COMMENTARY 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 06030- 3301, 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 adaptable means of bringing together. The wealth of the growing family of -interaction modules. By of genetic, biochemical and structural information binding with moderate affinity and selectivity to proline- available provides an intimate and detailed portrait of the rich ligands, these domains play critical roles in a wide domain, serving as a framework for understanding other variety of biological processes ranging from regulation of modular protein-interaction domains. Processes regulated enzymes by intramolecular interactions, increasing the by SH3 domains also raise important questions about the local concentration or altering the subcellular localization nature of specificity and the overall logic governing of components of signaling pathways, and mediating the networks of protein interactions. 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 Key words: Protein-protein interaction, Modular binding domains, that they provide the cell with an especially handy and Signal transduction

Introduction tyrosine kinases, the Crk adaptor protein, and phospholipase The idea that specific protein-protein interactions govern many C-γ (Mayer et al., 1988; Stahl et al., 1988). It was soon biological processes is by now so deeply ingrained that it is apparent that this ~60-residue region of similarity is present in easy to forget that it has only been a decade or so since this many proteins (at this point data from genomic sequencing was widely appreciated. In that time the Src homology 3 (SH3) projects suggest that 63 proteins in Drosophila, 55 in domain has become a standard-bearer for the protein Caenorhabditis elegans, and 25 in Saccharomyces cerevisiae interaction field, largely because it was one of the first modular contain at least one copy of the domain (Rubin et al., 2000); protein interaction domains identified, is one of the most for a graphical representation of the domain structure of the common and has served as a proving ground for new methods ~1000 proteins in the database containing SH3 domains, aimed at identifying partners or creating novel ligands through browse the database [http://pfam.wustl.edu/] (Bateman et chemical synthesis. Indeed, given the unrelenting attention to al., 1999)). Because the small size of the module seemed to such a modest domain, it seems hard to imagine that there preclude enzymatic activity, the search for function naturally could be much left to uncover. I will argue that, to the contrary, focused on potential protein interactions, and screening of SH3 domains actually serve to remind us just how much expression libraries using isolated SH3 domains soon remains to be learned. In fact their very omnipresence is one identified seemingly specific binding partners (Cicchetti et al., source of conceptual trouble - how can the cell reap meaningful 1992). information from networks based on such a common module, Early studies indicated that the region bound by SH3 whose affinity and selectivity is modest at best? Because SH3 domains is in all cases proline-rich and identified PxxP as a domains play a role in so many of the processes of interest to core conserved binding motif (Ren et al., 1993). A host of the cell biology and biochemical communities, it is without subsequent studies using alanine-scanning mutagenesis of doubt important that we get a handle on what they can and known binding sites, phage display, combinatorial chemistry cannot do, how we can use their properties to our advantage and high-resolution structure determination have revealed the and how we can disrupt them in vivo. Here, I very briefly specifics of binding in great detail (recently reviewed by Kay sketch out the general properties of SH3 domains, spend et al., 2000). What is clear is that the surface of the SH3 domain considerable time on recent advances in understanding the bears a relatively flat, hydrophobic ligand-binding surface, determinants of binding specificity (or lack of specificity) and which consists of three shallow pockets or grooves defined by its significance, and go on to discuss two specific recent conserved aromatic residues (Fig. 1). The ligand adopts an examples in which these domains appear to play complex and extended, left-handed helical conformation termed the varied roles in important biological processes. 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 A brief history domain. Two of the three ligand-binding pockets of the SH3 SH3 domains were first noted as regions of sequence similarity domain are occupied by two hydrophobic-proline (ΦP) between divergent signaling proteins such as the Src family of dipeptides in register on two adjacent turns of the helix, 1254 JOURNAL OF CELL SCIENCE 114 (7) A B C RT Class I ligand loop P P R Cα R N X X N Cα

δ Cβ C X X n-Src loop C Class I N

Class II ligand RT loop Cα X X R N X X R N Cα

Cδ Cβ P P n-Src loop N Class II C

