM
and Lpd, and Drosophila Pico [9–13]. Lpd contains a proline-
rich region harboring potential SH3-binding sites and a PH
previously that Lpd is required for lamellipodia formation and
that Lpd overexpression increases the speed of lamellipodial
protrusion in an Ena/VASP-dependent manner .
Lpd-dependent recruitment of Ena/VASP proteins to the
leading edge needs to be tightly regulated in order to precisely
control lamellipodia formation, but it is not known how this is
achieved. Studies in Drosophila have suggested that Abl
regulates Ena localization . Yet how the localization of
Ena/VASP proteins is controlled, and whether Abl plays a
role in this process in vertebrates, remains unclear.
Here we show that phosphorylation of Lpd by c-Abl
efficient recruitment of Mena and EVL to Lpd-positive lamelli-
podia requires Abl kinases. We provide evidence that Lpd and
Ena/VASP proteins regulate dorsal ruffling of fibroblasts upon
PDGF treatment and that Lpd function in this process is
Furthermore, we demonstrate that both Lpd and c-Abl
cooperate during axonal morphogenesis in an Ena/VASP-
dependent manner. Our data do not support the suggested
antagonistic roles of Abl and Ena, and we propose an alterna-
tive hypothesis that Abl kinases, via Lpd, positively regulate
Lamellipodin Is a Substrate for Abl Kinases
Tyrosine kinases play an essential role in the propagation of
signal transduction events . Interestingly, phosphorylation
site prediction software (Scansite) identified a putative c-Abl
phosphorylation site in Lpd. To test this, we overexpressed
GST-Lpd (Figure 1A) or HA-Lpd (see Figure S1A available on-
line) with wild-type, dominant-active, or kinase-inactive c-Abl
and found that Lpd is phosphorylated by wild-type and domi-
nant-active, but not kinase-inactive, c-Abl. We could not
detect any tyrosine phosphorylation of the Lpd-related protein
RIAM in the presence of c-Abl (data not shown), suggesting
that the posttranscriptional modification by Abl kinase is
specific for Lpd in the vertebrate MRL family.
or dominant-active but not kinase-inactive Arg also resulted in
phosphorylation of GST-Lpd (Figure 1B), indicating that Lpd is
a substrate of all members of the c-Abl family.
To identify the c-Abl phosphorylation sites in Lpd, we tested
the ability of purified c-Abl to phosphorylate 24 immobilized
peptides harboring all tyrosine residues within the Lpd amino
acid sequence. This analysis revealed that, in vitro, four Lpd
peptides harboring tyrosines (Y426, Y456, Y513, Y1226) are
highly phosphorylated, and eight additional peptides are
phosphorylated to a lesser extent (Figure 1C).
Three of the tyrosine residues were verified as c-Abl phos-
phorylation sites in full-length Lpd in cells, because non-
phosphorylatable single point mutations located in the PH
domain (Y426, Y456) or at the C terminus (Y1226) each
reduced c-Abl phosphorylation of Lpd (Figures 1D and 1E;
Figure S1B). However, we observed no reduction in phos-
phorylation of Y513 just behind the PH domain in the context
of full-length Lpd (Figure S1B), indicating that Y513 might not
be accessible for c-Abl phosphorylation in the folded protein
under the condition tested. Nevertheless, mutation of all four
tyrosines (GST-Lpd4YF) further reduced Lpd phosphoryla-
tion (Figures 1D and 1E), suggesting that Lpd is a novel
Abl family substrate harboring three major c-Abl phosphory-
Endogenous Lpd Is Phosphorylated by c-Abl upon PDGF
Because Abl kinases are activated upon PDGF receptor
ligation , we investigated whether endogenous Lpd is phos-
phorylated downstream of the PDGF receptor. We stimulated
serum-starved NIH 3T3 cells with PDGF for various time points
and immunoprecipitated endogenous Lpd from cell lysates.
Lpd phosphorylation peaked within 2 min of PDGF stimulation
and subsequently declined (Figures 2A and 2B).
To further investigate the phosphorylation of endogenous
Lpd, we generated phosphospecific antibodies against two
of the major phosphorylation sites, in the PH domain (anti-P-
Lpd-Y426) and in the C-terminal region (anti-P-Lpd-Y1226),
respectively. These antibodies detected phosphorylated Lpd
only when Lpd was cotransfected with wild-type but not with
kinase-inactive c-Abl (Figure 2E), or they detected endoge-
nous phosphorylated Lpd on western blots of PDGF-stimu-
lated NIH 3T3 cell lysates (Figure 2D).
To explore whether Abl kinases are required for the phos-
phorylation of Lpd upon PDGF stimulation, we pretreated
NIH 3T3 cells with a low concentration (1 mM) of the Abl kinase
inhibitor STI571 before stimulating the cells with PDGF for
2 min. Tyrosine phosphorylation of total Lpd (Figure 2C) and
at Lpd residues Y426 and Y1226 (Figure 2F) was abrogated
by Abl inhibitor treatment, suggesting that endogenous Lpd
is transiently phosphorylated by Abl kinases upon PDGF
c-Abl canbe activatedby interaction withits substrates ,
and we observed that wild-type c-Abl, as well as dominant-
active c-Abl (lacking the SH3 domain) and kinase-inactive
c-Abl, coprecipitated with GST-Lpd (Figure S2A). However,
we did not observe an increase in c-Abl kinase activity upon
cotransfection with Lpd in in vitro kinase assays (Figure S2B),
indicating that Lpd phosphorylation requires activation of c-
Abl by upstream signals such as PDGF receptor activation.
To test whether binding is mediated by the SH3 orSH2 domain
of c-Abl, we performed pull-down assays from NIH 3T3 cell
lysates with purified GST-Abl-SH3 or SH2 domains. As a posi-
tive control, we found that the Abl-SH3 domain pulled down
Mena as previously published . In contrast, Lpd and
RIAM did not interact with the Abl SH3 domain (Figure 2G).
