Molecular Cell, Vol. 11, 1215–1227, May, 2003, Copyright 2003 by Cell Press
Provides a Mechanism of Molecular Memory in WASP
rise to morphological features such as filopodia and
lamellipodia (Nobes and Hall, 1995). Both Cdc42 and
Rac elicit actin filament formation through activation of
proteins in the Wiskott-Aldrich syndrome protein (WASP)
family (Caron, 2002). These proteins, which include
WASP, its widely expressed homolog N-WASP, the
Scar/WAVE proteins, and Bee1, link Cdc42 and Rac
directly to the cellular actin nucleation machine, the
Arp2/3 complex (Higgs and Pollard, 2001; Pantaloni et
al., 2001). WASP and N-WASP, are effectors of Cdc42
and when bound to the activated GTPase can stimulate
the actin nucleating activity of Arp2/3 complex, leading
to rapid formation of new filaments (Higgs and Pollard,
analogously to stimulate Arp2/3 complex in response to
Rac but are activated through an alternative mechanism
involving GTPase-mediated destabilization of an inhib-
ited WAVE/Pir121/Nap1 complex rather than direct
binding (Eden and Kirschner, 2002). The dynamics and
localization of Cdc42/Rac signals, and their coordina-
tion with other pathways, are important determinants of
the structure and dynamics of actin filament networks,
and their resultant functions in biological processes.
Moreover, these signals likely exert their influence, at
of WASP/WAVE signals to Arp2/3 complex. However,
the relationship between WASP/WAVE activation dy-
namics and the nature of upstream signals is not well
WASP activity toward Arp2/3 complex can be stimu-
lated by a variety of other ligands in addition to Cdc42,
including PIP2, Nck, Grb2, WISH, and profilin (Caron,
2002). Of these, PIP2and Grb2 have been shown to act
could act in an approximately switch-like manner in vivo
in response to low-levels of multiple stimuli (Carlier et
lated on tyrosine in response to a number of different
signals, including overexpression of Hck, and stimula-
tion of B cell antigen, collagen, IgE, chemokine, and Trk
receptors in appropriate cell types (Baba et al., 1999;
Cory et al., 2002; Guinamard et al., 1998; Oda et al.,
1998; Okabe et al., 2002; Suetsugu et al., 2002). In a
mast cell line, phosphorylation by Btk or Lyn is greatly
potentiated by coexpression of an activated mutant of
Cdc42, indicating cooperativity between GTPase and
kinase pathways (Guinamard et al., 1998). In B cells,
macrophages, and PC12 cells, the site of WASP/
N-WASP phosphorylation has been mapped to Y291/
Y256, which lies in the conserved sequence IYDFI (Baba
et al., 1999; Cory et al., 2002; Suetsugu et al., 2002).
This sequence is a consensus substrate for Src family
in this group. Y291 lies in the region of WASP termed
the GTPase binding domain (GBD, residues 230–310;
Figure 1A), which mediates interaction with Cdc42
(Abdul-Manan et al., 1999; Rudolph et al., 1998). In free
WASP, the GBD is bound in intramolecular fashion to a
C-terminal segment termed the VCA (for verprolin ho-
Eduardo Torres1,2and Michael K. Rosen1,*
1Department of Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Boulevard
Dallas, Texas 75205
2Department of Physiology and Biophysics
Weill Medical College of Cornell University
New York, New York 10021
Cells can retain information about previous stimuli to
produce distinct future responses. The biochemical
mechanisms by which this is achieved are not well
understood. The Wiskott-Aldrich syndrome protein
whose activation leads to stimulation of the actin nu-
cleating assembly, Arp2/3 complex. We demonstrate
that efficient phosphorylation and dephosphorylation
of WASP at Y291 are both contingent on binding to
activated Cdc42. Y291 phosphorylation increases the
basal activity of WASP toward Arp2/3 complex and
enables WASP activation by new stimuli, SH2 domains
of Src-family kinases. The requirement for contin-
gency in both phosphorylation and dephosphorylation
enables long-term storage of information by WASP
following decay of GTPase signals. This biochemical
circuitry allows WASP to respond to the levels and
anisms to specifically achieve transient or persistent
actin remodeling, as well as long-lasting potentiation
of actin-based responses to kinases.
Dynamic regulation of the actin cytoskeleton is an inte-
gral feature of many eukaryotic cellular processes, in-
cluding migration, adhesion, and division (Borisy and
ing signals that drive these processes must be con-
trolled in their timing, duration, amplitude, and localiza-
tion. These parameters, often integrated through multiple
signaling pathways (Higgs and Pollard, 2001), cause de-
fined alterations in the basic biochemical properties of
actin filaments, such as polymerization/depolymeriza-
tion, branching, and bundling, resulting in functionally
specific cytoskeletal structures and dynamics.
GTPases in the Rho family, including Cdc42, Rac, and
Rho, are key components of signaling pathways that
control actin filament networks (Etienne-Manneville and
Hall, 2002). These proteins act as molecular switches in
vivo, alternating between an inactive GDP-bound state
and an active GTP-bound state in response to upstream
signals (Etienne-Manneville and Hall, 2002). Cdc42 and
Rac act in many processes to induce polymerization of
new actin filaments at the cell periphery, which give
Figure 1. Structural Domains of WASP and
(A) Domain structure of WASP and N-WASP.
Thick and thin black bars indicate homolo-
gous regions and sequence gaps, respec-
tively. Layers 1(WASP residues 230–276) and
2 (WASP residues 277–310) of the autoinhib-
ited fold, comprising the GBD, are boxed in
yellow and blue, respectively. The central re-
gion (WASP residues 466–476) of the VCA
segment is boxed in red.