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 there is solid evidence for specific biologically significant SH3- with a basic residue in the ligand distal to the ΦPxΦP core. ligand interactions. The first and most prominent example was Remarkably, different binding modules such as WW domains the role of Grb2 (or Sem-5 in C. elegans and DRK in and profilin have converged on very similar modes of Drosophila) in activating Ras. Grb2 contains an SH2 domain interaction with proline-rich, PPII-helical ligands (Kay et al., that binds to tyrosine-phosphorylated proteins, which is 2000; Zarrinpar and Lim, 2000). flanked by two SH3 domains that bind to the proline-rich tail As discussed below, the affinity of SH3 domains for their of Sos (as well as other proteins). Sos is a guanine-nucleotide- peptide ligands is quite low, and selectivity is generally exchange factor (GEF) for the small GTPase Ras. Genetic and marginal at best; so it is reassuring that in a number of cases biochemical evidence demonstrated that Grb2 recruits Sos to SH3 domains: complexity in moderation 1255 tyrosine-phosphorylated sites on the membrane, where Ras is amide nitrogen of the proline residue, and the carbonyl, α localized by virtue of its covalent lipid modification, and that carbon and side chain of the preceding residue (Fig. 1c) Grb2 thereby couples activation of Ras to changes in tyrosine (Nguyen et al., 1998). An x-P dipeptide is the only possible phosphorylation (McCormick, 1993). While this remains the sequence of naturally occurring amino acids in which two paradigm for SH3-mediated intermolecular interactions, there alkylated backbone atoms are separated by only a single are now several other examples for which there is solid carbon. evidence, such as recruitment of the Pak kinase by the Dock Given that almost all of the binding energy is provided by SH2-SH3 adaptor during retinal axon migration in Drosophila the two ΦP dipeptides and the basic residue, there wouldn’t (Hing et al., 1999). seem to be much room for selectivity among different SH3 The most prominent example of the importance of domains. Despite this, several groups have used phage display intramolecular SH3-mediated interactions comes from Src or combinatorial chemistry approaches to identify optimal itself. Src and its relatives are normally held in an inactive ligands for particular SH3 domains and in general have been conformation, in which a C-terminal phosphotyrosine engages able to find consensus sequences showing some specificity for in an intramolecular interaction with the SH2 domain of the a particular domain (Cestra et al., 1999; Cheadle et al., 1994; kinase. When the X-ray crystal structures of Src and its close Feng et al., 1994; Rickles et al., 1994; Sparks et al., 1994; relative Hck were solved, the previously under-appreciated Sparks et al., 1996). For synthetic peptide ligands, KD values SH3 domain took center stage. The SH3 domain was found to for ‘specific’ ligands tend to be around 10 µM, although in a bind to a linker region between the SH2 and catalytic domains few cases submicromolar affinities have been reported (Posern of the kinase, which adopted the typical PPII helical et al., 1998). Such studies have served as the foundation for an conformation of an SH3 ligand despite the absence of sequence algorithm based on sequence comparison, contacts identified similarity to known Src SH3-binding sites (Sicheri et al., 1997; in crystal structures, and phage-display binding data, which is Williams et al., 1997; Xu et al., 1997). High-affinity SH3 designed to predict specific binding sites for any SH3 domain domain ligands stimulate Hck activity in vitro (Moarefi et al., from its primary sequence (Brannetti et al., 2000). Until a much 1997; Nguyen et al., 2000), demonstrating that the more representative set of structural and binding data is intramolecular SH3-linker interaction seen in the crystal available, however, the predictive value of such programs is structures is critical for holding the protein in its repressed likely to be modest. conformation. This example illustrates two points: first, that Because the core ΦPxΦP scaffold (more commonly denoted biologically important SH3-mediated interactions involving PxxP) restricts the variability that can be used to generate suboptimal, low-affinity ligands can occur if the local specificity, recent efforts have addressed how selectivity can be concentration is high enough (e.g. when the two interacting achieved both in nature and in the test tube. One obvious regions are found on the same molecule); and second, that SH3 potential source of specificity is the third specificity pocket of domains can participate in complex regulatory interactions that the domain. Although most SH3 domains prefer an arginine depend on multiple contacts - in the case of the Src family residue at this site, the Crk SH3 has high selectivity for class kinases, a delicate conformational switch involving the SH2 II sites that instead contain a lysine residue (Wu et al., 1995). domain, the SH3 domain and phosphorylation of at least two The Abl SH3, which ironically was the first for which specific regulatory sites. 