Structure and Regulation of Src Family Kinases

Structure and regulation of Src family kinases

Titus J Boggon1,2 and Michael J Eck*,1,2

1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115, USA; 2Department of Cancer Biology, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115, USA

Src family kinases are prototypical modular signaling (Koegl et al., 1994; Resh, 1999). The SH4 region is proteins. Their conserved domain organization includes a followed by a ‘unique’ domain of 50–70 residues, which myristoylated N-terminal segment followed by SH3, SH2, is divergent among family members. and tyrosine kinase domains, and a short C-terminal tail. A hallmark of Src kinases is a short C-terminal tail, Structural dissection of Src kinases has elucidated the which bears an autoinhibitory phosphorylation site canonical mechanisms of phosphotyrosine recognition by (Tyrosine 527 in Src) (Cooper et al., 1986). Like most the SH2domain and proline-motif recognition by the SH3 protein kinases, Src family members require phosphor- domain. Crystallographic analysis of nearly intact Src ylation within a segment of the kinase domain termed kinases in the autoinhibited state has shown that these the activation loop for full catalytic activity.In Src, this protein interaction motifs turn inward and lock the kinase autophosphorylation site is Tyrosine 416 (for consis- in an inactive conformation via intramolecular interac- tency, we use chicken Src numbering throughout) tions. The autoinhibited Src kinase structures reveal a (Smart et al., 1981). In vivo, Src kinases are phosphory- mode of domain assembly used by other tyrosine kinases lated on either Tyr 416 (in their active state) or Tyr 527 outside the Src family, including Abl and likely Tec family (in the inactive state).The inactivating phosphorylation kinases. Furthermore, they illustrate the underlying on Tyr 527 is carried out by the Src-specific kinase Csk regulatory principles that have proven to be general (Nada et al., 1991) or its homolog Chk (Hamaguchi among diverse modular signaling proteins. Although there et al., 1996; Davidson et al., 1997). Phosphorylation of is considerable structural information available for the the C-terminal tail promotes assembly of the SH2, SH3, autoinhibited conformation of Src kinases, how they may and kinase domains into an autoinhibited conformation assemble into active signaling complexes with substrates maintained by intimate interactions among these and regulators remains largely unexplored. domains (Sicheri et al., 1997; Williams et al., 1997; Oncogene (2004) 23, 7918–7927. doi:10.1038/sj.onc.1208081 Xu et al., 1997). The discovery of the SH2 domain as a functionally Keywords: Src regulation; modular signaling; CD4/CD8a important, but noncatalytic segment of B100 residues conserved in v-Fps, Abl, and Src kinases (Sadowski et al., 1986) gave rise to the concept of ‘modular signaling domain’ (Pawson, 1995).Studies of the function and specificity of the SH2 and SH3 domains The domain structure of Src kinases of Src and of their effect on the activity of the adjacent kinase domain have provided a powerful paradigm for Src family nonreceptor tyrosine kinases are present in the functional dissection of a host of modular signaling essentially all metazoan cells, where their regulated domains (see Pawson (2004) for an excellent review and activation by diverse growth factor, cytokine, adhesion, historical perspective).Likewise, structural dissection of and antigen receptors is critical for generating an Src kinases into their component domains and eventual appropriate cellular response to external stimuli (Brown elucidation of the structure of the essentially intact Src and Cooper, 1996; Thomas and Brugge, 1997).The nine kinases have provided a paradigm for structural analysis members of the Src family include Src, Lck, Hck, Fyn, of many multidomain signaling proteins.In the follow- Blk, Lyn, Fgr, Yes, and Yrk.Src kinases share a ing paragraphs, we briefly describe the structure of the conserved domain structure consisting of consecutive isolated domains of Src kinases.In later sections, SH3, SH2, and tyrosine kinase (SH1) domains we consider in more detail the structure of autoinhibited (Figure 1).All family members also contain an SH4 Src kinases and the implications of the structure for Src membrane-targeting region at their N-terminus, which is function and regulation. always myristoylated and sometimes palmitoylated The SH2 domain *Correspondence: MJ Eck, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, MA, USA; The architecture of the SH2 domain and the structural E-mail: [email protected] basis for its recognition of phosphorylated tyrosine was Structure and regulation of Src kinases TJ Boggon and MJ Eck 7919

