Requirement of Nck adaptors for actin dynamics
and cell migration stimulated by platelet-derived
growth factor B
G. M. Rivera*, S. Antoku*, S. Gelkop†, N. Y. Shin‡, S. K. Hanks‡, T. Pawson†§, and B. J. Mayer*§
*Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology and Center for Cell
Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030;†Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
Toronto, ON, Canada M5G 1X5; and‡Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232
Contributed by T. Pawson, May 8, 2006
The Nck family of Src homology (SH) 2?SH3 domain adaptors
functions to link tyrosine phosphorylation induced by extracellular
signals with downstream regulators of actin dynamics. We inves-
tigated the role of mammalian Nck adaptors in signaling from the
activated platelet-derived growth factor (PDGF) receptor (PDGF?R)
to the actin cytoskeleton. We report here that Nck adaptors are
required for cytoskeletal reorganization and chemotaxis stimu-
lated by PDGF-B. Analysis of tyrosine-phosphorylated proteins
demonstrated that Crk-associated substrate (p130Cas), not the
activated PDGF?R itself, is the major Nck SH2 domain-binding
protein in PDGF-B-stimulated cells. Both Nck- and p130Cas-deficient
cells fail to display cytoskeletal rearrangements, including the
formation of membrane ruffles and the disassembly of actin
bundles, typically shown by their WT counterparts in response to
PDGF-B. Furthermore, Nck and p130Cascolocalize in phosphoty-
rosine-enriched membrane ruffles induced by PDGF-B in NIH 3T3
cells. These results suggest that Nck adaptors play an essential role
in linking the activated PDGF?R with actin dynamics through a
pathway that involves p130Cas.
Crk-associated substrate ? actin cytoskeleton ? SH2 domain ? SH3 domain ?
the actin cytoskeleton, i.e., the dynamic assembly and disassembly
of filamentous actin, governs essential aspects of cell motility such
as the formation of membrane protrusions and cell adhesion to
other cells or to the substrate (1). Extracellular signals can induce
remodeling of the actin cytoskeleton through changes in tyrosine
phosphorylation. For example, ligand-induced dimerization of
platelet-derived growth factor (PDGF) receptors (PDGF?R) stim-
ulates their intrinsic kinase activity, leading to the autophosphor-
ylation in trans of multiple intracellular tyrosine residues on the
molecules (4). Cellular effects mediated by signaling pathways
activated by PDGF?R involve proliferation, survival, actin reorga-
nization, and migration (3). Despite extensive efforts aimed at
characterizing effectors that associate with PDGF?R (2), the
dynamic assembly of signaling complexes induced by the activation
of this receptor and their relation to specific cellular responses are
still poorly understood.
Nck, a two-gene family in mammals, is an important link in
transducing signals from tyrosine phosphorylation to the cytoskel-
eton (5, 6). Nck proteins (termed Nck, Nck? or Nck1, and Nck? or
C-terminal SH2 domain. The SH2 domain of Nck can bind a
number of activated receptor tyrosine kinases and tyrosine-
phosphorylated docking proteins; on the other hand, the Nck SH3
domains engage proline-rich binding sites on a host of effectors
implicated in cytoskeletal regulation, including members of the
WASp?Scar family (reviewed in refs. 5 and 6). Nck adaptors have
ell motility is critically important in developmental processes
and in the pathogenesis of a variety of diseases. Remodeling of
been shown to play an important role during embryogenesis
potentially linked to cell motility and cytoskeletal organization (7).
can interact with the activated PDGF?R (8–10), their role in
signaling to the actin cytoskeleton downstream of this receptor
remains largely unknown. Here we report that Nck adaptors are
strictly required for cytoskeletal reorganization and chemotaxis
stimulated by PDGF-B. Furthermore, we provide mechanistic
of the activated PDGF?R by an indirect mechanism involving the
scaffolding protein p130Cas.
Nck Adaptors Are Required for Cytoskeletal Changes Induced by
PDGF-B. To determine the significance of Nck adaptors in cytoskel-
etal changes induced by PDGF-B, serum-starved, Nck-deficient
(both Nck genes inactivated; dKO), and WT mouse embryonic
fibroblasts (MEFs) were left untreated or were stimulated with
PDGF-B. Profound remodeling of the cytoskeleton, including the
disassembly of actin bundles and the formation of various types of
membrane protrusions, occurred in WT but not dKO cells after
PDGF-B stimulation (Fig. 1A and Fig. 7A, which is published as
supporting information on the PNAS web site). Occurrence of
‘‘peripheral’’ or ‘‘dorsal’’ ruffles (11) was compared between the
genotypes. Whereas dKO cells remained mostly in a ‘‘quiescent’’
state, the WT cells, in contrast, showed a dramatic increase in
peripheral and dorsal ruffling after PDGF-B stimulation (Fig. 7B).