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 Specificity residues found in other SH3 domains, which make specific The target specificity of particular SH3 domains is an contacts with the arginine residues of typical ligands (Ren et important issue, because if we understand the rules governing al., 1993; Weng et al., 1995). specificity we will be able to predict ligands for particular For some time, we have known that ligand residues outside proteins of interest and perhaps develop ways to inhibit specific the core binding motif can also contribute specificity by interactions in vivo. Because the PPII helix of an SH3 ligand interacting with surfaces on the SH3 domain outside the PPII is quite similar in overall structure when viewed from its N- or binding groove, such as the highly variable RT and N-Src loops C-terminus, ligands can bind in either of two orientations: so- (see Fig. 1) (Feng et al., 1995). For example, peptide-binding called class I ligands have the general consensus +xΦPxΦP sites for the second SH3 domain of the adaptor Nck have been and class II ligands have the general consensus ΦPxΦPx+ carefully mapped, showing a strong requirement for a serine (where x is any , and + is in most cases a basic residue downstream of the core class II binding motif residue, usually arginine) (Feng et al., 1994; Lim et al., 1994; (PxxPxRxxS); furthermore, phosphoserine, acidic and proline Mayer and Eck, 1995). This means that the invariant proline residues are not tolerated in positions immediately C-terminal residues of the ΦP dipeptides occupy different positions on the to the core motif (Zhao et al., 2000). surface of the SH3 domain relative to the helix axis, depending The binding of the SH3 domain of the on the orientation of the ligand. In other words, if one were to Hck to the HIV Nef protein illustrates how regions of a ligand look down the axis of the PPII helix of the ligand from the protein distant from the PPII helix can also modulate vantage point of the specificity pocket of the SH3 domain, the specificity. In this case, a single residue in the RT loop of the two essential prolines would be on the right side in the case of Hck SH3 confers high-affinity binding to native Nef, but not a class I ligand and on the left for a class II ligand (Fig. 1). to the isolated PxxP-containing peptide (Lee et al., 1995). This apparent paradox is resolved by the observation that the After screening a library of mutant Hck SH3 domains (in important determinant for binding to the two ΦP pockets which six RT loop residues were randomized) by phage appears to be a continuous surface presented by the alkylated display for binding to native Nef, Saksela and colleagues 1256 JOURNAL OF CELL SCIENCE 114 (7) were able to isolate mutant domains that have up to 40-fold tool and as a source of lead compounds for pharmaceutical higher affinity than the wild-type Hck SH3 (Hiipakka et al., development. 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. The larger specificity question - are we missing These results demonstrate that it is possible to generate SH3 something? domains that bind very selectively to a specific target protein. A central question that remains unresolved is this: do This might in some cases allow the experimental blocking of meaningful biological outputs necessarily depend on specific, particular SH3-binding sites, while leaving other, closely pairwise SH3-ligand interactions, or is it more realistic to think related sites untouched. This also raises the question of why of these interactions in a probabilistic way - i.e. does a naturally occurring SH3-mediated interactions tend to be of particular SH3 domain or ligand site simultaneously engage in relatively low affinity - why has evolution not taken full multiple interactions in dynamic equilibrium? The conceptual advantage of the potential affinity and selectivity available in problem arises from the fact that there are many different SH3 the system? There must be some evolutionary advantage to domains and potential ligand sites present simultaneously in maintaining relatively low affinity and selectivity for SH3- the cell – certainly many tens and probably hundreds of each. mediated interactions. From what we know about peptide-SH3 interactions, affinities A smattering of recent data indicate that SH3 domains can for natural ligands and selectivity for a particular SH3-ligand in some circumstances bind to ligands lacking the canonical pair versus potential competing interactions are relatively low. PxxP core binding motif – for example, the Pix SH3-binding Ladbury and Arold have argued that there is insufficient site in Pak (PPPVIAPRPETKS) (Manser et al., 1998), the Eps8 inherent specificity in most SH3-mediated interactions to SH3-binding consensus (PxxDY) (Mongiovi et al., 1999) and account for specific biological outputs without one having to the Hbp SH3-binding sites on UBPY (Px(V/I)(D/N)RxxKP) invoke additional mechanisms (Ladbury and Arold, 2000). (Kato et al., 2000). It can be argued that, for most of the phage Two extreme resolutions of this apparent paradox exist: display and combinatorial library experiments published to effective selectivity could be increased in vivo by a variety of date, non-PxxP ligands could never have been detected, means, such as compartmentalization of potential interaction because the original libraries were biased by fixing proline in partners, additive effects of multiple separate interactions the two ‘invariant’ positions. However, enough SH3 ligands between two partners, and the cooperative assembly of have been detected by direct filter binding or