Figure 1 The domain structure of Src family kinases.The Src kinase architecture consists of four domains: the unique region, which varies among family members, followed by the SH3, SH2, and tyrosine kinase domains.The approximate extent of each domain is indicated, with the unique, SH3, SH2, and kinase domains colored dark blue, salmon, light green, and light blue, respectively, here and throughout the review.The activation loop of the kinase domain is colored red, and the activating (Tyr 416) and autoinhibitory (Tyr 527) phosphorylation sites are indicated.Conserved residue Arg 175 in the SH2 domain is critical for phosphotyrosine recognition; Trp 260 at the extreme N-terminus of the kinase domain is important for autoinhibition (see text).In the autoinhibited form of Src kinases, the SH2 domain binds the phosphorylated C-terminal tail, and the SH3 domain binds the linker segment between the SH2 and kinase domain, which forms a polyproline type II helix (see Figure 3).By convention, amino-acid residues are numbered as in chicken Src.In the lower panel, Protein Data Bank (PDB) accession codes for selected Src family structures are given, and the approximate region included in each three-dimensional structure is indicated.PDB accession codes correspond to: Lck unique domain in complex with the CD4 (1Q68) and CD8a (1Q69) cytoplasmic tails (Kim et al., 2003); 1SHG, crystal structure of aspectrin SH3 domain (Musacchio et al., 1992); 1SHF, the Fyn SH3 domain (Noble et al., 1993); 1SRM, the NMR structure of Src SH3 domain (Yu et al., 1992); 1SHA, 1SPR, and 1LCJ, crystal structures of SH2 domains of Src and Lck, respectively (1SHA and 1LCJ are in complex with tyrosine- phosphorylated peptides) (Waksman et al., 1992, 1993; Eck et al., 1993); 1LCK and 1G83, crystal structures of SH2–SH3 fragments of Lck (Eck et al., 1994) and Fyn (Arold et al., 2001); 3LCK, Lck kinase domain in its active conformation, phosphorylated on the activation loop tyrosine (Yamaguchi and Hendrickson, 1996); 2SRC (Xu et al., 1999), 1FMK (Xu et al., 1997), 2HCK (Sicheri et al., 1997), Src and Hck crystal structures incorporating SH2-SH3-Kinase domains in the autoinhibited conformation

first revealed in near-simultaneous studies of the domains derived from Abl (Overduin et al., 1992), the p85 subunit of PI 3-OH kinase (Booker et al., 1992), and of Src itself (Waksman et al., 1992). The now-familiar fold consists of a central b-sheet, with a single helix packed against each side.These elements of secondary structure and the loops that connect them form two recognition pockets, one that coordinates phosphotyrosine, and the second on the opposite side of the central sheet that typically binds one or more hydrophobic residues just C-terminal to the phosphotyrosine (Eck et al., 1993; Waksman et al., 1993). The phosphotyrosyl recognition pocket is rather highly conserved among SH2 domains, and contains a universally conserved arginine residue (Arg 175 in Src) that forms requisite electrostatic interactions with the phosphorylated tyrosine (Waksman et al., 1992). Not surprisingly, the Figure 2 The Lck SH2 domain bound to a high-affinity C-terminal recognition pocket is much more divergent, phosphopeptide.The Lck SH2 domain binds the pYEEI -motif accounting for the differences among SH2 domains in phosphopeptide in a two-pronged manner.The phosphorylated recognition of specific phosphotyrosine-bearing se- tyrosine and the isoleucine at the pY þ 3 position insert into well- defined recognition pockets on the surface of the domain.The quences (Kuriyan and Cowburn, 1997).Historically, intervening glutamic acid residues extend across the surface of the phosphopeptide library-binding studies of SH2 specifi- domain.The Lck SH2 domain is shown as a solvent-accessible city established a paradigm for such studies of modular surface represented by white dots, the peptide is shown as a CPK signaling domains, and revealed distinct classes of model.Figure adapted from Eck et al.(1993) specificity for the three to five residues following the phosphorylated tyrosine (Songyang et al., 1993). Src- family SH2 domains bind preferentially to the pY-E-E-I The common-fold and phosphotyrosyl-binding prop- motif, coordinating the phosphotyrosine and isoleucine erties of the SH2 domain belie its versatility as a residues in the canonical recognition pockets (Figure 2). protein–protein recognition module.SH2 domains have Polar and electrostatic interactions favor glutamic acid evolved to function in diverse multidomain proteins, residues at the pY þ 1 and þ 2 positions, but many including transcription factors, ubiquitin ligases, other residues can be accommodated in these positions GTPase-activating proteins in addition to tyrosine in Src SH2 domains. kinases and phosphatases (Pawson and Nash, 2003).

Oncogene Structure and regulation of Src kinases TJ Boggon and MJ Eck 7920 In each of these contexts, the SH2 domain has evolved considerable variation in structure and binding modes critical interdomain interactions that confer particular (Mayer, 2001).For example, the SH3 domain of the recognition and/or regulatory properties.Thus, as with Gads T-cell adapter protein binds an RxxK motif rather most signaling domains, ‘modular’ does not imply than the characteristic PxxP motif (Berry et al., 2002; ‘interchangeable’.A more comprehensive discussion of Liu et al., 2003). Within the Src family, the Fyn SH3 can SH2 structure and speciﬁcity is beyond our scope, but bind the proline-independent motif RKxxYxxY found many excellent reviews are available (Sawyer, 1998; in the immune cell adaptor SKAP55 (Kang et al., 2000). Bradshaw and Waksman, 2002; Schlessinger and Additionally, the Fyn kinase is regulated by a non- Lemmon, 2003; Waksman and Kuriyan, 2004). proline ‘surface–surface’ interaction of its SH3 domain with the SH2 protein SAP (Chan et al., 2003; Latour The SH3 domain et al., 2003).

