(1998) 17, 1469 ± 1474  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc From Src Homology domains to other signaling modules: proposal of the ` recognition code'

Marius Sudol

The Department of Biochemistry, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029, USA

The study of has illuminated many aspects of domains have identi®ed a set of common features cellular signaling. The delineation and characterization which aids in their description. The domains are of protein modules exempli®ed by Src Homology composed of 40 ± 150 amino acids which fold to domains has revolutionized our understanding of the generate one or more -binding surfaces, known molecular events underlying path- as `recognition pockets'. Generally, conserved residues ways. Several well characterized intracellular modules of the domain are directly involved in mediating which mediate protein-protein interactions, namely SH2, contacts with the amino acids of the ligand and in SH3, PH, PTB, EH, PDZ, EVH1 and WW domains, maintaining the structure of the domain. Ligands are directly involved in the multitude of membrane, interact with their complementary domains through cytoplasmic and nuclear processes in multicellular and/or short sequence motifs (also called core motifs), unicellular organisms. The modular character of these composed of only 3 ± 6 amino acids (Figure 1). The protein domains and their cognate motifs, the univers- sequence of the core determines the selection of the ality of their molecular function, their widespread domains (Songyang and Cantley, 1995). The most occurrence, and the speci®city as well as the degeneracy `important' within the core is traditionally of their interactions have prompted us to propose the designated `0' and the remaining amino acids are concept of the `protein recognition code'. By a parallel designated `-1', `-2', and `1', `2', toward the amino- analogy to the universal genetic code, we propose here and carboxy-terminal directions, respectively. With that there will be a ®nite set of precise rules to govern one or two exceptions (Songyang et al., 1997), the and predict protein-protein interactions mediated by core motifs are surrounded by ¯anking amino acids at modules. Several rules of the `protein recognition code' the amino-(fN) and carboxy-terminal (fC) regions which have already emerged. dictate the speci®city of interaction within a given family of modules (Rickles et al., 1995). As expected, Keywords: protein modules; protein motifs; genetic variabilities in primary structures of domains also code; phosphotyrosine; polyproline contribute to the speci®city of interaction with the ligand. We propose to use a term, `epsilon determi- nant(s)' (e from the Greek word for speci®city: eidikotZta, pronounced edekohteta) for those amino The SH2 domain is a paradigm of small conserved acids within domains which determine ligand predilec- protein modules that mediate the formation of tions. The e determinant is represented by one or intracellular complexes in vivo and which participate several amino acid positions located mainly within the in a diverse array of signaling events (Pawson, 1995). conserved structure of the domain, and usually in the With the discovery and characterization of the SH2 ligand binding interface. Hydrophobic amino acids are domain, two important features became immediately represented by c (Psi) and aromatic amino acids by z apparent: First, its widespread occurrence and func- (Zeta). Any amino acid or an amino acid from a tion, and second, its speci®city of interaction with subset that can confer speci®city are indicated by x. In cognate protein ligands (Schlessinger, 1994). Subse- the latter case the x amino acids correlate with the e quent delineation of other intracellular signaling determinant (see Figures 2 and 3, and SH2 rules modules including SH3, PTB, PDZ, EVH1, and WW below). With the aid of this simple glossary, we will domains, as well as their binders, con®rmed these two brie¯y summarize the emerging rules of protein- characteristics (Cohen et al., 1995; Sudol, 1996a). protein interaction mediated by modules, using Intracellular protein modules are frequently referred several of the best characterized domains as exam- to as biological `Velcro', or as molecular adhesives. ples. Implicitly, the proposed formalization is meant However, the interactions that these modules mediate to unify several diverse descriptions currently in use in with their cognate ligands do not resemble a uniform order to facilitate discussion in the rapidly developing structure or a mechanism implicit in the analogy to ®eld of signaling by protein modules. molecular glue. Rather, each of the domains represents a unique three-dimensional structure that is comple- mentary to a speci®c sequence motif of its ligand (Kuriyan and Cowburn, 1997). SH2 rules

The SH2 (Src Homology 2) domain is widely General rules distributed among cytoplasmic , and the discovery of its ligands had a great impact on the Structural and functional studies of the domain-ligand understanding of cellular signaling (Sadowski et al., complexes for SH2, SH3, PTB, PDZ and WW 1986; Pawson, 1995). The domain functions primarily Protein-protein interaction by modules MSudolet al 1470