However, Lpd phosphorylated on both Y426 and Y1226
precipitated with the GST-Abl-SH2 domain from lysates of
PDGF-stimulated NIH 3T3 cells (Figure 2H), suggesting that
a complex between Lpd and c-Abl is formed upon growth
factor stimulation and that complex formation is mediated
via the Abl-SH2 domain.
Phosphorylation of Lpd by c-Abl Positively Regulates
A major function of Lpd is to recruit Ena/VASP proteins to the
leading edge, an event that needs to be tightly regulated for
controlled lamellipodia formation . In agreement with
a potential regulation of Lpd by c-Abl, we observed that Lpd
colocalized with endogenous c-Abl in clusters at the very
edge of lamellipodia in NIH 3T3 fibroblasts (Figure S3A) and
growth cones of primary hippocampal neurons (Figure S3B).
To test whether Lpd localization at the leading edge might
be regulated by Abl kinases, we used mouse embryonic fibro-
blasts lacking both c-Abl and Arg  and rescued this cellline
with YFP-tagged wild-type c-Abl (Figure S3C). Interestingly,
Lpd localized to lamellipodia in Abl2/2Arg2/2fibroblasts
(Figure 3D), as well as in c-Abl-expressing cells (Figure 3E),
indicating that Lpd localization is not regulated by Abl kinases.
Figure 1. Lpd Is a Substrate of c-Abl and Arg Kinases
(A and B) HEK293FT cells were transfected with GST-Lpd with wild-type
AblWT (A) or ArgWT (B), dominant-active AblDA (A) or ArgDA (B), kinase-
inactive c-Abl (AblKI) (A) or Arg (ArgKI) (B), or GST with AblWT or ArgWT
(negative control). GST pull-down from lysates was followed by western
blotting with anti-phosphotyrosine antibodies in (A), (B), and (D).
(C) 24 peptides representing all tyrosine residues in Lpd, immobilized on
amembrane,wereincubatedwithc-Ablkinase andg-32P-ATP andanalyzed
wild-type c-Abl (AblWT).
(E) Quantification of blots by densitometry from three independent experi-
ments. *p < 0.05; **p < 0.01 (one-way analysis of variance [ANOVA]). Error
bars represent standard error of the mean (SEM). See also Figure S1.
Current Biology Vol 20 No 9
To explore the possibility that phosphorylation of Lpd by
c-Abl may regulate Lpd-Ena/VASP interaction, we cotrans-
fected GST-Lpd with wild-type or kinase-inactive c-Abl and
tagged EVL, Mena, or VASP. Pull-down of GST-Lpd and
western blot for Ena/VASP proteins revealed that cotransfec-
tion of wild-type c-Abl increased the interaction between
Lpd and EVL or between Lpd and Mena (Figures 3A and 3B).
Consistently, cotransfection with kinase-inactive c-Abl de-
creased the interaction between Lpd and Mena (Figure 3B).
Interestingly, Lpd was less phosphorylated by c-Abl when
VASP was coexpressed. VASP coprecipitated with Lpd
regardless of whether kinase-inactive or wild-type c-Abl was
expressed (Figure 3C). However, cotransfection of wild-type
c-Abl increased the interaction between VASP and Lpd
compared to kinase-inactive c-Abl
together, this indicates that c-Abl positively regulates the
interaction between Lpd and Ena/VASP proteins.
(Figure 3C). Taken
Lpd Is Phosphorylated upon Netrin-1 Stimulation, and This
Positively Correlates with Increased Mena Coprecipitation
The Lpd and Ena/VASP C. elegans orthologs mig-10 and unc-
34 function downstream of the netrin receptor unc-40/DCC .
To explore whether netrin-1 stimulation regulates endogenous
Lpd-Mena interaction in vertebrates, we stimulated primary
cortical neurons with the axon guidance cue netrin-1 and
observed a transient increase in tyrosine phosphorylation
of Lpd within 5 min (Figure 3F). Interestingly, the amount of
Mena coprecipitating with Lpd also increased after 5 min of
netrin-1 stimulation (Figure 3F). To test
phosphorylation upon netrin-1 treatment is mediated by Abl
kinases, we stimulated STI571-treated cortical neurons with
netrin-1 and observed a loss of Lpd phosphorylation upon
Abl kinase inhibition (Figure 3G). Taken together, this
suggests that netrin/DCC signaling induces Abl kinase-
dependent transient phosphorylation of Lpd to increase
Leading-Edge Localization of Ena/VASP Proteins
Is Differentially Regulated by Abl Kinases
c-Abl-dependent phosphorylation of Lpd positively regulates
its interaction with Ena/VASP proteins (Figure 3). Therefore,
we investigated whether localization of Ena/VASP proteins to
the leading edge is regulated by Abl kinases. Abl2/2Arg2/2
edge regardless of the presence of Abl kinases (Figure S4B).
Surprisingly, Mena and EVL were not efficiently recruited to
Lpd-positive lamellipodia in Abl2/2Arg2/2fibroblasts but
localized to the leading edge in c-Abl-expressing Abl2/2
Arg2/2fibroblasts (Figures 4A and 4B; Figure S4C). This
Figure 2. Endogenous Lpd Is Phosphorylated at
Y426 and Y1226 by c-Abl upon Platelet-Derived
Growth Factor Stimulation
(A) Serum-starved NIH 3T3 cells were stimulated
with 50 ng/ml platelet-derived growth factor
(PDGF). Lpd was immunoprecipitated and immu-
noblotted with anti-phosphotyrosine antibodies.
(B) Densitometry: control values were subtracted
from values of phosphotyrosine bands in (A)
(three independent experiments) and repre-
sented as fold increase compared to 0 min.