(B) Molecular surface of the autoinhibited do-
main of WASP. Structural layers are colored
as in (A). The Tyr 291 side chain is shown in
purple as a stick model. Figure was created
with GRASP (Nicholls et al., 1991).
mology, central hydrophobic, and acidic), resulting in
autoinhibition toward Arp2/3 complex (Kim et al., 2000;
Miki et al., 1998; Rohatgi et al., 1999). The structures of
the GBD-VCA and GBD-Cdc42 complexes are mutually
incompatible (Kim et al., 2000), and binding of Cdc42
to the GBD causes global destabilization of the autoin-
hibited GBD-VCA domain (Buck et al., 2001; M. Buck et
al., submitted), leading to release of the VCA, facilitating
its activation of Arp2/3 complex. In free WASP, Y291
is ?90% buried in the fold of the autoinhibited domain
bilization by Cdc42 may be necessary to provide effi-
cient access to Y291 by kinases (Kim et al., 2000).
To date, the phosphorylation of WASP has been de-
scribed only phenomenologically in nearly all instances,
and the physiological significance of this modification
remains largely unexplored. However, very recently it
by Src family kinases increases the basal activity of
the proteins toward Arp2/3 complex (Cory et al., 2002;
Suetsugu et al., 2002). Moreover, Suetsugu et al. have
shown that this modification is necessary for NGF-
induced neurite outgrowth in PC12 cells (Suetsugu et
al., 2002). Interestingly, in this system, N-WASP phos-
phosphorylation for extended periods.
Here we have examined the control of WASP phos-
phorylation at Y291 by Cdc42, kinases, and phospha-
tases, as well as the functional significance of this cova-
lent modification. We demonstrate that WASP can be
efficiently phosphorylated at Y291 only in the presence
of activated Cdc42. Moreover, isolated p-Y291 WASP
is highly protected from tyrosine phosphatases. Phos-
phorylation can thus provide a long-lasting biochemical
record of coincident GTPase and kinase signals. This
record can be erased by a second round of Cdc42 acti-
vation, since dephosphorylation occurs readily in the
the basal activity of WASP toward Arp2/3 complex. In
addition, p-WASP can be activated by SH2 domains of
Src family kinases due to steric incompatibility between
the autoinhibited fold of WASP and SH2-pTyr interac-
tions. This biochemical scheme has important implica-
tions for the role of WASP phosphorylation in signaling
pathways that connect Src family kinases through
Cdc42 to the actin machinery. It provides mechanisms
to achieve differential actin-based outputs based on the
relative timing, duration, and amplitude of GTPase and
kinase signals, and suggests a means by which the cell
could retain a record of previous stimulation in order to
respond differently in the future.
Efficient Phosphorylation of WASP Proteins Is
Dependent on Binding to Activated Cdc42
The autoinhibited GBD-VCA domain of WASP is com-
posed of a ? hairpin and five helices, which form a
structure that can be considered in three layers (Kim et
al., 2000). The first layer, residues 230–276, consists of
the ? hairpin and ?1 helix, which are the minimal ele-
ments necessary to bind Cdc42 with high affinity. The
second layer, residues 277–310, consists of helices
?2–?4 and contains the remainder of the GBD. These
elements are disordered in GBD-Cdc42 complexes
(Abdul-Manan et al., 1999). Layer 3 consists of a single
helix, ?5, formed by the central region of the VCA (resi-
dues 466–479). Y291 is located near the center of helix
?3 in the second layer of structure. It is an integral part
of the interface between layers 1 and 2, and is nearly
buried by surrounding side chains of the ? hairpin and
the ?3 helix (Figure 1B). Less than 10% of the residue
surface area is accessible to solvent due to packing
interactions with the hydrophobic portions of the V249,
W252, K288, and D292 side chains. This organization
suggested that Y291 should be a poor substrate for
tyrosine kinases in the autoinhibited state of WASP.
However, since binding to Cdc42 destabilizes the GBD-
VCA domain (Buck et al., 2001; M. Buck et al., submit-
Molecular Memory in WASP
Figure 2. Active Cdc42 Increases the Phos-
phorylation of Autoinhibited
N-WASP Constructs but Not of the GBD
In all figures, upper panel is Coomassie blue
stained SDS-PAGE gel, lower panel is anti-
phosphotyrosine Western blot. Identical re-
sults were obtained in all cases with Abl, Lck,
and Lyn; selected data are shown.
(A) Phosphorylation of 5 ?M GBD by the Abl
kinase domain alone (lane 1), or in the pres-
ence of 5 ?M Cdc42-GMPPCP (lane 2) or
Cdc42-GDP (lane 3).
(B) Phosphorylation of 4 ?M GBD by Lck alone
(lane 1), or in the presence of 50 ?M VCA (lane
2), 50 ?M VCA ? 8 ?M Cdc42-GMPPCP (lane
3), or VCA ? 8 ?M Cdc42-GDP (lane 4).
(C) Phosphorylation of 5 ?M tWASP by Lck
alone (lane 1), or in the presence of 40 ?M
Cdc42-GMPPCP (lane 2) or Cdc42-GDP (lane
3). Identical results were obtained with phos-
phorylation by Abl or Lyn.
(D) Phosphorylation of 20 ?M N-GBD-VCA by
the Lyn kinase domain alone (lane 1), or in
the presence of 15 ?M Cd42-GMPPCP (lane
2) or Cdc42-GDP (lane 3).
ted), the GTPase should increase the accessibility of
Y291 to kinases, facilitating its phosphorylation.
In order to test these hypotheses, we examined phos-
phorylation of the WASP GBD by the Lck, Lyn, and Abl
kinases in the presence and absence of the VCA and
Cdc42. Identical results were obtained with all kinases,
indicating the effects of VCA and Cdc42 are due to
changes in Y291 accessibility rather than changes in
specificity of the GBD substrate. As shown in Figure
2A, the GBD peptide, which has no persistent tertiary
structure on its own, can be readily phosphorylated in
isolation. Phosphorylation is unaffected by Cdc42 in ei-
ther the GMPPCP- or GDP-bound states (the former
mimics the GTP-bound state). However, as shown in
Figure 2B, addition of the VCA, which induces folding
of the GBD (Kim et al., 2000), results in a dramatic de-
crease in phosphorylation efficiency. This decrease can
be reversed by addition of Cdc42-GMPPCP, but is only
slightly affected by Cdc42-GDP. Thus, induction of the
autoinhibited fold results in protection against phos-
phorylation, and destabilization of this structure by
Cdc42 enables covalent modification. This behavior is
reproduced in a C-terminal fragment of WASP, tWASP
(residues 230–502), where the GBD and VCA elements
are tethered by the natural proline-rich linker. As shown
in Figure 2C, this protein is phosphorylated poorly in
isolation and in the presence of Cdc42-GDP. But it is
phosphorylated well in the presence of Cdc42-GMPPCP.