yeast two-hybrid multiprotein complexes; alternatively, selectivity might not be approaches to suggest that, if non-PxxP ligands were common, strictly important in vivo - in this case, the central organizing they would have been detected much more often by now. It is principle would not be the linear pathway but a vast and ever- probably safe to assume that for most SH3 domains the PxxP shifting web of interactions, from which output is gauged by core is required, with the caveat that exceptions always exist. global changes in complex binding equilibria. One fascinating recent development is the chemical Certainly, in at least some situations, selectivity is enhanced synthesis of novel SH3 domain ligands that have much greater by multiple interactions - for example, the combination of the selectivity than do authentic peptide-binding sites found in intramolecular interaction of the Src SH3 domain with its own naturally ocurring proteins. Lim and colleagues proposed that linker peptide and multiple additional interactions among the the requirement for proline in the two ΦP dipeptides of the SH2, SH3 and catalytic domains. Grb2 provides a different ΦPxΦP core might be due at least in part to the unique type of example: its two SH3 domains are likely to engage structure of proline – it is the only N-substituted amino acid. multiple PxxP sites in the C-terminus of Sos simultaneously To test this idea they constructed synthetic peptoid ligands in (Simon and Schreiber, 1995). Other proteins that have multiple which one or both of the proline residues were replaced with SH3 domains, such as the Nck adaptor, presumably use a various non-natural N-substituted residues. Remarkably, not similar strategy to build selectivity through multiple, relatively only could some of these peptoid ligands bind to SH3 domains low-affinity interactions (Adler et al., 2000; Wunderlich et al., with higher affinity than known natural (proline-containing) 1999). A change in the subcellular localization of a domain or ligands, but they also found ligands that had greater selectivity ligand will certainly alter dramatically the repertoire of than natural peptides (Nguyen et al., 2000; Nguyen et al., proteins with which it can interact owing to changes in their 1998). For example, one compound, in which both prolines local concentration – this is the case for SH2-containing were replaced with synthetic N-substituted residues, bound to proteins that relocalize to sites of tyrosine phosphorylation the Crk SH3 domain with high affinity in the absence of during signaling. In this regard, the modest intrinsic affinity of detectable binding to either the Src or Grb2 SH3 domain SH3 domains for their ligands means that off-rates are fast, and (Nguyen et al., 2000). This implies that SH3 domains do not binding partners can exchange relatively rapidly upon generally recognize the entire ring structure of proline, but relocalization. Such plasticity is almost certainly advantageous instead recognize only a small portion of the ring near the in some contexts, allowing the rapid remodeling of interactions backbone nitrogen (Fig. 1c). As a consequence there is a great in response to changes in the environment. deal of structural variability among SH3 domains in the Despite such mechanisms, in many situations a particular vicinity of the ΦP pockets, which can be exploited if different SH3 or ligand will inevitably interact simultaneously with functional groups are appended to this nitrogen (by contrast, many different partners. Although we may not be comfortable the cell is limited in its ability to optimize binding by the fact with pathways that cannot be described in a linear (A→B→C) that proline is the only N-branched amino acid at its disposal). fashion, nonlinearity seems to be the logic of many biological The potential to inhibit specifically a particular SH3 domain networks. A combinatorial, probabilistic set of interactions (A while sparing others has obvious potential both as a research interacts with partners U, V, W, X, Y and Z; X, in turn, SH3 domains: complexity in moderation 1257 interacts with B, C, D, E and F in addition to A; and so forth) Discs Large (Dlg) are oncogenic, leading to epithelial could potentially generate far more specific information than overgrowth and malignant transformation (Woods et al., 1996). unique interactions, because the number of possible This observation, and the well-known role of SH3 domains as combinations of interactions is vastly greater than the actual protein interaction modules in other systems, led to a largely number of interacting proteins (Fig. 2). One can also easily unsuccessful hunt for ligands for the SH3 domains of various envision how the gradual process of evolution could weave and MAGUK family members. shape such a web of overlapping interactions: if a protein gains As it turns out, researchers did not have far to go for those an SH3 domain (or proline-rich binding site) by exon shuffling, ligands - they are located in the same protein. Several groups it will then engage in many new interactions that subtly alter independently found in yeast two-hybrid experiments that the the overall equilibrium of such interactions in the cell. There SH3 domains of various MAGUKs, including PSD-95, is