*p < 0.05 (Student’s t test). Error bars represent
(C) Serum-starved NIH 3T3 cells were treated
with dimethyl sulfoxide (DMSO) or 1 mM STI-571
for 2 hr prior to stimulation with PDGF. Lysates
were immunoblotted with anti-phosphotyrosine
(D) Serum-starved NIH 3T3 cells were stimulated
with 50 ng/ml PDGF and cell lysates were immu-
noblotted with Y426 and Y1226 phosphospecific
Lpd antibodies and anti-Hsc70 as loading
(E) HEK293FT cells were transfected with GST-
Lpd, wild-type (AblWT), or kinase-inactive (AblKI)
c-Abl. GST pull-down from lysates was followed
by immunoblotting with Y426 and Y1226 phos-
phospecific Lpd antibodies.
DMSO or 1 mM STI-571 for 2 hr and stimulated
with PDGF, and lysates were immunoblotted
with phosphospecific Lpd antibodies.
(G) GST-Abl-SH3-agarose was incubated with
lysates of NIH 3T3 cells and bound Lpd, RIAM,
or Mena detected in a western blot. GST-agarose
was used as a negative control.
(H) Lpd was pulled down from lysates of
PDGF-stimulated NIH 3T3 cells with GST-Abl-
SH2-agarose and was immunoblotted with anti-
used as a negative control.
three times. See also Figure S2.
Abl Positively Regulates Lpd-Ena/VASP Interaction
indicates that Mena and EVL, but not VASP recruitment to
lamellipodia, depend on c-Abl, and this may be mediated by
phosphorylation of Lpd.
Lpd and Ena/VASP Proteins Regulate PDGF-Induced
Abl kinases regulate dorsal ruffling of fibroblasts upon PDGF
stimulation . We observed that Lpd localizes to the rim of
PDGF-induced dorsal ruffles of NIH 3T3 fibroblasts where it
colocalizes with Mena (Figure 5A). Lpd overexpression signif-
icantly increased dorsal ruffling upon PDGF treatment
(Figure 5B). Conversely, Lpd knockdown decreased dorsal
ruffling (Figure 5C), suggesting that Lpd regulates this
To test the role of Ena/VASP for dorsal ruffle formation, we
employed a well-established strategy to artificially delocalize
all Ena/VASP proteins [19, 20]. This strategy relies on the
specific interaction of the EVH1 domain of Ena/VASP proteins
with a proline-rich motif (D/EFPPPPXD/ED/E) . Expressing
four EVH1-binding sites fused to a mitochondrial membrane
anchor (FP4-mito) delocalizes all Ena/VASP proteins to mito-
chondria, thereby rendering them nonfunctional. Mutation of
phenylalanine to alanine in the FP4 motif abolishes binding
to Ena/VASP proteins (AP4-mito, negative control) . Delo-
calization of all Ena/VASP proteins in NIH 3T3 fibroblasts
significantly reduced dorsal ruffling (Figure 5D), suggesting
that Ena/VASP function is important for this process.
Because Lpd might regulate dorsal ruffling by recruiting
Ena/VASP proteins, we tested whether the increase in dorsal
ruffling induced by Lpd overexpression was Ena/VASP depen-
dent. Coexpressing AP4-mito with GFP-Lpd was as efficient
as GFP-Lpd alone in increasing dorsal ruffling (Figure 5E).
Figure 3. Lpd Interaction with Ena/VASP Proteins Is Regu-
lated by c-Abl
(A–C) HEK293FT cells were transfected with GST-Lpd, EVL,
Mena, or VASP and AblWT or AblKI. GST pull-down on
lysates was followed by western blotting with anti-phospho-
tyrosine, Lpd, and EVL (A), Mena (B), or VASP (C) antibodies.
(D and E) Abl2/2Arg2/2and YFP-AblWT/Abl2/2Arg2/2cells
were fixed and stained with Lpd antibodies. Scale bar repre-
sents 30 mm.
(F and G) Primary cortical neurons were cultured for 36 hr
without (F) or with (G) 10 mM STI571 or DMSO, were stimu-
lated with 300 ng/ml netrin-1, and were lysed. Lpd was
immunoprecipitated and examined for tyrosine phosphory-
lation (F and G) and Mena coprecipitation (F) by western
blotting. Purified rabbit IgG was used for the control immu-
(A–G) All experiments have been repeated at least three
times. See also Figure S3.
However, coexpressing GFP-Lpd with FP4-mito
completely abolished this effect (Figure 5E),
Lpd-induced dorsal ruffling.
Lpd Function during Dorsal Ruffling
Is Regulated by c-Abl
Because c-Abl  and Lpd (Figure 5C) positively
affect dorsal ruffling, we investigated whether
c-Abl might regulate Lpd in this process. Coex-
pression of GFP-Lpd with c-Abl further increased
the GFP-Lpd overexpression phenotype (Fig-
ure 5F), indicating that c-Abl and Lpd cooperate
in this process. To test whether c-Abl kinase activity is
required for Lpd function, we pretreated GFP-Lpd-expressing
fibroblasts with the Abl inhibitor STI571. This treatment abol-
ished the GFP-Lpd-induced increase in dorsal ruffling
(Figure 5F), indicating that Lpd function requires phosphoryla-
tion by Abl kinases. In contrast to GFP-Lpd, overexpression of
GFP-Lpd4YF (Figure 1D) did not increase the dorsal ruffling
response (Figure 5G), suggesting that phosphorylation of
Lpd at the identified c-Abl sites is required for its function in
Lpd Regulates Axonal Morphogenesis
Abl kinases regulate neuronal morphogenesis and are
required for the integrin-dependent, laminin-induced increase
in axonal length and branching . To explore whether Lpd is
required for neuronal morphogenesis, we transfected primary
hippocampal neurons with two Lpd small interfering RNAs
siRNA (Figure S5A). Axons were detected by Tau-1 staining
(Figure S5B). The length, branching, and number of primary
dendrites per neuron were not significantly altered upon Lpd
knockdown (data not shown). Control siRNA-transfected
neurons formed long, highly branched axons when plated on
laminin for 2 days (Figure 6A). In contrast, the length of the
main axon was significantly shorter (reduced by 30%) in
neurons transfected with Lpd-specific siRNAs (Figures 6A
and 6B). The sum of the length of the main axon and all axonal
branches was further decreased (Figure 6C). Consistently,
axonal branching was highly impaired in the Lpd siRNA-trans-
fected neurons (Figure 6D). Coexpression of human GFP-Lpd
together with the mouse Lpd siRNA rescued the Lpd knock-
down phenotype, verifying that the observed phenotype was
Current Biology Vol 20 No 9
not due to an off-target effect (Figure 6E). Taken together,
these results suggest that Lpd regulates elongation and
branching of axons.