Similar behavior is also observed for an N-WASP con-
struct, N-GBD-VCA, containing the GBD (residues 192–
275) linked directly to theVCA (residues 393–505; Figure
2D). In all cases, mass spectrometric analyses demon-
strated that the WASP proteins are singly phosphory-
lated at Y291 (Y256 in N-WASP), the only tyrosine resi-
due in each construct (data not shown). These data
indicate that in WASP/N-WASP, the autoinhibited fold
protects Y291/Y256 against covalent modification by
kinases. Destabilization of the fold by active Cdc42 en-
ables ready phosphorylation. Thus, WASP and N-WASP
could function as logical AND gates through their phos-
phorylation at Y291/Y256, with modification possible
only in the presence of both activated Cdc42 and acti-
vated kinase. In this way, the proteins could act as coin-
ing a biochemical explanation for the cooperative
behavior observed in vivo (Guinamard et al., 1998).
Efficient Dephosphorylation Is Also Dependent
on Activated Cdc42
We next examined the effect of the autoinhibited fold
on the ability of tyrosine phosphatases to dephosphory-
late p-Y291-WASP proteins. Since the phosphatase(s)
that act on WASP in vivo are not known, we used two
enzymes in these studies: the catalytic domain of the
mammalian leukocyte antigen-related protein tyrosine
phosphatase, LAR-PTP, a typical eukaryotic phospha-
tase, and the highly active bacterial phosphatase, YOP
(Dixon, 1995). Similar results were obtained in both
cases. As shown in Figure 3A, p-GBD is rapidly dephos-
phorylated in the presence of 5 nM LAR-PTP (lane 1).
In preliminary quantitative analyses (data not shown),
LAR acts on p-GBD with kcat/Km? 1.3 ? 104M?1s?1,
Figure 3. Active Cdc42 Increases the Rate of
Dephosphorylation of Autoinhibited WASP
In all figures, upper panel is Coomassie blue-
stained SDS-PAGE gel, lower panel is anti-
phosphotyrosine Western blot.
(A) Time course of the dephosphorylation of
5?M p-GBD by 5 nM LAR phosphatase alone
(lane 1), with 15 ?M VCA (lane 2), with 15 ?M
15 ?M VCA ? 5 ?M Cdc42-GDP (lane 4).
(B) Dephosphorylation (30 min) of 5 ?M
p-tWASP by 5 nM LAR phosphatase alone
(lane 1), with 20 ?M Cdc42-GMPPCP (lane 2),
and with 20 ?M Cdc42-GDP (lane 3).
(C) Dephosphorylation (90 min) of 2 ?M
p-N-GBD-VCA by 5 nM LAR phosphatase
alone (lane 1), with 20 ?M Cdc42-GMPPCP
(lane 2), and with 20 ?M Cdc42-GDP (lane 3).
indicating catalytic efficiency only slightly below that
found with other substrates (kcat/Km? 2.5 ? 10 ? 104
M?1s?1[Cho et al., 1992]). Addition of VCA decreases
activity of LAR toward p-GBD substantially (lane 2). Ad-
dition of Cdc42-GMPPCP but not Cdc42-GDP, reverses
this inhibition and returns p-GBD to an efficient phos-
phatase substrate (lanes 3 and 4, respectively). Since
folded structure in the phosphorylated GBD, these re-
sults indicate that the autoinhibited fold protects p-WASP
against tyrosine phosphatases. These effects are also
reproducedin thetethered proteinstWASP andN-GBD-
VCA, which are dephosphorylated poorly in isolation,
but readily in the presence of Cdc42-GMPPCP (Figures
3B and 3C, respectively).
The combined data on kinase and phosphatase ac-
tions indicate that the phosphorylation status of WASP
at Y291 can only be efficiently altered in the presence
of activated Cdc42. This property results directly from
the structural and thermodynamic mechanisms of
WASP regulation. These give rise to steric occlusion of
the (p)Y291 side chain in the free protein, and increased
accessibility in the Cdc42-bound state. In the context
of signaling, following one round of GTPase and kinase
phosphorylated long after the initial stimulus has sub-
sided. Phospho-Y291 could thus serve as a long-lasting
biochemical record, or molecular memory, of previous
coincident signaling events.
Phosphorylation Increases the Basal Activity of WASP
Proteins toward Arp2/3 Complex
We originally speculated, based on the autoinhibited
GBD-VCA structure, that phosphorylation at Y291 might
leadto constitutiveactivationofWASP throughdestabi-
lization of the autoinhibited fold (Kim et al., 2000). To
test this, we examined the effects of phosphorylation
on affinity of GBD peptides for VCA peptides, and on
WASP protein activities in Arp2/3 complex-stimulated
pyrene-actin polymerization assays. Affinity was mea-
sured using isothermal titration calorimetry (ITC). Figure
4A shows calorimetric titrations of the N-WASP VCA
into pGBD and GBD. Fitting of the heats evolved as a
function of molar ratio yields a KDvalue of 2.8 ? 0.4 ?M
for the GBD, and 16.0 ? 0.2 ?M for p-GBD. Thus, while
p-GBD can bind the VCA, its affinity is decreased nearly
5-fold compared to the nonmodified protein.
To determine the functional significance of this de-
crease in affinity, we first examined the ability of GBD
peptides. Figure 4B shows polymerization of G-actin over
time (monitored by the increase in fluorescence of py-
rene-labeled [5%] actin). Polymerization of actin alone
is characterized by a significant lag phase and slow
Molecular Memory in WASP
Figure 4. Phosphorylation Decreases Affinity of GBD for VCA and Increases Basal Activity of WASP Constructs toward Arp2/3 Complex
(A) ITC trace of the titration of N-WASP VCA into p-GBD (left panel). Curve fits for the VCA into p-GBD and GBD titrations are shown in the
center and right panels, respectively. For p-GBD, KD? 16.0 ? 0.2 ?M, with stoichiometry of 0.97 ? 0.02: 1. For GBD, KD? 2.8 ? 0.4 ?M,
with stoichiometry of 0.99 ? 0.01: 1.