Figure 1 Schematic representation of widely distributed modular protein domains and their cognate ligands. Protein domains are structurally coherent, three-dimensional units that have the ability to fold independently (Siddiqui and Barton, 1995; Bork et al., 1996). They are composed of a minimum of 35 ± 40 amino acids. These protein domains include: SH2, PTB, PDZ, SH3, EVH1 and WW. They mediate a vast array of interactions by binding to unique sequences located in target proteins. The cognate sequences in the ligands are represented by short, conserved amino acid cores containing, for example, phospho-tyrosines (p- Y) or polyprolines. The formation of these complexes is the foundation of the intracellular network of signaling which Figure 2 Formalization of terms to describe protein-protein governs cell proliferation, di€erentiation, organiza- interaction mediated by modules using the SH2 domain and its tion, endocytosis, controlled protein degradation, and ligand as an example

by regulating various cellular events including For SH2 domains that belong to the same group, the e activity, substrate recruitment and protein localization determinant may reside in the EF1 position (the ®rst (Pawson, 1995). SH2 domains bind ligands containing amino acid in the loop between E [®fth] and F [sixth] b- phosphotyrosine residues within a speci®c sequence strands). A single amino acid change in the EF1 position (Schlessinger, 1994; Pawson, 1995). High anity of the Src SH2 domain from threonine to tryptophan binding is provided by the phosphotyrosine residue was shown to switch the ligand-binding speci®city to itself, and by residues carboxyterminal to it, p-Yxxc resemble that of the SH2 domain of Grb2 (Growth (Songyang and Cantley, 1995; Figure 2). Factor -Binding Protein 2) adaptor protein. Examination of several crystal and NMR (Nuclear The binding speci®city of the mutated domain correlated Magnetic Resonance spectroscopy) structures of SH2 well with the biological activity as the mutated Src SH2 domain-ligand complexes, as well as extensive analysis domain substituted the SH2 domain of the Grb2 protein of binding preferences of a variety of SH2 domains in activation of the Ras pathway in vivo (Marengere et using molecular repertoires of phosphopeptides, helped al., 1994). These two examples support the argument to classify the SH2 domain into four groups (Songyang that there is a relatively simple relationship between the e and Cantley, 1995). The e determinant for SH2 domains determinants and binding speci®city. is represented by the bD5 position (the notation The SH2 rules are being re®ned as more structural indicates ®fth amino acid in the fourth b-strand; D and functional data become available, but even in their indicates the fourth strand). The majority of known present form and with several well-documented SH2 domains fall into group I, which prefer hydrophilic exceptions, they provide a valuable tool to predict residues in the +1 position of the ligand. The e cognate pairs for SH2 domain-ligand complexes. determinant of the group I domains, bD5 position, is represented by aromatic residues, tyrosine or phenyla- lanine. Group III SH2 domains prefer hydrophobic PTB rules amino acids in the +1 position of the ligand and isoleucine or in the e determinant of the In general, PTB (Phosphotyrosine-binding) domains play domain. The e determinant for group II SH2 is a similar role to SH2 domains (Kavanaugh and Williams, threonine, whereas for group IV, it is methionine or 1994; Blaikie et al., 1994; Gustafson et al., 1995). Based on valine. ligand recognition, the PTB domains fall into two groups: The importance of the bD5 residue in determining Group I represents those PTBs which bind to ligands speci®cities of SH2 domains was documented experi- containing NP6p-Y (N-asparagine, P-proline) cores mentally by replacing the aliphatic residue of the group where tyrosine is phosphorylated (Van der Geer and III SH2 domain (phosphoinositide 3-kinase SH2 Pawson, 1995). The PTB domains of Shc (Src Homo- domain) with tyrosine of the group I SH2. The logous and Collagen) and IRS-1 ( resulting mutant acquired the speci®city of the group Substrate-1) belong to this class and bind ligands with I SH2 domain in terms of selectivity for phosphopep- c6NP6p-Yc and ccc66NP6p-Y consensus se- tide ligands (Songyang et al., 1995) quences, respectively (Van der Geer et al., 1995; He et Protein-protein interaction by modules MSudolet al 1471 al., 1995; Isako€ et al., 1996). The group II PTBs bind to the fC sequences include prolines, or the positively ligands with NP6Ycores where tyrosine is not necessarily charged amino acids lysine and/or arginine (Chen et phosphorylated (Zambrano et al., 1997). The e determi- al., 1997; Linn et al., 1997). of the nant for the latter group contains cysteine in the helix signature tyrosine in the ligand (PP6Y) prevents the aC12 position instead of . Another position formation of the complex (Chen et al., 1997). Group II of the e determinant for PTBs is an arginine residue in the WW domains bind ligands containing PPLP cores loop between the bF and bG strands. This arginine usually surrounded by at least three additional prolines interacts with the phosphotyrosine of the ligand for the distributed between fN and fC sequences (Bedford et al., group I PTBs (Shc for example) and is absent in several 1997; Ermekova et al., 1997). The e determinant here domains of the II group that bind non-phosphorylated allows for easy prediction between these two groups. NP6Y cores (Zhou et al., 1996; Zambrano et al., 1997). A For group I, the e determinant is an aliphatic amino common structural element in the NP6p-Y/NP6Y motif acid in the second b strand of the domain, position is that it forms a type I b-turn that is well accommodated bB6; whereas for the group II this position is occupied by the binding pocket of the PTB domain. by an aromatic amino acid (Sudol, 1996a; Macias et Recently, a new domain named EH after Eps al., 1996). It is important to note that in both groups Homology (Eps:EGF receptor phosphorylation sub- the bB6 residue is preceded by two consecutive strate) was identi®ed (Di Fiore et al., 1997). The aromatic amino acids. Interestingly, there is no cross- ligands to EH domains contain NPF cores, and the EH reactivity between domains of the two groups, domain-ligand complexes seem to be involved in the indicating a high degree of speci®city in ligand binding regulation of molecular events underlying endocytosis achieved by this small module. (Salcini et al., 1997). It is curious that the NP6Y Since its discovery and initial characterization, the sequence is a general internalization signal for proteins WW domain has attracted considerable attention (Bansal and Glerasch, 1991), and its similarity with the because the signaling complexes it mediates have been conserved cores of PTB, EH and WW ligands may not implicated in several human diseases including Liddle's be coincidental. syndrome of hypertension, muscular dystrophy and Alzheimer's disease (reviewed by Sudol, 1996b).