c-Abl Cooperates with Lpd in an Ena/VASP-Dependent
Manner in the Regulation of Axonal Morphogenesis
In primary neurons, overexpression of dominant-active c-Abl,
but not wild-type c-Abl, resulted in increased neurite growth
[21, 22]. Because Lpd is positively regulated by c-Abl and
contributes to axonal extension and branching, we explored
the possibility that Lpd and c-Abl may cooperate to regulate
this process. We transfected primary hippocampal neurons
either with empty EGFP vector, EGFP-Lpd, or wild-type
YFP-c-Abl and plated neurons after transfection onto poly-
D-lysine-coated coverslips, an adhesive substratum that
does not activate integrins. This induces only limited neurite
outgrowth and branching when compared to neurons plated
on laminin, thereby allowing us to investigate potential
increases in length and branching induced by overexpression.
Overexpression of wild-type c-Abl or EGFP-Lpd alone had no
effect on length and branching of either axons and dendrites
(Figures 7A–7D; data not shown). In contrast, co-overexpres-
sion of Lpd with wild-type c-Abl induced a significant increase
in length of the main axon (Figures 7A and 7B). The sum of the
Figure 4. Abl Kinases Regulate Leading-Edge
Localization of Mena and EVL
(A and B) Abl2/2Arg2/2and YFP-AblWT/Abl2/2
Arg2/2cells were plated, fixed, and stained with
Lpd (Figure S4C) and Mena (A) or EVL (B) anti-
bodies. Leading-edge localization of Mena and
EVL was quantified via line scans in cells with
Lpd leading-edge staining. Per cell type, n = 60
cells were analyzed from three independent
experiments. Values above the median were
The number of cells with positive leading-edge
as a percentage in comparison to YFP-AblWT/
Abl2/2Arg2/2cells. **p < 0.01 (Student’s t test).
Error bars represent SEM. See also Figure S4.
length of the main axon and all axonal
branches was further increased (Fig-
ure 7C). Consistently, branching was
doubled in the neurons transfected
with Lpd and c-Abl (Figures 7A and
7D), indicating that c-Abl and Lpd coop-
erate to positively regulate axonal length
To explore whether cooperation of
c-Abl with Lpd in axonal morphogenesis
is mediated by Ena/VASP proteins, we
co-overexpressed c-Abl and Lpd with
or without FP4-mito or AP4-mito con-
structs to delocalize all Ena/VASP pro-
teins. Co-overexpression of c-Abl and
EGFP-Lpd or of c-Abl, EGFP-Lpd, and
AP4-mito significantly increased the
length of the main axon, compared
to c-Abl with EGFP (Figures 7E and
7F). Importantly, co-overexpression of
c-Abl, EGFP-Lpd, and FP4-mito failed
to induce an increase in the length of
the main axon (Figures 7E and 7F), suggesting that axonal
elongation induced by cooperation of c-Abl and Lpd is medi-
ated by Ena/VASP proteins.
In this study we reveal Lpd as a novel substrate of Abl kinases
that is transiently phosphorylated upon PDGF and netrin-1
stimulation. We found that Lpd is phosphorylated by c-Abl at
its PH domain and at the C terminus. Phosphorylation by Abl
kinases at the PH domain does not regulate the interaction
of Lpd with the plasma membrane because Lpd localizes at la-
mellipodia in Abl2/2Arg2/2fibroblasts (Figure 3).
We observed that the interaction between Lpd and Ena/
VASP proteins is positively regulated by c-Abl. However,
by Abl kinases. The phosphorylation of Lpd might alter its
tertiary structure, thereby unmasking the Ena/VASP-binding
sites. Furthermore, we noticed differences in the biochemical
behavior of individual Ena/VASP proteins. We found that
wild-type, dominant-active, and kinase-inactive c-Abl can
coprecipitate with Lpd (Figure S2A). Wild-type and domi-
nant-active, but not kinase-inactive, c-Abl may bind directly
to phosphorylated Lpd via the c-Abl SH2 domain. In contrast,
kinase-inactive c-Abl may bind indirectly to Lpd via Ena/VASP,
Abl Positively Regulates Lpd-Ena/VASP Interaction
because Ena/VASP proteins can directly interact with the
c-Abl SH3 domain  and Ena/VASP can interact with FP4
motifs in Lpd .Furthermore, VASP localizationto theleading
edge is independent of Abl kinases (Figure S4B), suggesting
that its recruitment to this site can also occur without Lpd
phosphorylation by Abl kinases. In addition, only Lpd-Mena,
and not Lpd-EVL or Lpd-VASP interaction, is inhibited by
expression of kinase-inactive c-Abl, suggesting that c-Abl
may also regulate Mena directly. This might be achieved by
phosphorylation of Ena or Mena, because they are substrates
of Abl . We believe that this is unlikely because we did not
observe any tyrosine phosphorylation of Mena when we
co-overexpressed GST-Lpd, Mena, and c-Abl (data not
shown; Figure 3). Furthermore, direct phosphorylation of Ena
by D-Abl is not required for regulation of Ena function because
expression of a nonphosphorylatable Ena mutant rescued
axon guidance defects in the Drosophila ena mutant [5, 23].