(B) Polymerization of 4 ?M G-actin (5% pyrene labeled) in the presence of 10 nM Arp2/3 complex. N-WASP VCA peptides were added to 25
nM. N-WASP GBD peptides were added to 5 ?M. Similar effects were observed with WASP VCA and GBD peptides.
(C) Comparison of the ability of tWASP (dashed lines) and p-tWASP (solid lines) to stimulate polymerization of 2 ?M G-actin (5% pyrene
labeled) by 10 nM Arp2/3. The tWASP proteins were added to 10, 50, and 250 nM (green, blue, and red, respectively). Effects of 250 nM VCA
are shown in black for comparison.
rate. These parameters are not significantly altered by
addition of isolated Arp2/3 complex, consistent with
previous reports that the highly purified assembly is
inactive (Higgs and Pollard, 2001). Addition of 25 nM
VCA peptide causes a dramatic decrease in lag time
is greatly suppressed by addition of 5 ?M GBD, which
should sequester 64% of the VCA, based on a 2.8 ?M
KD(excluding effects of Arp2/3 complex on the GBD/
VCA binding equilibrium). In contrast, addition of 5 ?M
p-GBD (24% calculated VCA sequestration) has a much
weaker inhibitory effect on VCA-stimulated Arp2/3 com-
plex activity. At higher concentrations (data not shown)
p-GBD can block activity to a degree similar to GBD,
indicating that the effect of phosphorylation is largely
on affinity for VCA, rather than intrinsic ability to block
in tWASP. Figure 4C shows that, as previously reported,
tWASP is a poor activator of Arp2/3 complex at concen-
trations between 10 and 250 nM (Devriendt et al., 2001).
In contrast, p-tWASP shows significant activity toward
Arp2/3 complex that increases in a dose-dependent
manner in this same range.
The apparent discrepancy between resistance to
phosphatases (Figure 3; suggesting a stable GBD-VCA
complex) and activity toward Arp2/3 complex (Figures
4B and 4C; suggesting an unstable GBD-VCA complex)
can be reconciled by our recent observation that the
autoinhibited domain is modular in structure and stabil-
ity (Buck et al., 2001; M. Buck et al., submitted). That
of the domain result in appreciably larger decreases in
stability of the VCA helix, ?5. Thus, while phosphoryla-
tion may have only modest effects on the stability of ?3,
enabling protection from phosphatases, it could have a
larger destabilizing effect on ?5, enabling Arp2/3 com-
plex activation. To examine structural aspects of this
model, we determined the backbone13C chemical shifts
rately) with VCA at 25?C (data not shown). Values are
15N/13C-labeled p-GBD and GBD in complex (sepa-
nearly identical (rmsd, C? ? 0.22 ppm; rmsd, C? ? 0.22
ppm), indicating that helices ?1–?4 are preserved after
phosphorylation (Wishart and Sykes, 1994). Thermody-
namic aspects of the model are also consistent with our
lated GBD-VCA proteins fromWASP and N-WASP show
a single, high-temperature thermal unfolding transition
by circular dichroism (Tm? 60?C–80?C, depending on
linker length), the phosphorylated proteins show an ad-
ditional transition at approximately 35?C that may repre-
sent dissociation of the VCA helix from the ?1–?4 core.
lation of Y291/Y256 in the WASP/N-WASP GBD de-
the basal activity toward Arp2/3 complex. Thus, an im-
portant functional outcome of coincident Cdc42 and
kinase signals is conversion of transient WASP/Arp2/
3 complex activation, with lifetime dependent on the
nucleotide cycle of the GTPase, to persistent, limited-
tively). The poor chemical shift dispersion and broad
lines indicate that both proteins lack persistent tertiary
structure in isolation. In both cases, addition of excess
unlabeled VCA peptide (not observed in the spectra)
causes a dramatic increase in chemical shift dispersion
andnarrowing oflines,indicating inductionof thefolded
autoinhibited structure (Figures 5D and 5E). Addition of
excess SH2 domain to the GBD-VCA complex does not
cause changes in chemical shifts or line widths of the
GBD signals (Figure 5F). However, the SH2 domain
causes a dramatic collapse of p-GBD signals in the
shift dispersion and changes in line widths, indicating
disruption of the autoinhibited domain (Figure 5G). The
combined biochemical and NMR data demonstrate that
the Src SH2 domain is capable of binding p-GBD and
displacing it from VCA.
To determine if these effects are sufficient to activate
WASP toward Arp2/3 complex, we used pyrene-actin
polymerization assays. At low concentrations (1–2 ?M),
the Src SH2 domain has little effect on the ability of
p-GBD to inhibit VCA-mediated Arp2/3 complex activa-
tion (data not shown), consistent with decreased affinity
of the SH2 domain for p-GBD when the latter is in the
autoinhibited fold. The same sequestration of p-GBD by
VCA prevents catalytic concentrations of phosphatases
from acting on p-Y291 in autoinhibited WASP proteins.
However, as shown in Figure 6A, at high concentrations
(?10 ?M), the Src SH2 domain causes substantial acti-
vation of a p-GBD-VCA complex, demonstrating that
the physical displacement observed above leads to
functional activation. The same concentrations of SH2
domain have no effect on the nonphosphorylated GBD.