These observations suggested a critical role for Nck adaptors in
transducing signals from the activated PDGF?R to the actin
Expression of Nck Rescues the Response to PDGF-B in Nck-Deficient
Cells. Given the striking contrast in the response to PDGF-B
expression of Nck1 or Nck2 in dKO cells could rescue the respon-
siveness to PDGF-B. Cells were cotransfected with actin-GFP to
visualize cytoskeletal changes and a vector expressing Nck1, Nck2,
a loss-of-function mutant Nck1 (with inactivating mutation of all
three SH3 domains; Kall), or empty vector (EBB). No major
cytoskeletal changes were observed after PDGF-B stimulation in
serum-starved dKO cells cotransfected with a loss-of-function
mutant Nck1 (Fig. 1B, Kall) or with the empty vector (EBB in Fig.
site). In contrast, actin bundles disassembled and peripheral and
Conflict of interest statement: No conflicts declared.
Freely available online through the PNAS open access option.
Abbreviations: PDGF, platelet-derived growth factor; PDGF?R, PDGF receptor; SH, Src
homology; MEF, mouse embryonic fibroblast; PI3K, phosphatidylinositol 3-kinase; Fak,
focal adhesion kinase.
§To whom correspondence may be addressed. E-mail: email@example.com or bmayer@
© 2006 by The National Academy of Sciences of the USA
June 20, 2006 ?
vol. 103 ?
or Nck2 (Fig. 8 A and B) after PDGF-B stimulation. To further
evaluate the role of Nck in cytoskeletal dynamics induced by
PDGF-B, we performed live-cell imaging of Nck-deficient cells
cotransfected with actin-GFP and either empty vector or Nck1. As
shown in Movie 1, which is published as supporting information on
the PNAS web site, dKO cotransfected with the empty vector
fected with Nck1 exhibited formation of very dynamic peripheral
(Movie 2, which is published as supporting information on the
PNAS web site) and dorsal (Movie 3, which is published as
supporting information on the PNAS web site) membrane ruffles
after PDGF-B stimulation. Thus, these results demonstrate that
either Nck1 or Nck2 is required for PDGF-B-stimulated actin
rearrangements. Importantly, phosphorylation of the PDGF?R
and activation of p42?44Erk, cell proliferation-related signaling
molecules, was not affected in the absence of Nck adaptors (see
below). Actin dynamics is the cellular function most profoundly
altered in the absence of Nck adaptors.
Lack of Nck Adaptors Leads to Impaired Chemotactic and Haptotactic
genotypes and compared their ability to migrate using a conven-
tional transwell migration assay. Nck-deficient (dKO) cells had a
readily apparent migration disadvantage compared with their WT
counterparts (Fig. 9, which is published as supporting information
on the PNAS web site). As shown in Fig. 2, more WT MEFs
migrated in the presence of PDGF-B, fibronectin, or both than in
Nck1 or Nck2 abrogated the chemotactic response to PDGF (P ?
0.05). The haptotactic response to fibronectin was severely com-
promised in MEFs with inactivation of both Nck genes (dKO and
IRES). However, more (P ? 0.001) MEFs with only one of the two
Nck genes migrated in the presence of fibronectin than in the
presence of medium alone. Interestingly, Nck-deficient MEFs
rescued with a retrovirus expressing Nck (expression levels ?3-fold
higher than in WT cells) showed a slight, but significant (P ? 0.01),
increase in migration in response to PDGF and?or fibronectin
compared with cells exposed to medium alone. These results
strongly suggest that both Nck genes are required for normal
directed cell motility and that Nck1 and Nck2 are at least partially
Profile of Tyrosine Phosphorylation in Response to Activation of
PDGF?R. To begin to understand the mechanism by which Nck
eton, we examined tyrosine-phosphorylated proteins in cell lysates
obtained from serum-starved NIH 3T3 cells left untreated or
stimulated with PDGF-B. Western blot analysis using an antibody
against phosphotyrosine (Fig. 3, pY) showed that three major
proteins of ?185, 130, and 65 kDa underwent changes in their
phosphotyrosine levels in association with PDGF-B stimulation.
