COMMENTARY
Signal Transduction and Cellular Organization
1253
SH3 domains: complexity in moderation
Bruce J. Mayer
Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 060303301, USA
([email protected])
Journal of Cell Science 114, 1253-1263 © The Company of Biologists Ltd
Summary
The SH3 domain is perhaps the best-characterized member
of the growing family of protein-interaction modules. By
binding with moderate affinity and selectivity to prolinerich ligands, these domains play critical roles in a wide
variety of biological processes ranging from regulation of
enzymes by intramolecular interactions, increasing the
local concentration or altering the subcellular localization
of components of signaling pathways, and mediating the
assembly of large multiprotein complexes. SH3 domains
and their binding sites have cropped up in many hundreds
of proteins in species from yeast to man, which suggests
that they provide the cell with an especially handy and
Introduction
The idea that specific protein-protein interactions govern many
biological processes is by now so deeply ingrained that it is
easy to forget that it has only been a decade or so since this
was widely appreciated. In that time the Src homology 3 (SH3)
domain has become a standard-bearer for the protein
interaction field, largely because it was one of the first modular
protein interaction domains identified, is one of the most
common and has served as a proving ground for new methods
aimed at identifying partners or creating novel ligands through
chemical synthesis. Indeed, given the unrelenting attention to
such a modest domain, it seems hard to imagine that there
could be much left to uncover. I will argue that, to the contrary,
SH3 domains actually serve to remind us just how much
remains to be learned. In fact their very omnipresence is one
source of conceptual trouble - how can the cell reap meaningful
information from networks based on such a common module,
whose affinity and selectivity is modest at best? Because SH3
domains play a role in so many of the processes of interest to
the cell biology and biochemical communities, it is without
doubt important that we get a handle on what they can and
cannot do, how we can use their properties to our advantage
and how we can disrupt them in vivo. Here, I very briefly
sketch out the general properties of SH3 domains, spend
considerable time on recent advances in understanding the
determinants of binding specificity (or lack of specificity) and
its significance, and go on to discuss two specific recent
examples in which these domains appear to play complex and
varied roles in important biological processes.
A brief history
SH3 domains were first noted as regions of sequence similarity
between divergent signaling proteins such as the Src family of
adaptable means of bringing proteins together. The wealth
of genetic, biochemical and structural information
available provides an intimate and detailed portrait of the
domain, serving as a framework for understanding other
modular protein-interaction domains. Processes regulated
by SH3 domains also raise important questions about the
nature of specificity and the overall logic governing
networks of protein interactions.
Key words: Protein-protein interaction, Modular binding domains,
Signal transduction
tyrosine kinases, the Crk adaptor protein, and phospholipase
C-γ (Mayer et al., 1988; Stahl et al., 1988). It was soon
apparent that this ~60-residue region of similarity is present in
many proteins (at this point data from genomic sequencing
projects suggest that 63 proteins in Drosophila, 55 in
Caenorhabditis elegans, and 25 in Saccharomyces cerevisiae
contain at least one copy of the domain (Rubin et al., 2000);
for a graphical representation of the domain structure of the
~1000 proteins in the database containing SH3 domains,
browse the Pfam database [http://pfam.wustl.edu/] (Bateman et
al., 1999)). Because the small size of the module seemed to
preclude enzymatic activity, the search for function naturally
focused on potential protein interactions, and screening of
expression libraries using isolated SH3 domains soon
identified seemingly specific binding partners (Cicchetti et al.,
1992).
Early studies indicated that the region bound by SH3
domains is in all cases proline-rich and identified PxxP as a
core conserved binding motif (Ren et al., 1993). A host of
subsequent studies using alanine-scanning mutagenesis of
known binding sites, phage display, combinatorial chemistry
and high-resolution structure determination have revealed the
specifics of binding in great detail (recently reviewed by Kay
et al., 2000). What is clear is that the surface of the SH3 domain
bears a relatively flat, hydrophobic ligand-binding surface,
which consists of three shallow pockets or grooves defined by
conserved aromatic residues (Fig. 1). The ligand adopts an
extended, left-handed helical conformation termed the
polyproline-2 (or PPII) helix. The PPII helix has three residues
per turn; this means it is roughly triangular in cross-section,
and the base of this triangle sits on the surface of the SH3
domain. Two of the three ligand-binding pockets of the SH3
domain are occupied by two hydrophobic-proline (ΦP)
dipeptides in register on two adjacent turns of the helix,
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JOURNAL OF CELL SCIENCE 114 (7)
A
B
C
Class I ligand
RT
loop
P
X
P
X R
Cα
N
N
Cα
Cδ
Cβ
X
C
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n-Src
loop
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Class I
Class II ligand
RT
loop
X
X
X
X R
Cα
N
N
Cα
Cδ
P
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n-Src
loop
Class II
Cβ
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Fig. 1. Interaction of SH3 domains with their ligands. (A) Binding of class I (top) and class II (bottom) ligands to the surface of the SH3
domain is depicted diagrammatically. The specificity pocket that typically interacts with an arginine residue in the ligand is to the right, the two
Φ-P pockets to the left. The orientation of the ligand is indicated beneath each diagram. Approximate positions of the variable RT and N-Src
loops of the SH3 domain are indicated. (B) The Src SH3 domain bound to class I (top) and class II (bottom) ligands (adapted from Feng et al.,
1994). The surface of the SH3 domain was rendered with the program GRASP; ligands are represented in stick format. The overall view
corresponds to the diagram in A. (C) Schematic view of an X-P dipeptide of a class I (top) or class II (bottom) ligand on the surface of an SH3
domain (adapted from Nguyen et al., 1998). The view is down the axis of the PPII helix of the ligand from the perspective of the specificity
pocket of the SH3 domain. The surface of the SH3 domain is represented in green, and the portions of the X-P dipeptide that contact the surface
are represented in red. Note that the majority of the proline ring is not in contact with the surface of the SH3 domain and that it is the Cδ atom
of the proline ring (the atom directly bound to the main-chain nitrogen atom) that is in closest contact with the SH3 surface: N-substitution is
thus the main feature recognized by SH3 domains. Note that in either orientation the Cα atom of the proline is directed away from the SH3
surface and does not contribute to the binding energy.