SH3 rules PDZ rules The SH3 (Src Homology 3) domain is a representative of the rapidly growing family of modules that PDZ domains are present in cytosolic proteins, many recognize proline-rich ligands (Mayer et al., 1988; of which are located at specialized regions of cell-to- Stahl et al., 1988; Sudol, 1996a). Similar to SH2 cell contact. The name PDZ is derived from three domains, the SH3 domains regulate protein localiza- proteins that contain this domain: PSD-95 - post- tion, enzymatic activity and often participate in the synaptic density protein; Dlg - disc large tumor assembly of multicomponent signaling complexes suppressor; and ZO1 - a tight junction protein (Cho (Schlessinger, 1994; Mayer and Eck, 1995). The et al., 1992; Woods and Bryant, 1993; Ponting and minimal sequence requirement for the SH3 domain Phillips, 1995). Three groups of the PDZ domain are ligands is the P66 P motif (Ren et al., 1993), and known, and all recognize unique carboxyterminal structural analyses indicate that the ligands adopt a motifs (Kornau et al., 1995; Lau et al., 1996; left-handed polyproline II helix conformation (Yu et Niethammer et al., 1996; Songyang et al., 1997; al., 1994; Lim and Richards, 1994). The unique feature Sticker et al., 1997). Group I PDZ domains bind to of the SH3 domain-ligand interaction is that the ligand peptides with the consensus E(S/T)6(V/I) where V/I is binds the domain in one of two pseudosymmetrical the carboxy-terminal residue of the protein; group II orientations, class I and class II (Lim et al., 1994; Feng PDZ domains bind peptides with D6V triplet; and et al., 1994). The general consensus sequence for SH3 group III binds peptides with a zzc consensus (Figure domain ligands is cP6cP (Mayer and Eck, 1995). For 3). The presence of a hydrophobic carboxyterminal class I, the amino acids in the fN sequence, and for amino acid seems to be a rule for these three groups. class II the amino acids in the fC sequence dictate the Several amino acids constitute the e determinant for speci®city of interaction and also discriminate between PDZ domain, and among them are histidine, arginine, various SH3 domains. lysine, at the helix aB1 position for group I of PDZs. For group III of PDZ domains the aB1 position is occupied by valine, or aspartic acid (Cabral et WW rules al., 1996; Doyle et al., 1996; Songyang et al., 1997). For PDZ domains and in fact for other domains as Although structurally distinct, the WW domain is well, the e determinant is represented not by a single functionally related to the SH3 domain; it also binds but rather by multiple amino acid positions which are proline-rich ligands (Chen and Sudol, 1995; Macias et a part of binding pockets and/or re¯ect important al., 1996). The name `WW' refers to one of the structural determinants. For example, amino acids distinguishing features of the domain: the presence of present at bC4 and bC5 positions of PDZ domains two highly conserved tryptophan residues (W) which should also be considered as a part of the e are spaced 20 ± 22 amino acids apart (Sudol, 1996a). So determinant in the predictions of complementarities. far, two groups of WW domains have been identi®ed: In general, the complexity and variability of the e Group I WW domains bind ligands that contain a determinant correlates well with the structural diversity