no need to invoke the simultaneous evolution of both a chapsyn-110, SAP97, DLG, CASK and p55, all interact with unique binding domain and a unique ligand site for every their own GK regions (McGee and Bredt, 1999; Nix et al. biologically important interaction. Perhaps an apt analogy can 2000; Shin et al., 2000). Unsurprisingly, given the conservation be made to the webs of interactions among species: although of the SH3 and GK domains among MAGUK proteins, mice eat corn, and owls eat mice, the linear pathway of heterotypic interactions also occur: in some (but not all) owl→mouse→corn does not do justice to the facts that the owl cases, the SH3 domain of one MAGUK can bind to the will eat many other prey besides mice given the opportunity, GK domain of a second (as assayed by yeast two-hybrid, that mice have good reason to fear many predators beside owls, coimmunoprecipitation and GST-pulldown assays). In all cases and that mice will happily eat many things in addition to corn. examined, however, a MAGUK molecule that contains both an An owl’s nest in the barn might create a specific output (less SH3 and a GK domain cannot interact in trans with either the corn eaten by mice), but it would certainly be misleading to SH3 or GK domains of a second MAGUK molecule in vitro; attempt to define the system solely in terms of the ‘specificity’ this suggests that the intramolecular cis interaction is favored of owls for mice or mice for corn. 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 MAGUKs - controlling assembly of large complexes interaction in vivo, however, by co-immunoprecipitating The MAGUK (membrane-associated guanylate kinase) endogenous hCASK and hDLG from human colon cancer family of scaffold proteins illustrates the potential role of cell lysates and colocalizing the two proteins by both intramolecular and intermolecular SH3-mediated immunofluorescence at cell junctions in these and other interactions in assembly of macromolecular complexes. epithelial cells (Nix et al., 2000). Of course, whether this MAGUKs are a large family of related proteins consisting of apparent complex depends on SH3-GK interactions in trans 1-3 PDZ domains, an SH3 domain and a guanylate-kinase- remains to be proved, but, given that trans interaction of like (GK) domain (Fig. 3a); they are thought to play a role in isolated domains can be demonstrated in vitro, the full-length assembly of large protein complexes at specialized regions of proteins are likely to do so under some circumstances in vivo. the membrane such as synapses, neuromuscular junctions and Interestingly, not all MAGUK SH3-GK interactions are other cell-cell junctions (Dimitratos et al., 1999). The PDZ allowed - there is some specificity in which heterotypic domain, another protein-interaction module, typically binds interactions can occur. For example, the hDLG GK binds to its to sites at the extreme C-terminus of target proteins that have own SH3 domain, but not to those of hCASK or p55, whereas the consensus Exx(V/I) (Songyang et al., 1997). The GK the GK domains of hCASK and p55 bind equally well to SH3 region, although bearing all the sequence hallmarks of a domains of all three proteins (Nix et al., 2000); similarly, the catalytically active domain, appears not to have enzymatic SH3 domain of SAP 97 binds to the GK domains of all the activity but instead probably mediates protein-protein closely related MAGUKs in the PSD-95 family but not to interactions. In addition to the three highly conserved that of the more distantly related ZO-1 (Shin et al., 2000). domains, some members of the MAGUK family also contain Therefore, although all MAGUKs appear to be capable of additional motifs, including a region with similarity to the intramolecular (monomer) as well as homotypic (dimer or catalytic domain and calmodulin-binding site of calcium/ higher-order) interactions, only some heterotypic interactions calmodulin-dependent protein kinase (CaM kinase), and a so- will be favored. In some cases, heterotypic interactions are called HOOK domain that interacts with the band 4.1 protein allowed only in an asymmetric fashion: for example, hDLG- and its relatives (Dimitratos et al., 1999). hCASK heterodimers can form through interactions between Given their generous complement of protein-interaction the hDlg SH3 and the hCASK GK, but since the hCASK SH3 domains, MAGUK proteins are well-equipped to mediate the and the hDlg GK cannot bind to each other they are presumably assembly of large, multimolecular protein complexes through free to interact with other proteins (Nix et al., 2000). interactions with other proteins and self-assembly into One potential role for SH3-GK interactions might be to multimers. The PDZ domains interact with transmembrane or orient individual MAGUKs with respect to other proteins in membrane-associated proteins such as receptors and ion large, polymeric complexes. Individual MAGUKs, which can channels; clustering of these proteins presumably facilitates dock onto larger, assembling complexes through other protein- signaling by generating high local concentrations (Garner et interaction domains, such as the PDZ domains, would al., 2000). The role of the SH3 domains in scaffolding by effectively snap into place as the intramolecular SH3-GK MAGUK proteins has until recently been elusive. It has long interaction is out-competed by trans interactions once it has been known that the SH3 is somehow important, because in assumed the correct spatial orientation for proper assembly Drosophila, SH3 domain mutants of the MAGUK protein (Fig. 3b). The assembled complex would be stable despite the 1258 JOURNAL OF CELL SCIENCE 114 (7) A B input input