However, Abl kinase activity is required for its function in
axon guidance [24, 25], suggesting that D-Abl must phosphor-
ylate some other component. It was postulated that Abl
Figure 5. Lpd Regulates the PDGF-Induced Dorsal Ruffle
Response in an Ena/VASP-Dependent Manner
(A) Single confocal plane at the dorsal surface of PDGF
(5 min, 30 ng/ml)-stimulated NIH 3T3 cells stained with Lpd
and Mena antibodies. Scale bar represents 2 mm.
(B–G) NIH 3T3 cells were stimulated with 30 ng/ml PDGF for
5 min and fixed, and F-actin was stained with A568-phalloi-
din. Coverslips were automatically scanned, and transfected
cells with dorsal ruffles were quantified.
(B) GFP-Lpd overexpression increases the dorsal ruffle
(C) Lpd knockdown was achieved by transfection of
a plasmid with an Lpd small hairpin RNA or scrambled
control and puromycin resistance. After selection, cells
were serum starved and stimulated with 30 ng/ml PDGF.
(D) NIH 3T3 cells were transfected with mRFP1-FP4-mito to
delocalize all Ena/VASP proteins or mRFP1-AP4-mito as
(E) NIH 3T3 cells were transfected with GFP, GFP-Lpd alone,
or in combination with mRFP1-FP4-mito or mRFP1-AP4-
mito as control.
(F) NIH 3T3 cells were transfected with GFP or GFP-Lpd
alone, or in combination with wild-type c-Abl (AblWT) or
were pretreated for 2 hr with 1 mM STI571.
(G) NIH 3T3 cells were transfected with GFP, GFP-Lpd, or
GFP-Lpd4YF (mutated at Y426, Y456, Y513, Y1226). *p <
0.05; **p < 0.01 (B, C, D, and G: Student’s t test; E and F:
one-way ANOVA). Error bars represent SEM.
regulates Ena’s location in cells because Ena is
mislocalized in d-abl mutant flies . Taken
together, this suggests that Abl regulates Ena
localization indirectly by phosphorylating an
unknown protein. In this study we have discov-
ered that the interaction between Lpd and Ena/
VASP proteins is positively regulated by c-Abl.
Therefore, we suggest that Lpd is this hitherto
unknown intermediary between Abl and Ena/
VASP and that the differential formation of trimo-
lecular complexes between Lpd, c-Abl, and indi-
vidual Ena/VASP proteins allows for fine tuning
of signaling responses.
This positive regulation of Lpd and Ena/VASP
by Abl is surprising because it was postulated
that Drosophila d-abl negatively regulates ena
function [1, 2]. How can both be reconciled? Unexpectedly,
comparing known cellular functions of Abl kinases and Ena/
VASP proteins revealed that many functions are very similar
and not in opposition to each other. First, overexpression of
both Abl kinases and Ena/VASP proteins increases filopodia
formation [20, 22, 26]. Second, analysis of knockout fibro-
blasts lacking Ena/VASP proteins or Abl kinases revealed
that they migrated faster compared to cells re-expressing
physiological levels of the respective proteins [8, 19, 27, 28].
This indicates that both Abl kinases and Ena/VASP proteins
negatively regulate whole-cell migration [8, 19, 27, 29, 30].
Third, both Abl/Arg or Ena/VASP knockout mice have neurula-
tion defects during development and die of hemorrhage [18,
31–34]. Finally, both Drosophila ena and d-abl mutants have
defects in longitudinal and commissural axon tracts [2, 4, 6].
How can mutations in ena ameliorate abl phenotypes when
Abl is a positive regulator of Ena? We suggest that Abl posi-
tively regulates the correct subcellular localization of Ena/
VASP proteins, which is consistent with our observations
and with those in d-abl mutant flies . When D-Abl is absent,
Current Biology Vol 20 No 9
Ena accumulates ectopically where excess F-actin is ob-
served . Consistently, reduction of ena in abl mutant flies
restores viability [1, 2]. Therefore, rescue of viability can be
equally well explained by a positive regulation of Ena by Abl.
Lpd is transiently phosphorylated upon stimulation of
primary cortical neurons with netrin-1 and is accompanied
by an increased Lpd-Mena interaction. Treatment of neurons
5 to 10 min , which correlates well with the time course of
Lpd phosphorylation and increased interaction with Mena. In
C. elegans, mig-10 and unc-34/ena function together to
mediate unc-6/netrin-dependent axon guidance decisions
. Our data indicate that cooperation between Lpd and
Ena/VASP proteins downstream of the netrin receptor also
occurs in vertebrates.
receptor stimulation and found that Lpd and Ena/VASP
proteins contribute to the PDGF-induced dorsal ruffle
response of fibroblasts. Our data indicate that Lpd function
in the dorsal ruffle response is regulated by Abl kinases and
mediated by Ena/VASP proteins. In lamellipodia, Ena/VASP
actin filament capping . N-WASP and the Arp2/3 complex
facilitate actin nucleation off of the side of existing actin
Figure 6. Lpd Is Required for Axonal Morphogenesis
(A) Hippocampal neurons transfected with Lpd small inter-
fering RNAs (siRNAs) or control siRNA plated on laminin-
Tau-1 antibodies (Figure S5B). Scale bar represents 50 mm.
(B) Axons were identified with Tau-1 staining (Figure S5B),
and length of main axon and branches were manually quan-
tified with NeuronJ from anti-beta(III)-tubulin stainings.