We also determined the pattern of SH2-binding proteins by far-
from Nck1, Nck2, or phosphatidylinositol 3-kinase (PI3K) or GST
clearly observed in pY and PI3K blots soon after PDGF-B stimu-
lation but was undetectable in lysates of cells left untreated.
from Nck1 and Nck2, indicating that the activated PDGF?R is not
efficiently bound directly by Nck adaptors. In contrast, a single
major band of ?130 kDa was consistently detected in membranes
probed with Nck1 and Nck2 SH2 domains; this band was not seen
in the PI3K SH2 blot. The ?130-kDa band showed low but
starved, unstimulated cells and a modest (?3- to 5-fold) increase in
lysates obtained soon after PDGF-B stimulation. Interestingly, the
Shown are confocal images of serum-starved MEFs left untreated (time 0) or
(dKO) and WT cells fixed and stained with Texas red phalloidin and Hoechst
etal rearrangements in Nck-deficient cells coexpressing actin-GFP and WT
(Nck1) or a loss-of-function mutant Nck (Kall). Arrows and arrowheads indi-
cate dorsal and peripheral ruffles, respectively. (Scale bars: 25 ?m.)
Requirement of Nck in actin rearrangements induced by PDGF-B.
gration. Data show the number of migrating cells per microscopic field. Bars
represent means ? SD from eight pictures taken at random from each treat-
ment combination in each of two independent experiments. Genotypes com-
pared are WT, Nck1?/?Nck2?/?(Nck2), Nck1?/?Nck2?/?(Nck1), Nck?/?
means with a different letter over the bar are different (P ? 0.01).
Nck deficiency impairs PDGF-B- and fibronectin-stimulated cell mi-
Rivera et al. PNAS ?
June 20, 2006 ?
vol. 103 ?
no. 25 ?
ylation, with a rapid increase soon after PDGF-B stimulation (2–5
to a level lower than that in starved cells (Figs. 3 and 4 and Figs. 10
web site). Because the ?130-kDa Nck-interacting protein is likely
to mediate Nck recruitment to sites of actin rearrangements, we
sought to determine its identity in subsequent experiments.
p130CasIs the Major Tyrosine-Phosphorylated Protein That Binds Nck
in PDGF-B-Stimulated Cells. We investigated the identity of the
?130-kDa Nck SH2-binding protein by immunoprecipitation. Fo-
cal adhesion kinase (Fak) and p130Caswere likely candidates based
on their molecular weights and previous work suggesting possible
cells were subjected to immunoprecipitation with polyclonal anti-
p130Cas(Fig. 4) or anti-Fak antibodies (Fig. 10). Immunoprecipi-
tation with anti-Cas almost completely cleared the ?130-kDa Nck
SH2-binding protein from the lysates. In contrast, only a minor
fraction of the ?130-kDa protein was cleared from the lysates by
immunoprecipitation with anti-Fak, and this fraction did not bind
Nck SH2 domains (Fig. 10). Western blot analysis of the same
IP:Cas (Fig. 4) and IP:Fak (Fig. 10) filters (IP, immunoprecipita-
(Cas) and Fak (Fak), respectively, demonstrated the specificity of
The identity of the ?130-kDa Nck SH2 domain-binding protein
as p130Caswas confirmed by comparing the pattern of tyrosine-
phosphorylated proteins in Cas-deficient and WT MEFs or NIH
3T3 cells. As shown in Fig. 11, tyrosine phosphorylation of the
?130-kDa Nck SH2 domain-binding protein was absent in samples
from Cas-deficient cells, whereas it increased after PDGF-B stim-
ulation in cells containing p130Cas. Taken together, these results
show unambiguously that p130Casis the major tyrosine-
phosphorylated protein that can bind Nck SH2 domains in cells
stimulated with PDGF-B.
p130CasIs Required for Cytoskeletal Changes Induced by PDGF-B. The
striking finding that p130Casis the major Nck SH2 domain-binding
protein in fibroblasts stimulated with PDGF-B led us to test the
hypothesis that cells lacking p130Cas, or expressing p130Caslacking
the Nck SH2 binding sites, would fail to undergo cytoskeletal
rearrangements in response to PDGF-B. Accordingly, we com-
or p130Cas-deficient MEFs rescued with a retroviral vector express-
ing either WT p130Casor a phosphorylation-defective mutant
(Y3F substitutions of the 15 YXXP tyrosine residues of the
After PDGF-B stimulation, major cytoskeletal changes, including
the formation of membrane protrusions, were readily apparent in
WT and p130Cas-deficient MEFs reexpressing WT p130Cas(Fig. 5).