PP6Y core (Chen and Sudol, 1995). The fN sequences of a given domain class, re¯ecting evolutionary usually contain proline, lysine, cysteine or tyrosine, and changes. Protein-protein interaction by modules MSudolet al 1472

Figure 3 Modular protein domains and core sequences of their cognate ligands. SH2 and a subgroup of PTB domains recognize phospho-tyrosine in the context of speci®c sequences. Another subgroup of PTB domains, and the EH domain, bind ligands with NP6Y and NPF cores, respectively. SH3, WW, and EVH1 domains bind proline-rich sequences. Pro®lin also recognizes polyprolines (a minimum of six to eight prolines). Pro®lin is both a and an independent, functional protein of 13 ± 15 kiloDaltons molecular mass. PDZ domains evolved to recognize motifs at the carboxy-terminal ends of proteins. Many more intracellular and extracellular modular protein domains have been identi®ed (reviewed by Bork et al., 1996; Bork et al., 1997). However, their ligands are as yet unknown, and signi®cant portions of the newly described modules so far seem to be con®ned to a limited number of specialized proteins

`Protein recognition code' versus genetic code to be taken into account as well (Mayer et al., 1995; Alexandropoulos and Baltimore, 1996). Therefore, The `protein recognition code' is meant to represent a predictions of cognate interactions for modular set of precise rules that will govern and predict protein- protein domains in vivo will likely be described in protein interactions mediated by modules. The terms of probability rather than in absolute terms. proposed analogy to the principles of the genetic code Given the wealth of data accessible from the theoretical is general. In fact, there are many important and analysis of genomes and proteomes, and from contrasting di€erences between the two codes. The expression pro®ling by cDNAChip and ProteinChip genetic code is truly universal, whereas the `protein microarrays, such predictions do not seem to be far o€. recognition code' is only widespread. The protein modules are found in both and king- doms, including unicellular organisms, but overall the Degeneracy in the `protein recognition code' modules do not have a universal character. The genetic code is absolute and context-independent; a given One of the major features of the genetic code is a codon speci®es only one amino acid regardless of phenomenon known as degeneracy, which ultimately where it is in a message, or what organism it is found serves to minimize the deleterious e€ects of mutations. in. In contrast, the nature of modular protein binding There are several examples in the protein-protein is neither absolute nor context-independent. A given interaction network which suggest degeneracy in the ligand motif, for an SH3 domain for example, is able `protein recognition code'. For protein modules, one of to interact in vitro and in vivo with a number of SH3 the ®rst observations came from Leder and colleagues domains. The cognate interaction(s) in vivo for a who reported the interaction of a family of SH3 and randomly chosen module, SH3 domain, could be WW domains with a common proline-rich motif in predicted only if local concentrations of all possible formin, a protein implicated in the development of binding partners and their dissociation constants are kidney and limbs in mice (Chan et al., 1996). In known. The relative anity and speci®city of domain- addition to the SH3 and WW domains, there are two ligand interaction resides also at another level, namely, other modules also known to bind proline-rich motifs: at the level of conformation imposed by linking the EVH1 (Enabled, VASP, Homology 1) domain from multiple domains and ligands. For example, if the the VASP/Mena family of proteins, and pro®lin, a SH3 domain was a part of an adaptor protein protein that regulates dynamics (Gertler et al., containing other modular protein domains involved 1996; Niebuhr et al., 1997). Although the structure of in the interactions (in cis or in trans), this context has the EVH1-ligand complex is not yet known, it is likely Protein-protein interaction by modules MSudolet al 1473 that this domain will also bind its proline-rich ligand in The biological role of pro®lin appears to be more a way in which individual proline residues contact the complex than originally envisioned. In addition to domain directly as well as serve a sca€olding function regulating polymerization of actin ®laments, pro®lin to maintain the polyproline II helical structure, as was may provide a link between the cytoskeleton and the documented for ligands to SH3, WW and pro®lin (Yu signaling network (Mahoney et al., 1997). A cross-talk et al., 1994; Macias et al., 1996; Mahoney et al., 1997). between pro®lin and Mena connects the cytoskeleton The degeneracy among proline-binding protein to the signaling pathways of Abl and Src tyrosine modules arises from the identity or close similarity kinases (Gertler et al., 1996). Pro®lin also couples to among sequences of the ligands. Ligands for certain phosphatidylinositol 4,5-biphosphate pathways and to SH3 domains and ligands for group II WW domains, the signal transduction pathways mediated by the small share a PPLP core (Sudol, 1996b). In addition, many GTPase, Rho (Watanabe et al., 1997). Many of the physiological binders of pro®lin, including Mena, interactions between pro®lin and its numerous protein VASP, cdc12p, p140m Dia and Cappucino, all contain targets are mediated by identical or similar polyproline long runs of prolines interrupted or terminated by motifs and are well documented biochemically and/or leucine (Mahoney et al., 1997). It is the PPLP core(s) genetically. of Mena that interact(s) with the WW domain of the Another level of degeneracy exists speci®cally for FE65 adapter protein linked to the Alzheimer's beta proline-rich ligands that bind SH3 domains. It arises amyloid precursor (Ermekova et al., 1997). The same from the pseudosymmetry of the polyproline helical PPLP core interacts with selected SH3 domains structure and its ability to interact with the binding including those of Src and Abl, and in a longer pocket of SH3 domains in two orientations (Lim and context of ¯anking prolines, with pro®lin (Gertler et Richards, 1994; Lim et al., 1994). al., 1996; Figure 4). Homotypic interactions mediated by some PDZ domains (Brenman et al., 1995), formation of signaling dimers between SH2 domains of STATs (Darnell et al., 1994; Ihle, 1996), and strand interchange dimerizations between SH3 domains of Eps8 (Kishan et al., 1997) represent other examples of degeneracy and diversity in signaling by protein modules. In summary, for the `protein recognition code', degeneracy may primarily provide a mechanism for the convergence of multiple signaling pathways. Moreover, by creating a limited number of apparently redundant modules and redundant ligands, the `protein code' degeneracy may also contribute to the reduction of negative e€ects of mutations.