Three independent experiments were performed: control
siRNA, n = 128 neurons; Lpd siRNA-11, n = 174 neurons;
Lpd siRNA-12, n = 184 neurons.
(C and D) The total length of all axonal branches per neuron
(C) and the number of branch points per axon (D) were calcu-
(E) Axonal length of primary hippocampal neurons cotrans-
fected with either control siRNA and GFP, Lpd siRNA and
GFP, or Lpd siRNA and human GFP-Lpd was quantified.
Three independent experiments were performed: control
siRNA + EGFP, n = 102 neurons; Lpd siRNA-11+EGFP,
n = 92 neurons; Lpd siRNA-11+human Lpd-EGFP, n = 89
neurons. *p < 0.05; **p < 0.001 (one-way ANOVA). Error
bars represent SEM. See also Figure S5.
filaments, and cortactin stabilizes these actin
branches . N-WASP , cortactin , and
Lpd (this study) are positively regulated by Abl
kinases and function to regulate dorsal ruffling.
A fine balance of Arp2/3-mediated branching
and Ena/VASP-mediated elongation of actin
filaments regulates the actin ultrastructure during
dorsal ruffle formation. Therefore, positive regula-
tion by Abl kinases and cooperation between
Lpd, Ena/VASP, N-WASP, Arp2/3, and cortactin
might be a more general mechanism to modulate
the actin ultrastructure for different types of
In conclusion, we have identified Lpd as a
component of PDGF and netrin-1 signaling path-
ways. Lpd is a substrate of Abl kinases, and
c-AblcooperateswithLpdin anEna/VASP-dependent manner
during the PDGF-induced dorsal ruffle response and axonal
morphogenesis. Lpd’s interaction with Ena/VASP proteins is
positively regulated by Abl kinases. We propose that Lpd
is the hitherto unknown intermediary between Abl and Ena/
VASP. Our data donot support the suggested negative regula-
tory role of Abl for Ena, and we propose an alternative hypoth-
esis that Abl kinases, via Lpd, positively regulate Ena/VASP
Molecular Biology, Plasmids, and Reagents
See Supplemental Experimental Procedures.
NIH 3T3 (ATCC), HEK293FT (Invitrogen), and Abl2/2Arg2/2MEFs (a kind gift
from T. Koleske, Yale) were cultured in Dulbecco’s modified Eagle’s
medium, penicillin, streptomycin, 10% fetal bovine serum, or calf serum
(NIH 3T3) and transfected with Lipofectamine 2000 (Invitrogen). NIH 3T3
cells were serum starved (18 hr) before stimulation with 30 or 50 ng/ml
PDGF. pK1-YFP-c-Abl and pCL-Eco were cotransfected in HEK293FT to
generate retroviruses to transduce Abl2/2Arg2/2MEFs, and YFP-positive
cells were selected by fluorescence-activated cell sorting. See Supple-
mental Experimental Procedures for immunofluorescence analysis and
Abl Positively Regulates Lpd-Ena/VASP Interaction
Immunoprecipitations and Western Blotting
Primary cortical neurons were cultured for 36 hr before incubation with
S-transferase (GST) buffer (50 mM Tris-HCL, pH 7.4, 200 mM NaCl, 1%
NP-40, 2 mM MgCl2, 10% glycerol, NaF + Na3VO4, complete mini tablets
without EDTA, Roche). Precleared lysate was incubated with glutathione
beads (Amersham) or with primary antibody or control IgG followed by
protein A beads (Pierce) and was washed with GST buffer. Western blotting
was performed as described .
Peptide Array Assay
Custom-made immobilized peptide arrays (CR-UK) were incubated over-
night at room temperature in kinase buffer (50 mM Tris-Hcl, 10 mM MgCl2,
1mMEGTA, 2mMDTT,0.01% Brij 35) with 0.2 mg/ml bovine serum albumin
(BSA) and 10 mM NaCl, were blocked for 45 min at 30?C in kinase buffer
with 1 mg/ml BSA and 100 mM NaCl, and were incubated with kinase buffer
+ 0.2 mg/ml BSA + 120 units Abl kinase (NEB) + 24 mCi g-32P-ATP for 2 hr at
30?C. Washed membranes (10 3 15 min 1 M NaCl, 3 3 5 min H2O, 3 3 5 min
H3P04, 3 3 5 min H2O, 2 3 2 min ethanol) were dried and analyzed with
a Phosphorimager Typhoon 9200 (Amersham).
Supplemental Information includes SupplementalExperimentalProcedures
We thank F. Gertler (Massachusetts Institute of Technology) for Ena/VASP
plasmids and antibodies; T. Koleske (Yale) for the Abl2/2Arg2/2MEFs,
STI571, and Abl/Arg plasmids; and B. Eickholt, A. Jandke, and J. Leslie
(King’s College London) for critical reading of the manuscript. Special
thanks to E. Meijering for developing NeuronJ. M.M. is supported by a
Medical Research Council studentship. C.N. is supported by a European
Molecular Biology Organization long-term fellowship. A.V. is supported by
a Biotechnology and Biological Sciences Research Council grant to M.K.
(BB/G00319X/1). M.M., A.V., C.N., and M.K. are supported by funds from
a Wellcome Trust University Award (077429/Z/05/Z) to M.K.
Received: July 13, 2009
Revised: March 17, 2010
Accepted: March 17, 2010
Published online: April 22, 2010
1. Gertler, F.B., Doctor, J.S., and Hoffmann, F.M. (1990). Genetic suppres-
sion of mutations in the Drosophila abl proto-oncogene homolog.
Science 248, 857–860.