F F D E D E B C B C Fig. 2. Linear signaling vs complex networks. A group A A 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, W V W and there is minimal cross-reactivity. An input signal U V U (such as relocalization of protein A) will affect only its Y Z Y Z specific ligand (protein X). (B) Complex networks. Each X X 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. output output relatively low affinity of the individual SH3-GK interactions channel normally, is consistent with this idea (Shin et al., owing to the additive effect of many simultaneous interactions. 2000). The fact that Sheng and colleagues have identified a PSD-95 There are some interesting twists to the SH3-GK interaction SH3 domain mutant that cannot cluster the Kv1.4 potassium that highlight the dangers of overgeneralizing SH3 domain channel, and yet is targeted to the membrane and binds to the properties. One outstanding issue concerns the actual binding

A Fig. 3. Protein interactions in MAGUK family Receptors, channels, other ligands Other ligands? 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 PDZ PDZ PDZ SH3 GK 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 GK domains SH3 domains 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 B proximity to other MAGUK complexes. 2. MAGUK interacts with other MAGUKs on the membrane, Outside 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 1 is competed out by trans SH3-GK interaction, which snaps the new MAGUK subunit into place in the 2 appropriate position in the growing complex. Although a homotypic complex is depicted here, heterotypic complexes between different MAGUK 3 proteins are also possible, although only a subset of Cytosol 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. SH3 domains: complexity in moderation 1259 sites for the SH3s, which have yet to be mapped precisely. In interaction on other interactions must be considered. The the case of PSD-95, no canonical PxxP site is present in the C- kainate receptor is the only MAGUK SH3 ligand (other than terminal GK region. Although three proline residues do lie in GK domains) identified to date (Garcia et al., 1998), but it is potential degenerate class II sites (PxR), changing those possible that others exist. Their binding would depend on the residues to alanine individually or in combination did not disruption of intramolecular SH3-GK interactions to expose eliminate binding (Shin et al., 2000). Furthermore, the GK the SH3 domain and would necessarily expose the GK domain region required for SH3 binding extends beyond the borders of for interaction with other SH3 domains in trans. Proteins that the actual GK domain (defined by sequence similarity) both N- lack SH3 domains also bind to the GK domain, although in the and C-terminally, which suggests that a larger folded structure case of PSD-95 and its relatives binding of one of these, GKAP, is recognized by the SH3 domain (McGee and Bredt, 1999; to the GK domain is not affected by the intramolecular SH3- Shin et al., 2000). MAGUK family SH3 domains differ from GK interaction (Shin et al., 2000). Although this demonstrates the typical SH3 consensus at several highly conserved sites in that two independent binding sites are present in the GK the ligand-binding regions (see: http://pfam.wustl.edu/cgi- domain, the effects of SH3 binding on other GK-domain- bin/getdesc?name=SH3); their ligands might therefore also binding partners have yet to be examined. The possibility that differ from the canonical SH3-binding consensus. Mutation in a novel protein-binding interface is created by interaction of the PSD-95 SH3 of the invariant tryptophan (which defines the the SH3 and GK domains also needs to be explored. Clearly, ridge between the second ΦP pocket and the specificity pocket; the permutations of inter- and intra-molecular interactions that Mayer and Eck, 1995) apparently does not eliminate the can and do occur, and their effects on macromolecular intramolecular SH3-GK interaction, although it does eliminate assembly and interactions with other partners, are complex, binding to GK domains in trans. This tryptophan is generally and additional experiments will be required to sort these out. 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. WASP and friends − controlling actin Finally, the effect of the intra- or inter-molecular SH3-GK A second emerging story in which SH3 domains are implicated

GTP- Cdc42 PtdIns Arp2/3 -- nucleate (4,5)P2 actin filaments WASP EVH1 ++ PBD Proline-rich VC A G-actin Tyrosine- phosphorylated proteins Nck SH3 SH3 SH3 SH2

3 2 4

WIP/ V V Proline-rich verprolin bundling? 5 Arp2/3 -- nucleate actin filaments 1 Myosin motor domain SH3 A P

Pak PBD Kinase domain GTP- Cdc42