whereas the third ‘specificity’ pocket in most cases interacts
with a basic residue in the ligand distal to the ΦPxΦP core.
Remarkably, different binding modules such as WW domains
and profilin have converged on very similar modes of
interaction with proline-rich, PPII-helical ligands (Kay et al.,
2000; Zarrinpar and Lim, 2000).
As discussed below, the affinity of SH3 domains for their
peptide ligands is quite low, and selectivity is generally
marginal at best; so it is reassuring that in a number of cases
there is solid evidence for specific biologically significant SH3ligand interactions. The first and most prominent example was
the role of Grb2 (or Sem-5 in C. elegans and DRK in
Drosophila) in activating Ras. Grb2 contains an SH2 domain
that binds to tyrosine-phosphorylated proteins, which is
flanked by two SH3 domains that bind to the proline-rich tail
of Sos (as well as other proteins). Sos is a guanine-nucleotideexchange factor (GEF) for the small GTPase Ras. Genetic and
biochemical evidence demonstrated that Grb2 recruits Sos to
SH3 domains: complexity in moderation
tyrosine-phosphorylated sites on the membrane, where Ras is
localized by virtue of its covalent lipid modification, and that
Grb2 thereby couples activation of Ras to changes in tyrosine
phosphorylation (McCormick, 1993). While this remains the
paradigm for SH3-mediated intermolecular interactions, there
are now several other examples for which there is solid
evidence, such as recruitment of the Pak kinase by the Dock
SH2-SH3 adaptor during retinal axon migration in Drosophila
(Hing et al., 1999).
The most prominent example of the importance of
intramolecular SH3-mediated interactions comes from Src
itself. Src and its relatives are normally held in an inactive
conformation, in which a C-terminal phosphotyrosine engages
in an intramolecular interaction with the SH2 domain of the
kinase. When the X-ray crystal structures of Src and its close
relative Hck were solved, the previously under-appreciated
SH3 domain took center stage. The SH3 domain was found to
bind to a linker region between the SH2 and catalytic domains
of the kinase, which adopted the typical PPII helical
conformation of an SH3 ligand despite the absence of sequence
similarity to known Src SH3-binding sites (Sicheri et al., 1997;
Williams et al., 1997; Xu et al., 1997). High-affinity SH3
domain ligands stimulate Hck activity in vitro (Moarefi et al.,
1997; Nguyen et al., 2000), demonstrating that the
intramolecular SH3-linker interaction seen in the crystal
structures is critical for holding the protein in its repressed
conformation. This example illustrates two points: first, that
biologically important SH3-mediated interactions involving
suboptimal, low-affinity ligands can occur if the local
concentration is high enough (e.g. when the two interacting
regions are found on the same molecule); and second, that SH3
domains can participate in complex regulatory interactions that
depend on multiple contacts - in the case of the Src family
kinases, a delicate conformational switch involving the SH2
domain, the SH3 domain and phosphorylation of at least two
regulatory sites.
Specificity
The target specificity of particular SH3 domains is an
important issue, because if we understand the rules governing
specificity we will be able to predict ligands for particular
proteins of interest and perhaps develop ways to inhibit specific
interactions in vivo. Because the PPII helix of an SH3 ligand
is quite similar in overall structure when viewed from its N- or
C-terminus, ligands can bind in either of two orientations: socalled class I ligands have the general consensus +xΦPxΦP
and class II ligands have the general consensus ΦPxΦPx+
(where x is any amino acid, and + is in most cases a basic
residue, usually arginine) (Feng et al., 1994; Lim et al., 1994;
Mayer and Eck, 1995). This means that the invariant proline
residues of the ΦP dipeptides occupy different positions on the
surface of the SH3 domain relative to the helix axis, depending
on the orientation of the ligand. In other words, if one were to
look down the axis of the PPII helix of the ligand from the
vantage point of the specificity pocket of the SH3 domain, the
two essential prolines would be on the right side in the case of
a class I ligand and on the left for a class II ligand (Fig. 1).
This apparent paradox is resolved by the observation that the
important determinant for binding to the two ΦP pockets
appears to be a continuous surface presented by the alkylated
1255
amide nitrogen of the proline residue, and the carbonyl, α
carbon and side chain of the preceding residue (Fig. 1c)
(Nguyen et al., 1998). An x-P dipeptide is the only possible
sequence of naturally occurring amino acids in which two
alkylated backbone atoms are separated by only a single
carbon.