In contrast, the formation of membrane protrusions induced by
PDGF-B was severely compromised in p130Cas-deficient and
p130Cas-deficient cells reexpressing the phosphorylation-defective
the dorsal surface resembling disorganized dorsal ruffles. Quanti-
tative analysis showed that ?50% of the WT and p130Cas-deficient
MEFs reexpressing WT p130Casformed readily distinguishable
dorsal ruffles in response to PDGF-B stimulation whereas only
for various intervals were transferred to membranes and probed with anti-pTyr antibody (pY) or subjected to far-Western blot analysis using GST fusions of the
isolated SH2 domain from Nck1 (Nck1), Nck2 (Nck2), or PI3K p85 subunit and GST alone as a control. Apparent molecular weights (Mr? 10?3) of standards are
indicated. Arrows indicate a band corresponding to the activated PDGF?R, and arrowheads indicate a band of ?130 kDa in size that binds Nck SH2 domains.
Similar kinetics of tyrosine phosphorylation were observed in three independent experiments.
Pattern of tyrosine phosphorylation in response to activation of PDGF?R. Lysates from NIH 3T3 cells left untreated (time 0) or stimulated with PDGF-B
in cells stimulated with PDGF-B. Lysates obtained from NIH 3T3 cells left
untreated (time 0) or stimulated with PDGF-B for various intervals were
immunoprecipitated with polyclonal antibodies against p130Cas. Whole-cell
lysates (W) and supernatant (S) and pellet (P) fractions were subjected to
SDS?PAGE, and proteins were transferred to nitrocellulose filters. Pellet frac-
tions represent five times more lysate than whole-cell lysate or supernatant
fractions. Membranes were probed with GST fusions of the isolated SH2
domain from Nck1 (SH2) in a far-Western blot or with monoclonal anti-
Apparent molecular weights (Mr? 10?3) are indicated. Arrowheads indicate
a band of ?130 kDa in size corresponding to p130Cas, and the arrow indicates
p130Casis the major tyrosine-phosphorylated protein that binds Nck
www.pnas.org?cgi?doi?10.1073?pnas.0603786103Rivera et al.
?10–15% of the p130Cas-deficient and p130Cas-deficient cells re-
expressing the phosphorylation-defective p130Casmutant showed
actin foci on the dorsal surface (Fig. 12, which is published as
supporting information on the PNAS web site).
Nck and p130CasColocalize in Phosphotyrosine-Enriched Membrane
Ruffles Induced by PDGF-B. We first investigated the effects of
of either full-length Nck1 or its isolated SH2 domain in Nck-
deficient MEFs or NIH 3T3 cells. In Nck-deficient cells, full-length
Nck1-GFP was recruited to structures resembling peripheral and
dorsal ruffles (Fig. 13A Upper, which is published as supporting
information on the PNAS web site). Consistent with the failure of
the Nck SH3 domain mutant (Fig. 1B, Kall) to rescue the response
to PDGF-B, no structures resembling peripheral or dorsal ruffles
were detected in dKO cells transfected with Nck1 SH2-GFP after
PDGF-B stimulation (Fig. 13A Lower). In contrast, the GFP-Nck1
SH2 domain fusion localized strongly to membrane ruffles induced
by PDGF-B in NIH 3T3 cells where endogenous Nck is present
(Fig. 13B). This observation suggested that Nck recruitment de-
pends on SH2-domain-mediated interactions with tyrosine-
phosphorylated proteins enriched at sites of actin polymerization.
Next we analyzed by immunofluorescence the subcellular distri-
bution of tyrosine-phosphorylated proteins and Nck in relation to
stimulation in NIH 3T3 fibroblasts (Fig. 6). In Nck-deficient and
NIH 3T3 cells, respectively, ectopically expressed (Fig. 13C) and
on the PNAS web site) Nck displayed a diffuse cytoplasmic
distribution under serum starvation and localized strongly to
PDGF-B-induced membrane ruffles. In serum-starved NIH 3T3
cells, tyrosine-phosphorylated proteins accumulated in foci at the
cell periphery, presumably sites of cell-substrate adhesion that
coincided with tips of actin bundles. In sharp contrast, tyrosine-
phosphorylated proteins accumulated at the edges of membrane
ruffles in PDGF-B-stimulated cells, and, consistent with the dis-
assembly of actin bundles, the foci of phosphotyrosine proteins at
the cell periphery were no longer observed (Fig. 14A). Taken
together, these results suggest that PDGF-stimulated accumulation
of tyrosine-phosphorylated proteins recruits Nck to the membrane
through SH2 domain-mediated interactions, where it colocalizes
with p130Casat sites of active actin polymerization.