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 1996). As it turns out, the SH3 domains of Myo3p and Myo5p family of proteins. WASP was originally isolated as the gene bind the proline-rich region of Bee1p (Evangelista et al., 2000; mutated in Wiskott-Aldrich Syndrome, a severe X-linked Lechler et al., 2000). Surprisingly, Myo3p and Myo5p can also immunodeficiency (Derry et al., 1994). WASP and its relatives bind directly through their acidic tails to the Arp2/3 complex, play a pivotal role in organizing the actin cytoskeleton, a which suggests that, in a Bee1p-Myo3p or Bee1p-Myo5p process that must be exquisitely controlled in time and space complex, there is redundancy in Arp2/3 binding (Evangelista to allow directed movement (Higgs and Pollard, 1999; Mullins, et al., 2000; Lechler et al., 2000). Myosin motor activity is 2000; Ramesh et al., 1999; Takenawa and Miki, 2001; important for cortical actin structures (Lechler et al., 2000), Zigmond, 2000). Both WASP and its more ubiquitously perhaps to transport the complex to the barbed ends of actin expressed relative N-WASP contain several modular domains, filaments or to drive movement of actin filaments relative to including an EVH1 domain, a PtdIns(4,5)P2-binding site, a the membrane (Machesky, 2000). region that binds the Rho-family GTPase Cdc42, an extensive Phosphorylation of type 1 myosin at a specific site, proline-rich region, and a C-terminal VCA (for verprolin- presumably by the Pak family kinases Ste20p and Cla4p, is cofilin-acidic) region involved in regulating actin essential for Cdc42-stimulated actin assembly in yeast (Lechler polymerization. The EVH1 domain engages proline-rich et al., 2000), and phosphorylation of amoeboid type 1 myosin ligands, but the mode of recognition differs from that of SH3 by Pak-like kinases is essential for its activity (Brzeska and or WW domains (Prehoda et al., 1999). The Cdc42-binding Korn, 1996; Novak and Titus, 1998). Remarkably, in region binds to and inhibits the VCA region when Cdc42 is not mammalian cells Nck binds via its SH3 domains to Pak kinases bound; thus this region is a conditional switch that responds to (Bokoch et al., 1996; Galisteo et al., 1996; Lu et al., 1997), changes in GTP-Cdc42 levels (Abdul-Manan et al., 1999; Kim which suggests a mechanism whereby Nck might modulate the et al., 2000; Miki et al., 1998). The VCA region interacts with activity of unconventional associated with WASP- the Arp2/3 complex, which nucleates de novo actin filament Arp2/3 complexes. In Drosophila the Nck homolog Dock is formation and induces filament branching (Blanchoin et al., important for the directed migration of retinal axons, probably 2000; Mullins et al., 1998; Pantaloni et al., 2000), and the regulating localized actin polymerization in the growth cone in filament-nucleating activity of the Arp2/3 complex is greatly response to environmental cues (Garrity et al., 1996), and Pak increased when bound to the VCA region (Machesky et al., has been shown to function downstream of Dock in this system 1999; Rohatgi et al., 1999; Winter et al., 1999; Yarar et al., (Hing et al., 1999). 1999). Thus Cdc42 can activate Arp2/3-mediated actin The SH3 domains of Myo3p and Myo5p have also been polymerization by disrupting the intramolecular interaction shown to bind another proline-rich protein, verprolin, which is between the PBD and VCA regions (reviewed by Fawcett and also implicated in organization of the actin cytoskeleton Pawson, 2000). (Anderson et al., 1998; Evangelista et al., 2000; Geli et al., Where do SH3 domains fit into this picture? The long 2000). The mammalian homolog of verprolin, WIP, binds proline-rich region found in all WASP family members is an directly and with high affinity to the N-terminal EVH1 domain obvious potential SH3-binding site, and there is no shortage of of WASP (Ramesh et al., 1997), and this interaction is suspects for ligands. The Nck SH2/SH3 adaptor, for example, conserved in yeast (Naqvi et al., 1998); such a verprolin-Bee1p is localized on the surface of vaccinia virus particles and plays complex would thus contain multiple binding sites for type 1 a role in recruiting N-WASP and the Arp2/3 complex, thereby myosin SH3 domains. Furthermore, WIP is required for Nck serving to nucleate the formation of actin ‘comet tails’ that to recruit N-WASP to vaccinia virus particles, which suggests propel the virus in the cytosol (Frischknecht et al., 1999; that binding of the Nck SH3 domains to N-WASP is too weak Moreau et al., 2000). Because Nck has an SH2 domain, it could to be stable without the additional contacts provided by Nck- recruit and cluster WASP family members at sites of tyrosine WIP and WIP-N-WASP interactions (Anton et al., 1998; phosphorylation. Indeed, a phosphotyrosine-containing site Moreau et al., 2000). Finally, WIP might also function to whose phosphorylation is necessary for actin tail formation is bundle actin filaments together (N. Ramesh and R. Geha, present on the vaccinia surface and can bind to the Nck SH2 personal communication), thereby modulating the higher-order domain (Frischknecht et al., 1999). In T cells Nck helps to structure of filaments initiated by the Arp2/3 complex. mediate the phosphotyrosine-dependent assembly of a SH3 domains might also directly activate complexes complex containing WASP and Vav, a GEF for Rho family containing Arp 2/3 and WASP family members. Carlier