Given that almost all of the binding energy is provided by
the two ΦP dipeptides and the basic residue, there wouldn’t
seem to be much room for selectivity among different SH3
domains. Despite this, several groups have used phage display
or combinatorial chemistry approaches to identify optimal
ligands for particular SH3 domains and in general have been
able to find consensus sequences showing some specificity for
a particular domain (Cestra et al., 1999; Cheadle et al., 1994;
Feng et al., 1994; Rickles et al., 1994; Sparks et al., 1994;
Sparks et al., 1996). For synthetic peptide ligands, KD values
for ‘specific’ ligands tend to be around 10 µM, although in a
few cases submicromolar affinities have been reported (Posern
et al., 1998). Such studies have served as the foundation for an
algorithm based on sequence comparison, contacts identified
in crystal structures, and phage-display binding data, which is
designed to predict specific binding sites for any SH3 domain
from its primary sequence (Brannetti et al., 2000). Until a much
more representative set of structural and binding data is
available, however, the predictive value of such programs is
likely to be modest.
Because the core ΦPxΦP scaffold (more commonly denoted
PxxP) restricts the variability that can be used to generate
specificity, recent efforts have addressed how selectivity can be
achieved both in nature and in the test tube. One obvious
potential source of specificity is the third specificity pocket of
the domain. Although most SH3 domains prefer an arginine
residue at this site, the Crk SH3 has high selectivity for class
II sites that instead contain a lysine residue (Wu et al., 1995).
The Abl SH3, which ironically was the first for which specific
targets were identified, is atypical in that it prefers class I
ligands in which hydrophic residues contact the third
specificity pocket; this is because it lacks conserved acidic
residues found in other SH3 domains, which make specific
contacts with the arginine residues of typical ligands (Ren et
al., 1993; Weng et al., 1995).
For some time, we have known that ligand residues outside
the core binding motif can also contribute specificity by
interacting with surfaces on the SH3 domain outside the PPII
binding groove, such as the highly variable RT and N-Src loops
(see Fig. 1) (Feng et al., 1995). For example, peptide-binding
sites for the second SH3 domain of the adaptor Nck have been
carefully mapped, showing a strong requirement for a serine
residue downstream of the core class II binding motif
(PxxPxRxxS); furthermore, phosphoserine, acidic and proline
residues are not tolerated in positions immediately C-terminal
to the core motif (Zhao et al., 2000).
The binding of the SH3 domain of the Src family kinase
Hck to the HIV Nef protein illustrates how regions of a ligand
protein distant from the PPII helix can also modulate
specificity. In this case, a single residue in the RT loop of the
Hck SH3 confers high-affinity binding to native Nef, but not
to the isolated PxxP-containing peptide (Lee et al., 1995).
After screening a library of mutant Hck SH3 domains (in
which six RT loop residues were randomized) by phage
display for binding to native Nef, Saksela and colleagues
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JOURNAL OF CELL SCIENCE 114 (7)
were able to isolate mutant domains that have up to 40-fold
higher affinity than the wild-type Hck SH3 (Hiipakka et al.,
1999). They also generated mutant SH3 domains that have
very high affinity and selectivity for a Nef variant bearing a
point mutation in the region predicted to contact the RT loop.
These results demonstrate that it is possible to generate SH3
domains that bind very selectively to a specific target protein.
This might in some cases allow the experimental blocking of
particular SH3-binding sites, while leaving other, closely
related sites untouched. This also raises the question of why
naturally occurring SH3-mediated interactions tend to be of
relatively low affinity - why has evolution not taken full
advantage of the potential affinity and selectivity available in
the system? There must be some evolutionary advantage to
maintaining relatively low affinity and selectivity for SH3mediated interactions.
A smattering of recent data indicate that SH3 domains can
in some circumstances bind to ligands lacking the canonical
PxxP core binding motif – for example, the Pix SH3-binding
site in Pak (PPPVIAPRPETKS) (Manser et al., 1998), the Eps8
SH3-binding consensus (PxxDY) (Mongiovi et al., 1999) and
the Hbp SH3-binding sites on UBPY (Px(V/I)(D/N)RxxKP)
(Kato et al., 2000). It can be argued that, for most of the phage
display and combinatorial library experiments published to
date, non-PxxP ligands could never have been detected,
because the original libraries were biased by fixing proline in
the two ‘invariant’ positions. However, enough SH3 ligands
have been detected by direct filter binding or yeast two-hybrid
approaches to suggest that, if non-PxxP ligands were common,
they would have been detected much more often by now. It is
probably safe to assume that for most SH3 domains the PxxP
core is required, with the caveat that exceptions always exist.
One fascinating recent development is the chemical
synthesis of novel SH3 domain ligands that have much greater
selectivity than do authentic peptide-binding sites found in
naturally ocurring proteins. Lim and colleagues proposed that
the requirement for proline in the two ΦP dipeptides of the
ΦPxΦP core might be due at least in part to the unique
structure of proline – it is the only N-substituted amino acid.