We also examined the effect of the SH2 domain on
the tethered tWASP protein. As in the intermolecular
on the activity of p-tWASP, while high concentrations
were stimulatory (data not shown). However, the magni-
tude of this increase was modest, and required higher
and VCA peptides, due to the increased stability of the
autoinhibited fold imparted by the tether. The SH3 do-
proline-rich sequences of WASP and N-WASP, which
separate the GBD and VCA elements (Banin et al., 1996;
containing the SH3-SH2 module should be capable of
making bivalent interactions with p-tWASP. This multi-
valency should afford such constructs appreciably
higher affinity, and increased potency as p-tWASP acti-
vators than the SH2 domain alone. Viewed alternatively,
the SH3-proline interactions of an SH3-SH2 construct
should greatly increase the local concentration of the
SH2 domain toward p-Y291, enabling the latter to acti-
vate. As shown in Figure 6C, these predictions were
correct, and anSrc SH3-SH2 protein at1 ?M concentra-
tion activated p-tWASP to levels comparable to the free
VCA. This ability required multivalent interactions with
WASP, since a nonlinked mixture of SH3 and SH2 do-
main proteins at the same concentration had no effect.
The actions of the SH3-SH2 protein are dependent on
by Cdc42-GMPPCP, it is not affected by Src constructs
Src Family SH2 Domains Can Activate p-WASP
Proteins toward Arp2/3 Complex
The sequence surrounding Y291 is a consensus binding
site for Src family SH2 domains (Yaffe et al., 2001). In
the structuresof pTyrsubstrates boundto SH2domains
of this class, the peptide backbone is in an extended
conformation, with side chains of the pTyr and I?3 resi-
dues bound into two pockets that are well separated
on the protein surface (Waksman et al., 1993). In autoin-
hibited WASP, the Y291 motif is located in helix ?3 of
the domain, with the edge of the Y291 ring exposed to
solvent and I294 buried in the hydrophobic core of the
domain. Incompatibility between these conformations
suggested that SH2 domain binding to p-Y291 could,
analogousto Cdc42,destabilizethe autoinhibitedstruc-
ture, leading to physical separation of the GBD from the
VCA and activation toward Arp2/3 complex. We note
that the low affinity of phosphatases toward p-GBD
(Km? 155 ?M for LAR, see above) would prevent cata-
lytic concentrations of these enzymes from analogously
destabilizing the autoinhibited domain and accessing
To test this hypothesis, we first examined the ability
of SH2 domains to bind p-GBD and displace it from VCA
peptides. Immobilized Src SH2 domain can bind p-GBD
with reasonable affinity, but is unable to bind nonphos-
phorylated GBD. As described in Experimental Proce-
of p-WASP proteins throughout this work. Preliminary
isothermal titration calorimetry measurements of the af-
finity of p-GBD for the Src SH2 domain indicate a KDof
approximately 2 ?M (data not shown). To determine if
this interaction is incompatible with binding of p-GBD
to VCA, we used two assays. Figure 5A shows that both
GBD and p-GBD can be retained by immobilized GST-
VCA. However, incubation of the resin with excess solu-
ble SH2 domain resulted in substantial elution of p-GBD
but not GBD, demonstrating that SH2 domains can de-
stabilize the WASP autoinhibitory interactions. We also
examined this issue by NMR spectroscopy. Figure 5
p-GBD (Figures 5B, 5D, and 5F; 5C, 5E, and 5G, respec-
1H/15N HSQC spectra of
15N-labeled GBD and
Molecular Memory in WASP
Figure 5. Src SH2 Domain Can Physically Disrupt the Interaction between p-GBD but Not GBD and VCA
(A) GST-VCA was immobilized on glutathione-Sepharose beads and incubated with the Src SH2 domain and either p-GBD (left panels) or
GBD (right panel). Proteins bound to the beads (lanes 1 and 3) and remaining in solution (lanes 2 and 4) were analyzed by Coomassie blue-
stained SDS-PAGE gels and anti-phosphotyrosine Western blotting.
(B and C)1H/15N HSQC spectra of 100 ?M15N-labeled GBD and p-GBD, respectively.
(D and E)1H/15N HSQC spectra of 100 ?M15N-labeled GBD and p-GBD in the presence of 250 ?M and 400 ?M unlabeled VCA, respectively.
(F and G)1H/15N HSQC spectra recorded after 200 ?M unlabeled Src SH2 domain was added to samples from (D) and (E), respectively.
SH3-SH2 module (Figure 6B). Thus, steric incompatibil-
of the p-Y291 motif results in physical displacement of
p-GBD from VCA by SH2 domains, leading to activation
toward Arp2/3 complex. These data demonstrate that
a second functional consequence of WASP phosphory-
lation at Y291 is sensitivity to a new input signal, binding
of Src-family SH2 domains, which has no effect on the
(p)Y291 to modifying enzymes. Once phosphorylated at
Y291, WASP becomes partially activated toward Arp2/3
complex, likely because of moderate destabilization of
the GBD-VCA contacts in the autoinhibited fold. In addi-
tion, since the helical conformation of the p-Y291 motif
in autoinhibited, WASP is incompatible with that in SH2-
peptide complexes, the autoinhibited domain of the
phosphorylated protein can also be disrupted by Src
family SH2 domains, leading to activation.
These properties generate a potentially powerful cir-
cuit governing the cellular response to Cdc42 and Src
family kinase signals. The output of this circuit, manifest
by the level of phosphorylated WASP (p-WASP), in re-
constant phosphatase activity, is described schemati-
cally in Figure 7A. When Cdc42 or kinase is active inde-
pendently (periods a and b), WASP is not phosphory-
lated (third trace), although it is active toward Arp2/3
complex for the duration of the GTPase signal (fourth
We have demonstrated here that both phosphorylation
and dephosphorylation of WASP at Y291 are dependent
VCA complex sterically protects the (p)Y291 side chain
from both kinases and phosphatases in the free state
of the protein. Binding to Cdc42 substantially destabi-
lizes the autoinhibited domain, enabling access of
Figure 6. The Src SH2 Domain Can Activate p-WASP toward Arp2/3 Complex
(A) Polymerization of 4 ?M G-actin (5% pyrene labeled) in the presence of 10 nM Arp2/3 complex. GBD curves are dashed; p-GBD curves
(B and C) Polymerization of 2 ?M G-actin (5% pyrene labeled) in the presence of 10 nM Arp2/3 complex.