and GTPases, and dominant-negative experiments suggest that colleagues have shown that SH3 domains from the Grb2 disruption of this complex inhibits localized actin adaptor enhance the activity of the N-WASP−Arp2/3 complex polymerization following T cell receptor stimulation (Bubeck in vitro (Carlier et al., 2000). We find that Nck SH3 domains Wardenburg et al., 1998). Furthermore, a close relative of Nck, are more effective than those of Grb2, and multiple Nck SH3 Nckβ, is implicated in actin rearrangements downstream of domains are required for optimal activation (R. Rohatgi, B. J. PDGF receptor stimulation (Chen et al., 2000). Mayer, and M. Kirschner, unpublished). This requirement for Other potential partners for WASP family members are the multiple SH3 domains raises the possibility that simultaneous unconventional SH3-containing myosins. In yeast the WASP engagement of two or three SH3-binding sites on N-WASP can relative Bee1p/Las17p is essential for proper formation and fix it in an open, activated conformation in which its VCA localization of cortical actin patches and for endocytosis (Li, region is exposed. In these in vitro assays GTP-Cdc42 and Nck 1997; Naqvi et al., 1998). Loss of both type I myosins Myo3p seem to play redundant roles in the activation of N-WASP, in and Myo5p, or mutation of their SH3 domains, leads to severe both cases synergizing with PtdIns(4,5)P2 (R. Rohatgi, B. J. defects in the actin cytoskeleton (Anderson et al., 1998; Mayer, and M. Kirschner, unpublished). A Cdc42-independent Evangelista et al., 2000; Geli et al., 2000; Goodson et al., mode to nucleate actin polymerization in the cell through high SH3 domains: complexity in moderation 1261 local concentrations of Nck (or other SH3-containing proteins) O., Siminovitch, K. A. and Rosen, M. K. (1999). Structure of Cdc42 in might therefore exist. This indeed seems to be the case for complex with the GTPase-binding domain of the ‘Wiskott-Aldrich vaccinia actin tail formation, which appears to be independent syndrome’ protein. Nature 399, 379-383. Adler, C. E., Miyoshi-Akiyama, T., Aleman, L. M., Tanaka, M., Smith, J. of GTP-Cdc42 (Moreau et al., 2000). M. and Mayer, B. J. (2000). Abl family kinases and Cbl cooperate with the What emerges from these studies is a picture in which a vital Nck adaptor to modulate Xenopus development. J. Biol. Chem. 275, 36472- function, nucleation of new actin filaments at the appropriate 36478. time and place in the cell, is controlled by a multicomponent Anderson, B. L., Boldogh, I., Evangelista, M., Boone, C., Greene, L. A. complex of proteins assembled through SH3-mediated and and Pon, L. A. (1998). The Src homolgy domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of other protein-protein interactions (Fig. 4). The individual actin polarization. J. Cell Biol. 141, 1357-1370. components of the complex provide multiple functional outputs Anton, I. M., Lu, W., Mayer, B. J., Ramesh, N. and Geha, R. S. (1998). (filament nucleation, filament bundling and transport along The Wiskott Aldrich Interacting Protein (WIP) binds to the adaptor protein filaments), multiple levels of control (by guanine nucleotide Nck. J. Biol. Chem. 273, 20992-20995. Bateman, A., Birney, E., Durbin, R., Eddy, S. R., Finn, R. D. and exchange, phosphorylation and local concentration), and Sonnhammer, E. L. L. (1999). Pfam 3.1: 1313 multiple alignments and functional redundancy. Of course many other functions can be profile HMMs match the majority of proteins. Nucl. Acids Res. 27, 260-262. envisioned. The central role played by SH3 domains promotes Blanchoin, L., Amann, K. J., Higgs, H. N., Marchand, J. B., Kaiser, D. A. flexibility in possible interactions, because their relatively low and Pollard, T. D. (2000). Direct observation of dendritic actin filament affinity and modest specificity allows for the assembly of large networks nucleated by Arp2/3 complex and WASP/Scar proteins. Nature 404, 1007-1011. but ‘informal’ complexes that can also disassemble rapidly in Bokoch, G., Wang, Y., Bohl, B. P., Sells, M. A., Quilliam, L. A. and Knaus, response to changes in the environment. U. G. (1996). Interaction of the Nck adapter protein with p21-activated kinase (PAK1). J. Biol. Chem. 271, 25746-25749. 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(1999). synthetic peptoid compounds that have high affinities and Signaling pathways are focused at specialized regions of the plasma selectivities might eventually fulfil the longstanding dream of membrane by scaffolding proteins of the MAGUK family. BioEssays 21, intervening in SH3-mediated interactions for the treatment of 912-921. human disease. We have come a long way in the past ten years, Evangelista, M., Klebl, B. M., Tong, A. H. Y., Webb, B. A., Leeuw, T., but our progress has brought a more mature appreciation of just Leberer, E., Whiteway, M., Thomas, D. Y. and Boone, C. (2000). A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and how much there is left to do. the Arp2/3 complex. J. Cell Biol. 148, 353-362. Fawcett, J. and Pawson, T. (2000). Signal transduction: N-WASP regulation I thank T. Kirchhausen, A. Musacchio, N. Ramesh, R. Rohatgi, and - the sting in the tail. Science 290, 725-725. K. 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