To test this idea they constructed synthetic peptoid ligands in
which one or both of the proline residues were replaced with
various non-natural N-substituted residues. Remarkably, not
only could some of these peptoid ligands bind to SH3 domains
with higher affinity than known natural (proline-containing)
ligands, but they also found ligands that had greater selectivity
than natural peptides (Nguyen et al., 2000; Nguyen et al.,
1998). For example, one compound, in which both prolines
were replaced with synthetic N-substituted residues, bound to
the Crk SH3 domain with high affinity in the absence of
detectable binding to either the Src or Grb2 SH3 domain
(Nguyen et al., 2000). This implies that SH3 domains do not
generally recognize the entire ring structure of proline, but
instead recognize only a small portion of the ring near the
backbone nitrogen (Fig. 1c). As a consequence there is a great
deal of structural variability among SH3 domains in the
vicinity of the ΦP pockets, which can be exploited if different
functional groups are appended to this nitrogen (by contrast,
the cell is limited in its ability to optimize binding by the fact
that proline is the only N-branched amino acid at its disposal).
The potential to inhibit specifically a particular SH3 domain
while sparing others has obvious potential both as a research
tool and as a source of lead compounds for pharmaceutical
development.
The larger specificity question - are we missing
something?
A central question that remains unresolved is this: do
meaningful biological outputs necessarily depend on specific,
pairwise SH3-ligand interactions, or is it more realistic to think
of these interactions in a probabilistic way - i.e. does a
particular SH3 domain or ligand site simultaneously engage in
multiple interactions in dynamic equilibrium? The conceptual
problem arises from the fact that there are many different SH3
domains and potential ligand sites present simultaneously in
the cell – certainly many tens and probably hundreds of each.
From what we know about peptide-SH3 interactions, affinities
for natural ligands and selectivity for a particular SH3-ligand
pair versus potential competing interactions are relatively low.
Ladbury and Arold have argued that there is insufficient
inherent specificity in most SH3-mediated interactions to
account for specific biological outputs without one having to
invoke additional mechanisms (Ladbury and Arold, 2000).
Two extreme resolutions of this apparent paradox exist:
effective selectivity could be increased in vivo by a variety of
means, such as compartmentalization of potential interaction
partners, additive effects of multiple separate interactions
between two partners, and the cooperative assembly of
multiprotein complexes; alternatively, selectivity might not be
strictly important in vivo - in this case, the central organizing
principle would not be the linear pathway but a vast and evershifting web of interactions, from which output is gauged by
global changes in complex binding equilibria.
Certainly, in at least some situations, selectivity is enhanced
by multiple interactions - for example, the combination of the
intramolecular interaction of the Src SH3 domain with its own
linker peptide and multiple additional interactions among the
SH2, SH3 and catalytic domains. Grb2 provides a different
type of example: its two SH3 domains are likely to engage
multiple PxxP sites in the C-terminus of Sos simultaneously
(Simon and Schreiber, 1995). Other proteins that have multiple
SH3 domains, such as the Nck adaptor, presumably use a
similar strategy to build selectivity through multiple, relatively
low-affinity interactions (Adler et al., 2000; Wunderlich et al.,
1999). A change in the subcellular localization of a domain or
ligand will certainly alter dramatically the repertoire of
proteins with which it can interact owing to changes in their
local concentration – this is the case for SH2-containing
proteins that relocalize to sites of tyrosine phosphorylation
during signaling. In this regard, the modest intrinsic affinity of
SH3 domains for their ligands means that off-rates are fast, and
binding partners can exchange relatively rapidly upon
relocalization. Such plasticity is almost certainly advantageous
in some contexts, allowing the rapid remodeling of interactions
in response to changes in the environment.
Despite such mechanisms, in many situations a particular
SH3 or ligand will inevitably interact simultaneously with
many different partners. Although we may not be comfortable
with pathways that cannot be described in a linear (A→B→C)
fashion, nonlinearity seems to be the logic of many biological
networks. A combinatorial, probabilistic set of interactions (A
interacts with partners U, V, W, X, Y and Z; X, in turn,
SH3 domains: complexity in moderation
interacts with B, C, D, E and F in addition to A; and so forth)
could potentially generate far more specific information than
unique interactions, because the number of possible
combinations of interactions is vastly greater than the actual
number of interacting proteins (Fig. 2). One can also easily
envision how the gradual process of evolution could weave and
shape such a web of overlapping interactions: if a protein gains
an SH3 domain (or proline-rich binding site) by exon shuffling,
it will then engage in many new interactions that subtly alter
the overall equilibrium of such interactions in the cell. There
is no need to invoke the simultaneous evolution of both a
unique binding domain and a unique ligand site for every
biologically important interaction. Perhaps an apt analogy can
be made to the webs of interactions among species: although
mice eat corn, and owls eat mice, the linear pathway of
owl→mouse→corn does not do justice to the facts that the owl
will eat many other prey besides mice given the opportunity,
that mice have good reason to fear many predators beside owls,
and that mice will happily eat many things in addition to corn.
An owl’s nest in the barn might create a specific output (less
corn eaten by mice), but it would certainly be misleading to
attempt to define the system solely in terms of the ‘specificity’
of owls for mice or mice for corn.
MAGUKs - controlling assembly of large complexes
The MAGUK (membrane-associated guanylate kinase)
family of scaffold proteins illustrates the potential role of
both intramolecular and intermolecular SH3-mediated
interactions in assembly of macromolecular complexes.