Concluding remarks

With the impressive progress in genome/proteome analyses and in structural biology, and consequently in the identi®cation of new protein modules (Bork et al., 1997; Evangelista et al., 1996; Schultz et al., 1998), we anticipate that the complete blueprint of the `protein recognition code' will be formulated in the near future. At present, there are many exceptions to the established and emerging rules of the protein-protein interaction discussed here. It is likely that these exceptions may soon give rise to new rules. Nevertheless, we expect a uni®ed picture of the `protein recognition code' in which we will see patterns of order and logic, perhaps reminiscent of Nature's design imprinted on the periodic table of elements and the genetic code.

Figure 4 Examples of degeneracy among modular protein domains recognizing proline-rich sequences. The upper panel shows complexes assembled on the proline-rich segment of Mena, Acknowledgements a protein implicated in the control of micro®lament dynamics. Src Supported by grants from the National Institutes of Health and Abl SH3 domains, pro®lin, and the WW domain of the FE65 and Muscular Dystrophy Association. Special thanks go to adapter protein, recognize identical (or closely overlapping) PPLP Peter K Vogt for the much needed encouragement to cores in Mena (Ermekova et al., 1997; Gertler et al., 1996). The propose the concept of the `protein code', to Mariano polyproline motifs in Mena represent potential points of Barbacid and Saburo Hanafusa for valuable advice on our convergence for several signal transduction pathways. The lower study of the WW module, and to Dina Alexandropoulos, panel shows putative complexes assembled on a polyproline Steve Almo, Mark Bedford, Martyn Bot®eld, Giulio sequence of a binder to the SH3 domain of Src (Alexandropoulos et al., 1995). Considering the consensus for a ligand to the EVH1 Draetta, Xavier Espanel, Siegmund Fischer, Frank Ger- domain, (D/E)(F/W/L/PPPP) we speculate that the Src SH3 ligand tler, Steve Hanes, Hillary Linn, Eugene Koonin, Bruce (especially with the serine residue decorated by phosphorylation) Mayer, Tom Sakmar and Richard Stanley for helpful could also interact with proteins containing EVH1 domains comments on the manuscript. For J. Protein-protein interaction by modules MSudolet al 1474 References

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