2. Gertler, F.B., Comer, A.R., Juang, J.L., Ahern, S.M., Clark, M.J., Liebl,
E.C., and Hoffmann, F.M. (1995). enabled, a dosage-sensitive
suppressor of mutations in the Drosophila Abl tyrosine kinase, encodes
an Abl substrate with SH3 domain-binding properties. Genes Dev. 9,
3. Pula, G., and Krause, M. (2008). Role of Ena/VASP proteins in homeo-
stasis and disease. Handb Exp Pharmacol (186), 39–65.
4. Drees, F., and Gertler, F.B. (2008). Ena/VASP: Proteins at the tip of the
nervous system. Curr. Opin. Neurobiol. 18, 53–59.
5. Krause, M., Dent, E.W., Bear, J.E., Loureiro, J.J., and Gertler, F.B.
(2003). Ena/VASP proteins: Regulators of the actin cytoskeleton and
cell migration. Annu. Rev. Cell Dev. Biol. 19, 541–564.
6. Bradley, W.D.,and Koleske, A.J. (2009). Regulation of cell migration and
morphogenesis by Abl-family kinases: Emerging mechanisms and
physiological contexts. J. Cell Sci. 122, 3441–3454.
7. Sini, P., Cannas, A., Koleske, A.J., Di Fiore, P.P., and Scita, G. (2004).
Abl-dependent tyrosine phosphorylation of Sos-1 mediates growth-
factor-induced Rac activation. Nat. Cell Biol. 6, 268–274.
Figure 7. Lpd Cooperates with c-Abl in an Ena/VASP-Dependent Manner in
the Regulation of Axonal Morphogenesis
(A–D) Hippocampal neurons transfected with EGFP control, Lpd-EGFP,
wild-type YFP-c-Abl+EGFP control, or Lpd-EGFP + wild-type YFP-c-Abl
plated on poly-D-lysine-coated coverslips were stained with anti-beta(III)-
tubulin (A) and Tau-1(Figure S5C) antibodies. Scale bars in(A) and (E) repre-
sent 50 mm.
(B and F) Axons were identified with Tau-1 staining (Figure S5C), and length
of main axon and branches were manually quantified with NeuronJ from
anti-beta(III)-tubulin stainings. Three independent experiments were per-
formed: control EGFP, n = 137 neurons; Lpd-EGFP, n = 134 neurons;
EGFP+c-Abl, n = 161 neurons; Lpd-EGFP+c-Abl, n = 133 neurons.
(C and D) The total length of all axonal branches per neuron (C) and the
number of branch points per axon (D) were calculated.
(E and F) Hippocampal neurons transfected with wild-type YFP-c-
Abl+EGFP control, Lpd-EGFP + wild-type YFP-c-Abl with mRFP1-FP4-
mito, or AP4-mito and plated onto poly-D-lysine-coated coverslips were
stained with anti-beta(III)-tubulin and Tau-1 antibodies (Figure S5D).
(F) Data from three independent experiments: EGFP+c-Abl, n = 140
neurons; Lpd-EGFP+c-Abl, n = 94 neurons; Lpd-EGFP+c-Abl+FP4-mito,
n = 121 neurons; Lpd-EGFP+c-Abl+AP4-mito, n = 90 neurons. *p < 0.05;
**p < 0.001 (one-way ANOVA). Error bars represent SEM.
Current Biology Vol 20 No 9
8. Bear, J.E., Svitkina, T.M., Krause, M., Schafer, D.A., Loureiro, J.J., Download full-text
Strasser, G.A., Maly, I.V., Chaga, O.Y., Cooper, J.A., Borisy, G.G., and
Gertler, F.B. (2002). Antagonism between Ena/VASP proteins and actin
filament capping regulates fibroblast motility. Cell 109, 509–521.
9. Krause, M., Leslie, J.D., Stewart, M., Lafuente, E.M., Valderrama, F.,
Jagannathan, R., Strasser, G.A., Rubinson, D.A., Liu, H., Way, M.,
et al. (2004). Lamellipodin, an Ena/VASP ligand, is implicated in the
regulation of lamellipodial dynamics. Dev. Cell 7, 571–583.
10. Manser, J., Roonprapunt, C., and Margolis, B. (1997). C. elegans cell
migration gene mig-10 shares similarities with a family of SH2 domain
proteins and acts cell nonautonomously in excretory canal develop-
ment. Dev. Biol. 184, 150–164.
11. Jenzora, A., Behrendt, B., Small, J.V., Wehland, J., and Stradal, T.E.
(2005). PREL1 provides a link from Ras signalling to the actin cytoskel-
eton via Ena/VASP proteins. FEBS Lett. 579, 455–463.
12. Lafuente, E.M., van Puijenbroek, A.A., Krause, M., Carman, C.V.,
Freeman, G.J., Berezovskaya, A., Constantine, E., Springer, T.A.,
Gertler, F.B., and Boussiotis, V.A. (2004). RIAM, an Ena/VASP and
Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced
adhesion. Dev. Cell 7, 585–595.
13. Lyulcheva, E., Taylor, E., Michael, M., Vehlow, A., Tan, S., Fletcher, A.,
Krause, M., and Bennett, D. (2008). Drosophila pico and its mammalian
ortholog lamellipodin activate serum response factor and promote cell
proliferation. Dev. Cell 15, 680–690.
14. Grevengoed, E.E., Fox, D.T., Gates, J., and Peifer, M. (2003). Balancing
different types of actin polymerization at distinct sites: Roles for
Abelson kinase and Enabled. J. Cell Biol. 163, 1267–1279.
15. Huber, A.B., Kolodkin, A.L., Ginty, D.D., and Cloutier, J.F. (2003).
Signaling at the growth cone: Ligand-receptor complexes and the
16. Filippakopoulos, P., Kofler, M., Hantschel, O., Gish, G.D., Grebien, F.,
Salah, E., Neudecker, P., Kay, L.E., Turk, B.E., Superti-Furga, G., et al.