MAGUKs are a large family of related proteins consisting of
1-3 PDZ domains, an SH3 domain and a guanylate-kinaselike (GK) domain (Fig. 3a); they are thought to play a role in
assembly of large protein complexes at specialized regions of
the membrane such as synapses, neuromuscular junctions and
other cell-cell junctions (Dimitratos et al., 1999). The PDZ
domain, another protein-interaction module, typically binds
to sites at the extreme C-terminus of target proteins that have
the consensus Exx(V/I) (Songyang et al., 1997). The GK
region, although bearing all the sequence hallmarks of a
catalytically active domain, appears not to have enzymatic
activity but instead probably mediates protein-protein
interactions. In addition to the three highly conserved
domains, some members of the MAGUK family also contain
additional motifs, including a region with similarity to the
catalytic domain and calmodulin-binding site of calcium/
calmodulin-dependent protein kinase (CaM kinase), and a socalled HOOK domain that interacts with the band 4.1 protein
and its relatives (Dimitratos et al., 1999).
Given their generous complement of protein-interaction
domains, MAGUK proteins are well-equipped to mediate the
assembly of large, multimolecular protein complexes through
interactions with other proteins and self-assembly into
multimers. The PDZ domains interact with transmembrane or
membrane-associated proteins such as receptors and ion
channels; clustering of these proteins presumably facilitates
signaling by generating high local concentrations (Garner et
al., 2000). The role of the SH3 domains in scaffolding by
MAGUK proteins has until recently been elusive. It has long
been known that the SH3 is somehow important, because in
Drosophila, SH3 domain mutants of the MAGUK protein
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Discs Large (Dlg) are oncogenic, leading to epithelial
overgrowth and malignant transformation (Woods et al., 1996).
This observation, and the well-known role of SH3 domains as
protein interaction modules in other systems, led to a largely
unsuccessful hunt for ligands for the SH3 domains of various
MAGUK family members.
As it turns out, researchers did not have far to go for those
ligands - they are located in the same protein. Several groups
independently found in yeast two-hybrid experiments that the
SH3 domains of various MAGUKs, including PSD-95,
chapsyn-110, SAP97, DLG, CASK and p55, all interact with
their own GK regions (McGee and Bredt, 1999; Nix et al.
2000; Shin et al., 2000). Unsurprisingly, given the conservation
of the SH3 and GK domains among MAGUK proteins,
heterotypic interactions also occur: in some (but not all)
cases, the SH3 domain of one MAGUK can bind to the
GK domain of a second (as assayed by yeast two-hybrid,
coimmunoprecipitation and GST-pulldown assays). In all cases
examined, however, a MAGUK molecule that contains both an
SH3 and a GK domain cannot interact in trans with either the
SH3 or GK domains of a second MAGUK molecule in vitro;
this suggests that the intramolecular cis interaction is favored
under normal circumstances, which might be expected given
the high local concentration of the two domains on the same
molecule. Nix et al., have provided evidence for heterotypic
interaction in vivo, however, by co-immunoprecipitating
endogenous hCASK and hDLG from human colon cancer
cell lysates and colocalizing the two proteins by
immunofluorescence at cell junctions in these and other
epithelial cells (Nix et al., 2000). Of course, whether this
apparent complex depends on SH3-GK interactions in trans
remains to be proved, but, given that trans interaction of
isolated domains can be demonstrated in vitro, the full-length
proteins are likely to do so under some circumstances in vivo.
Interestingly, not all MAGUK SH3-GK interactions are
allowed - there is some specificity in which heterotypic
interactions can occur. For example, the hDLG GK binds to its
own SH3 domain, but not to those of hCASK or p55, whereas
the GK domains of hCASK and p55 bind equally well to SH3
domains of all three proteins (Nix et al., 2000); similarly, the
SH3 domain of SAP 97 binds to the GK domains of all the
closely related MAGUKs in the PSD-95 family but not to
that of the more distantly related ZO-1 (Shin et al., 2000).
Therefore, although all MAGUKs appear to be capable of
intramolecular (monomer) as well as homotypic (dimer or
higher-order) interactions, only some heterotypic interactions
will be favored. In some cases, heterotypic interactions are
allowed only in an asymmetric fashion: for example, hDLGhCASK heterodimers can form through interactions between
the hDlg SH3 and the hCASK GK, but since the hCASK SH3
and the hDlg GK cannot bind to each other they are presumably
free to interact with other proteins (Nix et al., 2000).
One potential role for SH3-GK interactions might be to
orient individual MAGUKs with respect to other proteins in
large, polymeric complexes. Individual MAGUKs, which can
dock onto larger, assembling complexes through other proteininteraction domains, such as the PDZ domains, would
effectively snap into place as the intramolecular SH3-GK
interaction is out-competed by trans interactions once it has
assumed the correct spatial orientation for proper assembly
(Fig. 3b). The assembled complex would be stable despite the
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A
B
input
F
E
D
V
U
X
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U
Z
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W
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Y
X
output
output
relatively low affinity of the individual SH3-GK interactions
owing to the additive effect of many simultaneous interactions.
The fact that Sheng and colleagues have identified a PSD-95
SH3 domain mutant that cannot cluster the Kv1.4 potassium
channel, and yet is targeted to the membrane and binds to the
C
B
A
W
Y
F
E
D
C
B
A
Fig. 2. Linear signaling vs complex networks. A group
of six proteins containing SH3 domains (labeled A
through F) and six SH3 ligands (labeled U through Z)
are represented as spheres. (A) Linear signaling. Each
SH3 domain binds specifically to one of the six ligands,
and there is minimal cross-reactivity. An input signal
(such as relocalization of protein A) will affect only its
specific ligand (protein X). (B) Complex networks. Each
SH3 domains binds to all six ligands, and there is
roughly comparable affinity. An input signal affecting
protein A will affect all six of the ligands. Furthermore,
any significant change in the binding activity or local
concentration of any one component will shift the
binding equilibria of all twelve components.