trace). When Cdc42 and kinase are active simultane-
ously, both in time and location (periods c and e), WASP
is rapidly phosphorylated in its GTPase complex. If the
kinase signal subsides prior to the Cdc42 signal, WASP
will be returned to its nonphosphorylated state by the
WASP complex (period d). If, however, the Cdc42 signal
terminates prior to the kinase (period f), p-WASP will
fold to the autoinhibited, phosphatase-resistant confor-
mation and remain phosphorylated for potentially long
periods. During this time, partial destabilization of the
autoinhibited domain imparts increased basal activity
on the protein. The p-Y291 switch can ultimately be
reset (periodg) bysubsequent activationof Cdc42inde-
pendent of kinase. Decay of this second GTPase signal
(period h) enables WASP to return to its completely
inactive state. This circuit ultimately arises because of
dictate that the efficiency of WASP as a substrate for
properties of WASPresult in a circuitthat senses coinci-
dence of GTPase and kinase signals. Moreover, the cir-
cuit can function as a memory device, maintaining a
long-lasting record of this event even after the initiating
signals are terminated.
Suetsugu et al. have reported that PIP2can enhance
phosphorylation of N-WASP at Y256 by Fyn (Suetsugu
et al., 2002), analogous to our findings with Cdc42. Al-
though these authors did not examine whether PIP2in-
creases the intrinsic activity of Fyn, one possible expla-
nation of their data is that the phospholipid increases
accessibility of Y256 in the autoinhibited fold to kinases.
This commonality would indicate that deconstruction of
by both Cdc42 and PIP2. Thus, cooperativity between
GTPase and phospholipid, and perhaps others other
activators as well, is likely to involve not only WASP
activity, as has been reported by several groups (Higgs
and Pollard, 2000; Prehoda et al., 2000; Rohatgi et al.,
2000), but also WASP phosphorylation. The existence
ofmultiple pathwaysthatenhance phosphorylationmay
phorylation induced by overexpression of Hck, which
Molecular Memory in WASP
Figure 7. Dynamic Responses of WASP to
Cdc42 and Src Family Kinase Signals
(A) Phosphorylation status and activity of
WASP/N-WASP in response to Cdc42 and
(B) Dynamic features of feedforward loop be-
tween Src family kinases and WASP. Top
panel shows initial response of the system,
where GTPase and kinase must both be acti-
vated to achieve WASP phosphorylation.
Open arrowheads indicate indirect pathway
from kinase to WASP phosphorylation, filled
arrowhead indicates direct pathway. Inde-
pendent GAP activation (red arrow) results in
WASP phosphorylation that persists follow-
ing decay of the kinase signal. Bottom panel
shows response whenWASP is initially phos-
phorylated by a previous round of coincident
GTPase and kinase signals.
activates diverse pathways, is not strongly affected by
coexpression of dominant-negative or constitutively ac-
tive Cdc42 mutants.
Pathways connecting Src family kinases to Cdc42 to
ber of biological processes. In HeLa cells and macro-
phages, constitutively activated Hck signals through
Cdc42 and Rac to stimulate the formation of actin-rich
membrane protrusions (Carreno et al., 2002). Macro-
phage phagocytosis mediated by the Fc? receptor
(Fc?R) appears to use an analogous pathway (Green-
lation leads to activation of Hck and Lyn (Carreno et al.,
2002; Kedzierska et al., 2001; Suzuki et al., 2000), and
recruitment (and presumably activation) of Cdc42
(Caron and Hall, 1998), WASP/N-WASP (Castellano et
al., 2001; Lorenzi et al., 2000; May et al., 2000), and
Arp2/3complex (Mayetal., 2000)tothe phagocyticcup,
providing actin rearrangements necessary for efficient
phagocytosis. Cdc42activation atthis sitemay proceed
through Hck-mediated phosphorylation and activation
of a guanine nucleotide exchange factor (GEF) in the
Vav family (Coppolino et al., 2001; English et al., 1997),
whose members have been shown to act on Cdc42 in
vivo and in vitro (Abe et al., 2000). A similar pathway
ulation of the T cell antigen receptor (TCR) leads to
activation of the tyrosine kinases Lck, Fyn, and ZAP-
70, which phosphorylate Vav1 and SLP-76, leading to
localized activation of Cdc42, WASP, and Arp2/3 com-
plex (Cannon and Burkhardt, 2002; Cannon et al., 2001;
Dustin and Cooper, 2000; Krause et al., 2000; Krawczyk
et al., 2002). These interactions appear to be critical to
the polymerization and dynamics of actin during forma-
tion of the immunological synapse (Dustin and Cooper,
2000). Finally, PDGF stimulation of NIH 3T3 fibroblasts
causes activation of Src, which phosphorylates Vav,
leading to activation of Cdc42 and Rac, ultimately in-
creasing expression of the c-myc transcription factor
(Chiariello et al., 2001). In both macrophage phagocyto-
sis and T cell activation, strong evidence exists for colo-
calization of kinase, Cdc42, and WASP/N-WASP (Can-
non et al., 2001; Castellano et al., 1999; Coppolino et
al., 2001; Greenberg and Grinstein, 2002; Krause et al.,
2000; Stinchcombe et al., 2001). Thus, the biochemistry
we have described indicates it is highly likely that WASP
will belocally phosphorylatedon Y291during thesepro-
cesses. The biochemical interactions we have de-
scribed, when present in these biological contexts,
would thus create the coherent feedforward circuit
shown in Figure 7B. The indirect arm of the circuit be-
tween the Src family kinase and WASP (open arrow-
heads) would be delayed relative to the direct arm (filled
arrowheads) due to additional intermediates between
ciate from RhoGDI and migrate to membranes during
activation (Olofsson, 1999). In addition, phosphorylation
of Vav at multiple sites appears to be necessary to
achieve high-level activation of GEF activity (Bustelo,
2000), a feature that could give rise to hypersensitivity
(Ferrell, 1999). Analogous coherent feedforward circuits
are highly abundant in both prokaryotic and eukaryotic
transcriptional regulatory networks, as well as in the
neuronal synaptic connectivity network in C. elegans
(Milo et al., 2002; Shen-Orr et al., 2002). Mathematical
modeling and experimental investigations have demon-
strated that such circuits act to reject short duration/
low amplitude input stimuli, producing positive output
(i.e., WASP phosphorylation in the present case) only
upon persistent, strong stimulation (Shen-Orr et al.,
2002). These circuits also produce rapid decay of the
signal followinginput termination (Shen-Orr etal., 2002).