Actin Dynamics Is the Cellular Function Most Profoundly Altered in the
Absence of Nck Adaptors. To analyze whether other aspects of
PDGF?R signaling were affected in the absence of Nck adaptors,
ulation. Shown are confocal images of WT, p130Cas-deficient (???), and
p130Cas-deficient MEFs reexpressing either WT (??? WT) or a phosphoryla-
tion-deficient mutant (??? F1–15) p130Cas. Cells were left untreated (Starva-
(Scale bar: 25 ?m.)
p130Casis required for membrane ruffling induced by PDGF-B stim-
induced by PDGF-B. Shown are confocal images of serum-starved NIH 3T3
fibroblasts left untreated (Starvation) or stimulated with PDGF for 10 min
(PDGF-B). Fixed cells were stained with Texas red phalloidin and anti-Nck and
anti-p130Casantibodies. In the merged images, filamentous actin, Nck, and
p130Casare colored red, green, and blue, respectively. Arrows indicate dorsal
ruffles. (Scale bar: 25 ?m.)
Recruitment of endogenous Nck and p130Casto membrane ruffles
Rivera et al. PNAS ?
June 20, 2006 ?
vol. 103 ?
no. 25 ?
we compared the overall patterns of tyrosine phosphorylation,
p42?44Erk activation, and p130Casphosphorylation of Nck-
deficient vs. Nck-deficient cells rescued with Nck1 (Fig. 15, which
is published as supporting information on the PNAS web site). The
overall pattern of tyrosine phosphorylation did not differ signifi-
cantly between dKO and IRES?Nck1 cells. Importantly, the ex-
pression and functionality of PDGF?R are not affected in cells
autophosphorylation in dKO and IRES?Nck1 cells (pY blot).
Furthermore, PDGF-B-dependent activation of the cell prolifera-
tion-related signaling molecules p42?44Erk did not differ between
dKO and IRES?Nck1 cells.
Tyrosine phosphorylation of proteins in the ?120- to 130-kDa
range showed similar basal levels under starvation conditions and
an ?3- to 5-fold increase after PDGF-B stimulation (Fig. 15). In
IRES?Nck1 cells, tyrosine phosphorylation of these proteins fol-
lowed a biphasic pattern that resembled that observed in WT NIH
3T3 cells. Interestingly, in cells lacking Nck adaptors (dKO cells),
phosphorylation of proteins in the ?120- to 130-kDa range ap-
peared to increase linearly after PDGF-B stimulation (i.e., down-
regulation was not apparent in this time scale). Last, the phosphor-
ylation of p130Casin response to PDGF-B increased to the same
extent, as detected by far-Western blotting with SH2 domains from
Nck, in dKO and IRES?Nck1 cells (Fig. 15, SH2 blot). Taken
together, these observations suggest that actin dynamics, but not
other aspects of PDGF signaling, is the cellular function most
profoundly altered in the absence of Nck adaptors.
Using a combination of genetics, cell biology, and biochemistry we
phosphorylation induced by activation of the PDGF?R, remodel-
ing of the actin cytoskeleton, and directed cell motility. Further-
more, our studies provide mechanistic insights suggesting that Nck
adaptors transduce signals to the cytoskeleton downstream of the
PDGF?R through a signaling pathway that involves p130Cas.
Inactivation of both Nck1 and Nck2 results in profound defects
in development of mesoderm-derived tissues and the formation of
Here we report that MEFs lacking functional Nck proteins fail to
show the typical cytoskeletal changes observed in their WT coun-
terparts in response to PDGF-B stimulation and have seriously
compromised directional motility. Importantly, the responsiveness
are entirely consistent with an essential role of Nck adaptors in
promoting the dynamic assembly of actin in response to tyrosine
kinase signals and are in agreement with previous studies demon-
strating that the Nck?Dock adapter protein is required in photo-
receptor axon guidance and target recognition in the Drosophila
visual system (reviewed in ref. 15).