(2008). Structural coupling of SH2-kinase domains links Fes and Abl
substrate recognition and kinase activation. Cell 134, 793–803.
17. Gertler, F.B., Niebuhr, K., Reinhard, M., Wehland, J., and Soriano, P.
(1996). Mena, a relative of VASP and Drosophila Enabled, is implicated
in the control of microfilament dynamics. Cell 87, 227–239.
18. Koleske, A.J., Gifford, A.M., Scott, M.L., Nee, M., Bronson, R.T., Miczek,
K.A., and Baltimore, D. (1998). Essential roles for the Abl and Arg
tyrosine kinases in neurulation. Neuron 21, 1259–1272.
19. Bear, J.E., Loureiro, J.J., Libova, I., Fa ¨ssler, R., Wehland, J.,and Gertler,
F.B. (2000). Negative regulation of fibroblast motility by Ena/VASP
proteins. Cell 101, 717–728.
20. Lebrand, C., Dent, E.W., Strasser, G.A., Lanier, L.M., Krause, M.,
Svitkina, T.M., Borisy, G.G., and Gertler, F.B. (2004). Critical role of
Ena/VASP proteins for filopodia formation in neurons and in function
downstream of netrin-1. Neuron 42, 37–49.
21. Zukerberg, L.R., Patrick, G.N.,Nikolic, M., Humbert, S., Wu, C.L., Lanier,
L.M., Gertler, F.B., Vidal, M., Van Etten, R.A., and Tsai, L.H. (2000).
Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphoryla-
tion, kinase upregulation, and neurite outgrowth. Neuron 26, 633–646.
22. Woodring, P.J., Litwack, E.D., O’Leary, D.D., Lucero, G.R., Wang, J.Y.,
and Hunter, T. (2002). Modulation of the F-actin cytoskeleton by c-Abl
tyrosine kinase in cell spreading and neurite extension. J. Cell Biol.
23. Comer, A.R., Ahern-Djamali, S.M., Juang, J.L., Jackson, P.D., and
Hoffmann, F.M. (1998). Phosphorylation of Enabled by the Drosophila
Abelson tyrosine kinase regulates the in vivo function and protein-
protein interactions of Enabled. Mol. Cell. Biol. 18, 152–160.
24. Henkemeyer, M., West, S.R., Gertler, F.B., and Hoffmann, F.M. (1990).
A novel tyrosine kinase-independent function of Drosophila abl
correlates with proper subcellular localization. Cell 63, 949–960.
25. Wills, Z., Bateman, J., Korey, C.A., Comer, A., and Van Vactor, D. (1999).
The tyrosine kinase Abl and its substrate enabled collaborate with the
receptor phosphatase Dlar to control motor axon guidance. Neuron
26. Radha, V., Rajanna, A., Mitra, A., Rangaraj, N., and Swarup, G. (2007).
C3G is required for c-Abl-induced filopodia and its overexpression
promotes filopodia formation. Exp. Cell Res. 313, 2476–2492.
27. Peacock, J.G., Miller, A.L., Bradley, W.D., Rodriguez, O.C., Webb, D.J.,
and Koleske, A.J. (2007). The Abl-related gene tyrosine kinase acts
through p190RhoGAP to inhibit actomyosin contractility and regulate
focal adhesion dynamics upon adhesion to fibronectin. Mol. Biol. Cell
28. Miller, A.L., Wang, Y., Mooseker, M.S., and Koleske, A.J. (2004). The
Abl-related gene (Arg) requires its F-actin-microtubule cross-linking
activity to regulate lamellipodial dynamics during fibroblast adhesion.
J. Cell Biol. 165, 407–419.
29. Frasca, F., Vigneri, P., Vella, V., Vigneri, R., and Wang, J.Y. (2001).
Tyrosine kinase inhibitor STI571 enhances thyroid cancer cell motile
response to Hepatocyte Growth Factor. Oncogene 20, 3845–3856.
30. Kain, K.H., and Klemke, R.L. (2001). Inhibition of cell migration by Abl
family tyrosine kinases through uncoupling of Crk-CAS complexes.
J. Biol. Chem. 276, 16185–16192.
K.L., Mason, C.A., Fassler, R., and Gertler, F.B. (2004). Mena and
vasodilator-stimulated phosphoprotein are required for multiple actin-
dependent processes that shape the vertebrate nervous system. J.
Neurosci. 24, 8029–8038.
32. Lanier, L.M., Gates, M.A., Witke, W., Menzies, A.S., Wehman, A.M.,
Macklis, J.D., Kwiatkowski, D., Soriano, P., and Gertler, F.B. (1999).
Mena is required for neurulation and commissure formation. Neuron
33. Furman, C., Sieminski, A.L., Kwiatkowski, A.V., Rubinson, D.A., Vasile,
E., Bronson, R.T., Fa ¨ssler, R., and Gertler, F.B. (2007). Ena/VASP is
required for endothelial barrier function in vivo. J. Cell Biol. 179,
34. Kwiatkowski, A.V., Rubinson, D.A., Dent, E.W., Edward van Veen, J.,
Leslie, J.D., Zhang, J., Mebane, L.M., Philippar, U., Pinheiro, E.M.,
Burds, A.A., et al. (2007). Ena/VASP is required for neuritogenesis in
the developing cortex. Neuron 56, 441–455.
35. Pollard, T.D. (2007). Regulation of actin filament assembly by Arp2/3
complex and formins. Annu. Rev. Biophys. Biomol. Struct. 36, 451–477.
36. Boyle, S.N., Michaud, G.A., Schweitzer, B., Predki, P.F., and Koleske,
A.J. (2007). A critical role for cortactin phosphorylation by Abl-family
kinases in PDGF-induced dorsal-wave formation. Curr. Biol. 17,
Abl Positively Regulates Lpd-Ena/VASP Interaction