Fig. 3. Protein interactions in MAGUK family
members. (A) A prototypical MAGUK protein is
depicted, the positions of PDZ, SH3 and GK
domains being indicated schematically. PDZ
domains interact with a variety of ligands, including
receptors and ion channels. SH3 domains can bind in
cis to the GK domain of the same molecule or in
trans to GK domains of other molecules or to other
proline-rich ligands. GK domains can similarly
interact in cis or in trans with SH3 domains or with
other ligands. (B) Hypothetical mechanism for
assembly of MAGUK-mediated complexes on the
membrane. 1. SH3 and GK domains interact in cis,
preventing interaction with other proteins; PDZ
domains interact with various ligands, at least one of
which recruits the complex to the membrane in
proximity to other MAGUK complexes. 2. MAGUK
interacts with other MAGUKs on the membrane,
eventually attaining an orientation in which its SH3
domain is in close proximity to a free GK domain on
a neighboring MAGUK molecule. 3. Cis interaction
is competed out by trans SH3-GK interaction, which
snaps the new MAGUK subunit into place in the
appropriate position in the growing complex.
Although a homotypic complex is depicted here,
heterotypic complexes between different MAGUK
proteins are also possible, although only a subset of
GK-SH3 interactions are permitted in trans.
Interactions between PDZ domain ligands, and
between other regions of the MAGUK proteins
themselves, probably contribute to the assembly of
the complex.
input
channel normally, is consistent with this idea (Shin et al.,
2000).
There are some interesting twists to the SH3-GK interaction
that highlight the dangers of overgeneralizing SH3 domain
properties. One outstanding issue concerns the actual binding
Receptors, channels,
other ligands
PDZ
PDZ
Other ligands?
GK
SH3
PDZ
GK domains
SH3 domains
B
Outside
1
2
3
Cytosol
SH3 domains: complexity in moderation
sites for the SH3s, which have yet to be mapped precisely. In
the case of PSD-95, no canonical PxxP site is present in the Cterminal GK region. Although three proline residues do lie in
potential degenerate class II sites (PxR), changing those
residues to alanine individually or in combination did not
eliminate binding (Shin et al., 2000). Furthermore, the GK
region required for SH3 binding extends beyond the borders of
the actual GK domain (defined by sequence similarity) both Nand C-terminally, which suggests that a larger folded structure
is recognized by the SH3 domain (McGee and Bredt, 1999;
Shin et al., 2000). MAGUK family SH3 domains differ from
the typical SH3 consensus at several highly conserved sites in
the ligand-binding regions (see: http://pfam.wustl.edu/cgibin/getdesc?name=SH3); their ligands might therefore also
differ from the canonical SH3-binding consensus. Mutation in
the PSD-95 SH3 of the invariant tryptophan (which defines the
ridge between the second ΦP pocket and the specificity pocket;
Mayer and Eck, 1995) apparently does not eliminate the
intramolecular SH3-GK interaction, although it does eliminate
binding to GK domains in trans. This tryptophan is generally
assumed to be essential for binding to canonical PxxP ligands,
and so the milder effect in the case of PSD-95 is again
consistent with the idea that its ligand is atypical.
Finally, the effect of the intra- or inter-molecular SH3-GK
1259
interaction on other interactions must be considered. The
kainate receptor is the only MAGUK SH3 ligand (other than
GK domains) identified to date (Garcia et al., 1998), but it is
possible that others exist. Their binding would depend on the
disruption of intramolecular SH3-GK interactions to expose
the SH3 domain and would necessarily expose the GK domain
for interaction with other SH3 domains in trans. Proteins that
lack SH3 domains also bind to the GK domain, although in the
case of PSD-95 and its relatives binding of one of these, GKAP,
to the GK domain is not affected by the intramolecular SH3GK interaction (Shin et al., 2000). Although this demonstrates
that two independent binding sites are present in the GK
domain, the effects of SH3 binding on other GK-domainbinding partners have yet to be examined. The possibility that
a novel protein-binding interface is created by interaction of
the SH3 and GK domains also needs to be explored. Clearly,
the permutations of inter- and intra-molecular interactions that
can and do occur, and their effects on macromolecular
assembly and interactions with other partners, are complex,
and additional experiments will be required to sort these out.
WASP and friends − controlling actin
A second emerging story in which SH3 domains are implicated
GTPCdc42
PtdIns
(4,5)P2
++
EVH1
WASP
Arp2/3 -- nucleate
actin filaments
PBD
Proline-rich
VC A
G-actin
SH3
Nck
SH3
3
WIP/
verprolin
SH3
Tyrosinephosphorylated
proteins
SH2
2
4
V
V
Proline-rich
bundling?