One of the potential mechanisms by which Nck adaptors could
couple to the PDGF?R is by direct binding to autophosphorylated
docking sites induced on the receptor upon ligand-mediated acti-
vation. Indeed, it has been shown that Nck1 (8) and Nck 2 (10) can
bind in vitro, through SH2 domain-mediated interactions, to phos-
photyrosine residues (Tyr-751 and Tyr-1009, respectively) in the
ligand-activated PDGF?R. Our data, however, support a model in
which Nck proteins could couple to PDGF?R through an indirect
mechanism. We used an SH2 domain-based far-Western blot
analysis (16) to determine Nck SH2 domain-binding partners in
SH2 domains from both Nck1 and Nck2 consistently detected a
single band of ?130 kDa that was unambiguously identified as
PDGF?R, was readily detected by anti-pY and PI3K SH2 domains
but not Nck SH2 domains. In previous studies (8) the presence of
tyrosine-phosphorylated proteins in addition to the PDGF?R in
GST-Nck pull-down fractions was not investigated, and thus the
possibility of an indirect interaction was not ruled out.
Crk-associated substrate (p130Cas) is implicated in cytoskeletal
regulation and cell migration, forms a complex with cell-matrix
adhesion-associated proteins, and is phosphorylated upon integrin
tin promoted p130Casphosphorylation via Fak?Src and the forma-
tion of a complex between phosphorylated p130Casand Nck (12).
Consistent with the PDGF-B-stimulated phosphorylation of
p130Caspreviously reported (18), our results point to a complex
linking the activated PDGF?R with the actin cytoskeleton. Several
converging observations lend support to this notion: (i) the rapid
increase (3- to 5-fold) in phosphorylation of p130Casafter stimu-
lation with PDGF-B; (ii) the change in subcellular distribution of
endogenous p130Casand Nck from cytoplasmic?focal adhesion-
brane ruffles induced by PDGF-B; (iii) the preferential binding of
Nck SH2 domain to p130Casin far-Western blot analysis; (iv) the
strikingly similar cytoskeletal phenotypes, i.e., the absence of actin
rearrangements after PDGF-B stimulation, in Nck- and p130Cas-
deficient cells; and (v) the inability of a phosphorylation-defective
mutant of p130Cas(which cannot bind Nck SH2 domains) to rescue
the response to PDGF-B in p130Cas-deficient cells. Interestingly,
unpublished work from our laboratory shows that specific down-
regulation by small interfering RNA interference of Crk adaptors
(Crk and Crk L), strong binding partners of p130Cas, does not alter
consistent with our observation of increased phosphorylation of
PDGF-B stimulation. Thus, the above observations suggest that a
molecular complex involving p130Casand Nck, but not Crk adap-
actin cytoskeleton. Future studies will be necessary to elucidate
the molecular mechanism linking the activated PDGF?R with the
formation of a complex between p130Casand Nck, including the
mechanisms of p130Casphosphorylation and relocalization.
p130Casexhibited a biphasic pattern of phosphorylation, with a
rapid increase soon after PDGF-B stimulation (2–5 min) followed
by an equally rapid and sustained decrease thereafter. This obser-
vation suggests the existence of a limiting or ‘‘off’’ mechanism,
presumably mediated by a protein tyrosine phosphatase, that could
be important in fine-tuning actin rearrangements induced by
PDGF-B. Further studies will be necessary to uncover the molec-
ular mechanisms underlying the dynamics of p130Casphosphory-
lation and its physiological consequences in the context of
PDGF?R activation. Using a very sensitive probe, such as the SH2
domains of the regulatory subunit of PI3K, we could detect
PDGF-B stimulation (Fig. 3). We recently found a similarly rapid
increase in the GTP loading of Rac (G.M.R. and B.J.M, unpub-
after (3.5 min) PDGF-B stimulation (Figs. 7A and 8A). During T
cell receptor activation, a rapid increase in tyrosine phosphoryla-
tion recruits Nck into a signaling complex that subsequently mi-
grates peripherally and accumulates at a ring-shaped actin-rich
structure (19). It is likely that highly dynamic processes similar to
those occurring during T cell receptor engagement also occur
during activation of the PDGF?R. Our data are consistent with a
model in which p130Casserves to recruit Nck to the membrane to
initiate actin polymerization and that these complexes are remod-
eled over time as additional proteins are recruited and?or phos-
Nck can signal to the actin cytoskeleton by interaction of its SH3
domains with a variety of downstream effectors. PAK-1 (p21-
activated kinase 1) is recruited by the middle SH3 domain of Nck
(20, 21), and Nck-mediated targeting of PAK to the plasma
membrane is sufficient for its activation by members of the Rho
www.pnas.org?cgi?doi?10.1073?pnas.0603786103Rivera et al.