Although as described above, if the Cdc42 signal is
terminated prior to kinase, for example by independent
activation of a GTPase-activating protein (GAP; red
arrow in Figure 7B), the memory function of the circuit
is activated and WASP can remain phosphorylated for
interactions we have described could be used during
Cdc42/actin-dependent processes such as macro-
phage phagocytosis or T cell activation, to reject low-
in response to long-lasting, high-level stimulation.
Mechanisms leading to activation of most GEFs for
Rho GTPases, including those specific for Cdc42, are
not yet known. Many will certainly involve pathways that
are independent of Src family kinases. In these cases,
p-WASP could also function as the output of circuits
that detect the simultaneous activation of independent
Cdc42 and kinase pathways. Experimental testing of
this idea must await a better understanding of signaling
mechanisms that control Cdc42.
We have shown that the functional consequences of
WASP phosphorylation at Y291 are 2-fold. First, p-WASP
has significantly higher basal activity than WASP. Sec-
ond, phosphorylation enables WASP to respond to a
new signal, SH2 domain binding. Potential implications
of these effects will be discussed separately, although
both could be important in particular systems. In sys-
tems/processes where the enhanced basal activity of
p-WASP is sufficient to mediate signaling, phosphoryla-
tion would effectively convert transient WASP activa-
tion, with a lifetime governed by the GTPase nucleotide
cycle, to persistent activation. In a biological context,
this could serve either to impart new functionality to
WASP signals, or to extend the lifetime of existing func-
tions. In the former case, the requirement of Cdc42/
kinase coincidence would enhance the specificity of
of N-WASP phosphorylation in neurite outgrowth could
be an example of such behavior (Suetsugu et al., 2002).
In the latter case, the biochemistry we have described
could provide a mechanism for the cell to sample a
WASP-dependent processes transiently, under control
of the GTPase clock, and then commit to that process
when appropriate, independent kinase signals are re-
ceived. Interestingly, Wu ¨lfing and colleagues (C. Wu ¨lf-
ing, personal communication) have recently demon-
strated that stimulation of primary T cells with limiting
antigen (presented by antigen presenting cells, APCs)
in the presence of costimulatory blockade (antibodies
to the CD28 and LFA-1 ligands, ICAM-1 and B7) results
in only transient actin accumulation at the T cell-APC
interface. In contrast, sustained actin accumulation,
which is necessary for accumulation and organization
of the signaling and adhesion receptors at the immuno-
logical synapse, requires high levels of antigen and co-
stimulation of CD28. The biochemistry we have de-
scribed could, in principle, provide circuitry that
contributes to such behaviors.
The ability of Src family SH2 domains to bind and
sequences. First, it could extend the lifetime of the actin
polymerization signal. That is, since Src proteins both
phosphorylate and bind WASP, it seems likely that after
phosphorylation, the kinase will remain bound to
p-WASP through bivalent interactions of its SH2 and
SH3 domains. This would lead to high-level activation
of WASP in the absence of Cdc42. Second, SH2 domain
binding would also extend the lifetime of the p-Y291
itself, since the phosphate group would be protected
from phosphatase both when bound to SH2 and when
free. Third, in the context of the circuit in Figure 7B,
a mechanism for potentiating the system toward ki-
nases. When WASP is not phosphorylated, high levels
of kinase are necessary to initiate actin rearrangements
since the signal must pass through GEF and Cdc42
before reaching WASP (see above). In contrast, once
WASP is phosphorylated by an initial intense signal,
ule of the kinase can activate p-WASP directly without
thelags inherentintheGTPase pathway.Alternatively,if
kinase are distinct, modification of WASP could provide
a mechanism for actin rearrangements in response to
an entirely new stimulus. Thus, phosphorylation of
WASP can serve as a general mechanism to sensitize
a system to actin rearrangements in response to Src
family kinase pathways. This behavior could allow the
cell to be maintained in an inert state, relatively resistant
to stimuli, but converted to a responsive state by an
initial, committing, high-intensity burst of input signal.
The biochemical process we have discovered for
WASP provides a mechanism for cells to retain informa-
tion for time periods of intermediate duration: longer
than the rapid rise and decay of many signaling events,
butlikely shorterthantimescharacteristic oflarge-scale
as differentiation. Analogous hysteresis can be gener-
ated on a cellular basis by feedback elements within
multicomponent signaling networks, as demonstrated
Ferrell, 2002). The information storage described here
is achieved by a distinct mechanism, which is based on
the biophysical construction of WASP. The behavior of
WASP, and its ultimate causes are in many respects
similar to those of Ca2?/calmodulin-dependent protein
kinase II (CamKII), an extensively studied protein postu-
lated to function as a molecular memory device in neu-
rons (Hudmon and Schulman, 2002; Lisman et al., 2002).
ylation requires an initial conformational change to
transform the protein into an efficient kinase substrate.