5
Myosin 1
Myosin motor domain
Arp2/3 -- nucleate
actin filaments
SH3
A
P
Pak
PBD
Kinase domain
GTPCdc42
Potential roles of SH3-mediated interactions in regulation of WASP activity:
1. help recruit WASP to sites of tyrosine phosphorylation; directly activate WASP-Arp2/3
complex in absence of GTP-Cdc42
2. recruit myosin motors and additional Arp2/3-binding sites to sites of actin nucleation
3. recruit Pak kinase to sites of actin nucleation--activate myosin
4. indirectly recruit WASP to Nck (sites of tyrosine phosphorylation)
5. couple myosin to actin-bundling activity; indirectly recruit myosin motors and additional
Arp2/3-binding sites to WASP
Fig. 4. Protein interactions implicated in nucleation of actin filaments by WASP family members. Wasp, Nck, WIP/verprolin, class 1 myosin and
Pak are depicted approximately to scale; important domains and regions are shown as labeled boxes. Interactions implicated in regulation are
indicated by arrows; SH3-mediated interactions are depicted in red. Sites of interaction indicated by arrows are intended to be approximate only.
1260
JOURNAL OF CELL SCIENCE 114 (7)
is the regulation of actin dynamics by members of the WASP
family of proteins. WASP was originally isolated as the gene
mutated in Wiskott-Aldrich Syndrome, a severe X-linked
immunodeficiency (Derry et al., 1994). WASP and its relatives
play a pivotal role in organizing the actin cytoskeleton, a
process that must be exquisitely controlled in time and space
to allow directed movement (Higgs and Pollard, 1999; Mullins,
2000; Ramesh et al., 1999; Takenawa and Miki, 2001;
Zigmond, 2000). Both WASP and its more ubiquitously
expressed relative N-WASP contain several modular domains,
including an EVH1 domain, a PtdIns(4,5)P2-binding site, a
region that binds the Rho-family GTPase Cdc42, an extensive
proline-rich region, and a C-terminal VCA (for verprolincofilin-acidic) region involved in regulating actin
polymerization. The EVH1 domain engages proline-rich
ligands, but the mode of recognition differs from that of SH3
or WW domains (Prehoda et al., 1999). The Cdc42-binding
region binds to and inhibits the VCA region when Cdc42 is not
bound; thus this region is a conditional switch that responds to
changes in GTP-Cdc42 levels (Abdul-Manan et al., 1999; Kim
et al., 2000; Miki et al., 1998). The VCA region interacts with
the Arp2/3 complex, which nucleates de novo actin filament
formation and induces filament branching (Blanchoin et al.,
2000; Mullins et al., 1998; Pantaloni et al., 2000), and the
filament-nucleating activity of the Arp2/3 complex is greatly
increased when bound to the VCA region (Machesky et al.,
1999; Rohatgi et al., 1999; Winter et al., 1999; Yarar et al.,
1999). Thus Cdc42 can activate Arp2/3-mediated actin
polymerization by disrupting the intramolecular interaction
between the PBD and VCA regions (reviewed by Fawcett and
Pawson, 2000).
Where do SH3 domains fit into this picture? The long
proline-rich region found in all WASP family members is an
obvious potential SH3-binding site, and there is no shortage of
suspects for ligands. The Nck SH2/SH3 adaptor, for example,
is localized on the surface of vaccinia virus particles and plays
a role in recruiting N-WASP and the Arp2/3 complex, thereby
serving to nucleate the formation of actin ‘comet tails’ that
propel the virus in the cytosol (Frischknecht et al., 1999;
Moreau et al., 2000). Because Nck has an SH2 domain, it could
recruit and cluster WASP family members at sites of tyrosine
phosphorylation. Indeed, a phosphotyrosine-containing site
whose phosphorylation is necessary for actin tail formation is
present on the vaccinia surface and can bind to the Nck SH2
domain (Frischknecht et al., 1999). In T cells Nck helps to
mediate the phosphotyrosine-dependent assembly of a
complex containing WASP and Vav, a GEF for Rho family
GTPases, and dominant-negative experiments suggest that
disruption of this complex inhibits localized actin
polymerization following T cell receptor stimulation (Bubeck
Wardenburg et al., 1998). Furthermore, a close relative of Nck,
Nckβ, is implicated in actin rearrangements downstream of
PDGF receptor stimulation (Chen et al., 2000).
Other potential partners for WASP family members are the
unconventional SH3-containing myosins. In yeast the WASP
relative Bee1p/Las17p is essential for proper formation and
localization of cortical actin patches and for endocytosis (Li,
1997; Naqvi et al., 1998). Loss of both type I myosins Myo3p
and Myo5p, or mutation of their SH3 domains, leads to severe
defects in the actin cytoskeleton (Anderson et al., 1998;
Evangelista et al., 2000; Geli et al., 2000; Goodson et al.,
1996). As it turns out, the SH3 domains of Myo3p and Myo5p
bind the proline-rich region of Bee1p (Evangelista et al., 2000;
Lechler et al., 2000). Surprisingly, Myo3p and Myo5p can also
bind directly through their acidic tails to the Arp2/3 complex,
which suggests that, in a Bee1p-Myo3p or Bee1p-Myo5p
complex, there is redundancy in Arp2/3 binding (Evangelista
et al., 2000; Lechler et al., 2000). Myosin motor activity is
important for cortical actin structures (Lechler et al., 2000),
perhaps to transport the complex to the barbed ends of actin
filaments or to drive movement of actin filaments relative to
the membrane (Machesky, 2000).