family of GTPases (22). Under these conditions PAK may also Download full-text
serve as an adaptor to recruit guanine nucleotide exchange factors
Nck-mediated recruitment of PAK-1 to dorsal ruffles and to the
edges of lamellipodia (24). Alternatively, Nck adaptors could
stimulate actin polymerization through interactions with members
of the WASp?WAVE family and their binding partners. We have
targeted Nck SH3 domains is sufficient to trigger localized actin
polymerization through a pathway that requires N-WASp (25).
invadopodia, and circular dorsal ruffles (26). Interestingly, inva-
dopodium formation in metastatic carcinoma cells downstream of
epidermal growth factor receptor requires Nck-mediated recruit-
ment of the N-WASp-Arp2?3 complex (27). Also, Nck adaptors
studies will be necessary to clarify the role of Nck adaptors in
activation and targeting of the various downstream effectors upon
In summary, our results demonstrate that Nck adaptors are
strictly required for cytoskeletal reorganization and chemotaxis
stimulated by PDGF-B. Furthermore, these studies provide mech-
cytoskeleton downstream of the PDGF?R through a signaling
adaptors may constitute an important target of intervention in
altered actin dynamics, and aberrant cell migration.
Materials and Methods
Cell Culture, Transfections, PDGF-B Stimulation, and Migration Assay.
NIH 3T3 cells and MEF lacking Nck (7) or p130Cas(29) were
cultured in DMEM supplemented with antibiotics and 10% calf
serum or 10% FBS, respectively. Transient transfections were
carried out by using the Lipofectamine reagent according to
instructions provided by the manufacturer. Cells were serum-
starved (DMEM plus 0.1% FBS) for 24 h before stimulation with
30 ng?ml PDGF-BB (Upstate Biotechnology). Migration assay of
assay (Boyden chamber) as detailed in Supporting Text, which is
published as supporting information on the PNAS web site.
was performed as previously described (25), and further details are
provided in Supporting Text. The affinity-purified polyclonal anti-
Nck antibody was raised in rabbits immunized with a GST fusion
of full-length human Nck1. The anti-phosphotyrosine antibody was
from Cell Signaling Technology (P-Tyr-100, catalog no. 9411), and
the polyclonal anti-Fak antibody was from Santa Cruz Biotechnol-
ogy (C-20, sc-558). The monoclonal anti-Cas (6G11) antibody was
described previously (30). In some experiments, cells were stained
with Texas red phalloidin (Molecular Probes) and Hoechst 33342
dye to visualize the actin cytoskeleton and nuclei, respectively.
Fluorescent images were collected on a Zeiss LSM 510 confocal
microscope with a ?63 NA 1.25 Plan-NEOFLUAR oil-immersion
objective. Live images were obtained on a Nikon TE2000 inverted
details are provided in Supporting Text.
Far-Western Blot Analysis. Serum-starved or PDGF-B-treated cells
were harvested, and lysates were subjected to immunoprecipitation
with polyclonal anti-Fak or anti-Cas (Cas-B) antibodies (30). Pro-
cedural details are described in Supporting Text. Pull-down assays
were performed with glutathione-Sepharose beads precomplexed
with GST-Nck SH2 domains. Western immunoblotting was carried
out by using a monoclonal anti-phosphotyrosine antibody (dilution
1:5,000), a monoclonal anti-Cas antibody (dilution 1:1,000), or a
polyclonal anti-Fak antibody (dilution 1:1,000). We used GST
procedures is provided in Supporting Text.
We thank Amy Bouton (University of Virginia School of Medicine,
Charlottesville) for reagents including anti-Cas antibodies and for crit-
ically reading the manuscript. We are grateful to Drs. A. Cowan and W.
Mohler for expert advice in imaging techniques and Dr. Kazuya Machida
for helping with the far-Western blot analysis. This work was supported
by National Institutes of Health Grant CA82258 (to B.J.M.) and a
postdoctoral fellowship from the American Heart Association (to
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