In WASP, binding of Cdc42 to the GBD destabilizes the
autoinhibited domain, enabling modification of Y291. In
of Ca2?/calmodulin to one kinase subunit reveals its site
of autophosphorylation (T286), enabling modification by
a neighboringsubunit (Hudmon and Schulman,2002). In
mediated activity to be retained for long periods. In
from phosphatases. In CamKII, slow dissociation of cal-
modulin from phosphorylated subunits (Schulman et al.,
subunit to phosphorylate its neighbor in the presence
of basal Ca2?levels (Lisman et al., 2002; Lisman and
Zhabotinsky, 2001; Zhabotinsky, 2000), both provide
mechanisms to prolong activity and duration of phos-
phorylation. Finally, phosphorylation can potentiate the
activation of both WASP and CamKII by later stimuli by
lowering the threshold levels of Src family kinase (see
tion (De Koninck and Schulman, 1998; Hanson et al.,
1994; Meyer et al., 1992; Shen et al., 2000). In both
Molecular Memory in WASP
Experiments were performed in an Omega titration calorimeter (Mi-
croCal, Inc). Titrations were performed by injecting 25 ? 10 ?l of
1.6 mM VCA at 3 min intervals into a 1.4 ml solution of 100 ?M GBD
or p-GBD at 25?C in 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM
EDTA, and 2 mM DTT.
proteins, autoinhibitory regulation is central to these
biochemical functions (Hudmon and Schulman, 2002;
Kim et al., 2000). Large structural rearrangements, in-
volving domain separation and extensive exposure of
buried surface area, are inherent in such designs. These
features make autoinhibited proteins ideally suited to
integrate multiple signals through contingent covalent
modification. In fact, such behavior has now been ob-
served directly and indirectly in other autoregulated
GTPase effectors, including the Pak and Raf kinases
(Renkema et al., 2002; Stokoe et al., 1994). Our studies
with WASP, and the large body of biochemical work on
CamKII, show that when autoregulation appears in an
appropriate structural and thermodynamic context, it
can also lead to mechanismsof biochemical memory. In
CamKII, this functionality is thought to make important,
nance of long-term potentiation, a neuronal property
that plays important roles in learning and memory (Lis-
man et al., 2002). However, in other systems, including
speculative. As our understanding of these potentially
powerful biochemistries develops, discovery of how
will represent a critical avenue for future research.
Actin Polymerization Assays
Detailed procedures are described in Higgs et al. (1999). Reactions
contained 2 or 4 ?M Mg-ATP actin (5% pyrene labeled), 10 mM
imidazole (pH 7.0), 50 mM KCl, 1 mM EGTA, and 1 mM MgCl2in G
buffer-Mg (2 mM Tris-HCl [pH 8.0], 0.5 mM DTT, 0.1 mM ATP, 0.1
MgCl2). Pyrene fluorescence (?ex ? 365 nm, ?em? 407 nm) was
monitored with a Fluorolog-3 spectrafluorimeter (JY Horiba). Fluo-
rescence was normalized to the final value observed at steady state
within each experiment.
GST-VCA was immobilized on glutathione Sepharose beads at a
concentration of 5 mg/ml resin. Beads (20 ?l) were incubated 10
min at 25?C with 100 ?l of 100 ?M GBD or p-GBD, plus 20 ?l of 170
?M Src SH2 domain in PBS buffer. Supernatant was removed and
beads were washed three times with 120 ?l of PBS. Proteins bound
to beads were eluted with SDS sample buffer. Supernatant and
bound samples were analyzed by SDS-PAGE.
NMR experiments were carried at 25?C on a Varian Inova 600 MHz
spectrometer using a gradient and sensitivity enhanced
HSQC pulse sequence (Kay et al., 1992). All protein samples were
in 25 mM phosphate (pH 7.0), 100 mM NaCl, 2 mM EDTA, 2 mM
DTT, and 10% D2O.
Human N-WASP GBD amino acids 192–275, human WASP GBD
amino acids 230–310, Src SH2 amino acids 144–249, and human
Cdc42 amino acids 1–179 were cloned into pET11a. N-WASP VCA
amino acids 393–505, WASP VCA amino acids 420–502, Src SH3
amino acids 83–144, and Src SH3SH2 amino acids 83–249 were
cloned into pGEX-2T. Truncated WASP (tWASP), amino acids 230–
502, was cloned into pET16b. N-WASP GBD-VCA (N-GBD-VCA),
was cloned into pET11a. All proteins were expressed in E. coli strain
BL21(DE3) except for tWASP, which was expressed in BL21-Co-
donPlus (Stratagene). Lck tyrosine kinase was overexpressed in Sf9
insect cells. Expression and purification protocols for all proteins
are available from the authors. Cdc42 was loaded with GMPPCP
as described (Abdul-Manan et al., 1999). The Abl kinase domain,
and the LAR and YOP phosphatases were purchased from NEB.
LynA was purchased from Biomol.
Bovine Arp2/3 complex was purified from calf thymus as de-
scribed (Higgs et al., 1999). Actin was purified from rabbit muscle
and labeled with pyrene as described (Pollard and Cooper, 1984;
Spudich and Watt, 1971).
N-WASP GBD, WASP GBD, N-GBD-VCA, and tWASP were phos-
phorylated on preparative scale by Lck or Abl kinase (New England
BioLabs) in the presence of a 5-fold excess of Cdc42-GMPPCP.
Phosphorylation was monitored by anti-p-Tyr Western blotting.
Phosphoproteins were purified on a GST-Src SH2 domain affinity
column eluted with 10 mM phospho-tyrosine in 50 mM Tris-HCl, pH
7.0. Single-site phosphorylation of p-GBD of WASP and N-WASP,
Each construct contains only a single tyrosine, Y291/Y256 for
We thank Dr. Annette Kim for first recognizing the potential impor-
tance of WASP phosphorylation atY291. We thank Drs. Harry Higgs,
Don Kaiser, and Tom Pollard for advice regarding purification of
bovine Arp2/3 complex and actin; Dr. Gaya Amarasinghe for Lck
expression in Sf9 cells; Ivan Haller and Bikash Pramanik for per-
structs; Dr. William Lowry for performing preliminary WASP phos-
phorylationexperiments;Drs. RamaRanganathan,UriAlon, andJan
Burkhardt for exciting discussions regarding biochemical circuits
and lymphocyte signaling; Dr. Yuh Min Chook for critical reading of
the manuscript; and Dr. Christoph Wu ¨lfing for communicating re-
sults prior to publication. This work was supported by grants from
the NIH (RO1 GM56322) and the Welch Foundation (Chemical Re-
search Grant #I1544). Preliminary studies were performed at the
Howard Hughes Medical Institute at the Memorial Sloan-Kettering
Received: November 22, 2002
Revised: February 7, 2003
Accepted: March 6, 2003
Published: May 22, 2003
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