Phosphorylation of type 1 myosin at a specific site,
presumably by the Pak family kinases Ste20p and Cla4p, is
essential for Cdc42-stimulated actin assembly in yeast (Lechler
et al., 2000), and phosphorylation of amoeboid type 1 myosin
by Pak-like kinases is essential for its activity (Brzeska and
Korn, 1996; Novak and Titus, 1998). Remarkably, in
mammalian cells Nck binds via its SH3 domains to Pak kinases
(Bokoch et al., 1996; Galisteo et al., 1996; Lu et al., 1997),
which suggests a mechanism whereby Nck might modulate the
activity of unconventional myosins associated with WASPArp2/3 complexes. In Drosophila the Nck homolog Dock is
important for the directed migration of retinal axons, probably
regulating localized actin polymerization in the growth cone in
response to environmental cues (Garrity et al., 1996), and Pak
has been shown to function downstream of Dock in this system
(Hing et al., 1999).
The SH3 domains of Myo3p and Myo5p have also been
shown to bind another proline-rich protein, verprolin, which is
also implicated in organization of the actin cytoskeleton
(Anderson et al., 1998; Evangelista et al., 2000; Geli et al.,
2000). The mammalian homolog of verprolin, WIP, binds
directly and with high affinity to the N-terminal EVH1 domain
of WASP (Ramesh et al., 1997), and this interaction is
conserved in yeast (Naqvi et al., 1998); such a verprolin-Bee1p
complex would thus contain multiple binding sites for type 1
myosin SH3 domains. Furthermore, WIP is required for Nck
to recruit N-WASP to vaccinia virus particles, which suggests
that binding of the Nck SH3 domains to N-WASP is too weak
to be stable without the additional contacts provided by NckWIP and WIP-N-WASP interactions (Anton et al., 1998;
Moreau et al., 2000). Finally, WIP might also function to
bundle actin filaments together (N. Ramesh and R. Geha,
personal communication), thereby modulating the higher-order
structure of filaments initiated by the Arp2/3 complex.
SH3 domains might also directly activate complexes
containing Arp 2/3 and WASP family members. Carlier and
colleagues have shown that SH3 domains from the Grb2
adaptor enhance the activity of the N-WASP−Arp2/3 complex
in vitro (Carlier et al., 2000). We find that Nck SH3 domains
are more effective than those of Grb2, and multiple Nck SH3
domains are required for optimal activation (R. Rohatgi, B. J.
Mayer, and M. Kirschner, unpublished). This requirement for
multiple SH3 domains raises the possibility that simultaneous
engagement of two or three SH3-binding sites on N-WASP can
fix it in an open, activated conformation in which its VCA
region is exposed. In these in vitro assays GTP-Cdc42 and Nck
seem to play redundant roles in the activation of N-WASP, in
both cases synergizing with PtdIns(4,5)P2 (R. Rohatgi, B. J.
Mayer, and M. Kirschner, unpublished). A Cdc42-independent
mode to nucleate actin polymerization in the cell through high
SH3 domains: complexity in moderation
local concentrations of Nck (or other SH3-containing proteins)
might therefore exist. This indeed seems to be the case for
vaccinia actin tail formation, which appears to be independent
of GTP-Cdc42 (Moreau et al., 2000).
What emerges from these studies is a picture in which a vital
function, nucleation of new actin filaments at the appropriate
time and place in the cell, is controlled by a multicomponent
complex of proteins assembled through SH3-mediated and
other protein-protein interactions (Fig. 4). The individual
components of the complex provide multiple functional outputs
(filament nucleation, filament bundling and transport along
filaments), multiple levels of control (by guanine nucleotide
exchange, phosphorylation and local concentration), and
functional redundancy. Of course many other functions can be
envisioned. The central role played by SH3 domains promotes
flexibility in possible interactions, because their relatively low
affinity and modest specificity allows for the assembly of large
but ‘informal’ complexes that can also disassemble rapidly in
response to changes in the environment.
Perspectives
The WASP-mediated actin nucleation system is likely to be an
excellent paradigm for the role of the ubiquitous SH3 domain
in many other complex cellular processes. They will play
varied and important roles wherever it is advantageous to bring
together proteins with moderate specificity and affinity.
Proteomics and genomics will soon provide us with the entire
compendium of interactions that can occur among all SH3containing proteins and their ligands. The overriding challenge
will then be to tease out specific functional effects from this
‘probability cloud’ of potential interactions. Recent progress in
constructing mutant SH3 domains that have very high
selectivities for specific targets, or synthetic ligands that have
high selectivities for specific SH3 domains, might allow us to
inhibit discrete subsets of interactions without disturbing the
entire equilibrium (although one suspects it will be difficult to
perturb any aspect of the system significantly without some
global effects). New approaches designed to direct the specific,
pairwise association of proteins in the absence of other
competing interactions might allow the consequences of any
particular protein-protein interaction to be assessed. Advances
in imaging techniques such as fluorescence resonance energy
transfer (FRET) should allow us to visualize SH3-mediated
interactions as they occur in the living cell. Furthermore,
synthetic peptoid compounds that have high affinities and
selectivities might eventually fulfil the longstanding dream of
intervening in SH3-mediated interactions for the treatment of
human disease. We have come a long way in the past ten years,
but our progress has brought a more mature appreciation of just
how much there is left to do.
I thank T. Kirchhausen, A. Musacchio, N. Ramesh, R. Rohatgi, and
K. Saksela for helpful comments on this manuscript, and N. Ramesh
for communicating results before publication. I am especially grateful
to A. Musacchio for kindly providing Figs 1a and 1b. Work in B. J.
M.’s laboratory was supported by National Institutes of Health grant
CA82258.
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