The SH3 domain has a b-barrel architecture consisting The tyrosine kinase domain of five antiparallel b-strands and two prominent loops, termed the RT and n-Src loops (Musacchio et al., 1992; Src kinases share the bilobal protein kinase fold Yu et al., 1992; Noble et al., 1993). These loops lie at (Knighton et al., 1991a) characteristic of all tyrosine either end of a surface composed of aromatic and kinases and ser/thr kinases (Figure 3).The N-terminal hydrophobic residues that comprise the recognition site (or small) lobe is composed of five b-strands and a single for proline-rich sequences bearing the ‘PxxP’ motif. a-helix, termed the C helix, which is an important These sequences adopt a polyproline type II helical component of the regulatory mechanism deployed in Src conformation in complex with the SH3 domain.The kinases.The C-terminal (or large) lobe is predominantly PPII helix has a triangular cross section, the conserved a-helical, and contains the regulatory activation loop, prolines lie on the base of the triangle and intercalate which is the site of activating tyrosine phosphorylation with aromatic side chains on the SH3 surface.In the in Src and other kinases.Nucleotide binding and case of Src-family SH3 domains, additional specificity phosphotransfer occur in the cleft between the two and binding affinity are conferred by coordination of a lobes.The adenine moiety of the bound nucleotide is lysine or arginine residue just N- or C-terminal to the coordinated largely by interactions with the N lobe and PxxP core.The pseudosymmetry of the PPII helix allows a short hinge segment that connects the two lobes. two high-affinity-binding modes, which differ in the Bound nucleotide phosphates are in part coordinated by N- to C-terminal orientation of the bound peptide. the glycine-rich G loop (also termed the P-loop, for Src SH3 domains bind class I ligands with the motif phosphate binding) (Taylor et al., 1992; Hubbard and RxxPxxP in an orientation opposite to that of class II Till, 2000). ligands with the motif XpxxPxR (Feng et al., 1994, 1995; The first direct structural information for an Src- Lim et al., 1994). As with the SH2 domain, structural family tyrosine kinase domain was obtained from the analysis of numerous SH3 domains has revealed kinase domain of Lck in an active conformation, with

Figure 3 The three-dimensional structure of Src family tyrosine kinases.Ribbon diagrams representing crystal structures of autoinhibited c-Src and the active, Tyr 416-phosphorylated Lck kinase domain are shown on the left and right, respectively.In the assembled, autoinhibited structure, the SH3 domain makes intramolecular interactions with the linker and the N-terminal lobe of the kinase, while the SH2 domain binds the phosphorylated C-terminal tail.These interactions help to stabilize an inactive conformation in which the active site is disrupted by expulsion of the C helix.Also, the activation loop folds into a short helix with unphosphorylated tyrosine 416 oriented towards the active site cleft and buried.This activation loop arrangement further stabilizes the inactive conformation of the kinase domain and may also protect tyrosine 416 from inappropriate phosphorylation.In the active conformation of a Src catalytic domain, phosphorylation of tyrosine 416 stabilizes a very different conformation in the activation loop and also allows the C helix to assume its active position.Ribbon diagrams drawn from the coordinates of PDB depositions 2SRC (autoinhibited Src) and 3LCK (Lck kinase domain).Note that Lck residues are numbered as in Src for simplicity

Oncogene Structure and regulation of Src kinases TJ Boggon and MJ Eck 7921 Tyr 394 phosphorylated (the equivalent of Tyr 416 in (Kim et al., 2003) reveals that they share a similar zinc- Src) (Yamaguchi and Hendrickson, 1996).This struc- binding core, composed of a b-hairpin in Lck and a ture showed how phosphorylation of Tyr 394 pinned the short extended segment in CD4 or CD8a (Figure 4).In activation loop in an active conformation very similar the CD4 complex, this core region is augmented by to that observed in cyclic AMP-dependent kinase hydrophobic interactions between two helices, one in (Knighton et al., 1991a, b), but quite different from that Lck and the other in CD4.Interestingly, this interaction observed in the autoinhibited insulin receptor kinase buries a dileucine motif that is critical for internalization (Figure 3, right panel) (Hubbard et al., 1994). Further- of CD4 by the clathrin adapter protein AP-2 (Shin et al., more, it has provided an important reference point for 1990).Internalization of CD4 is known to require comparisons with the conformation of the inactive phosphorylation of Ser 408 in CD4, followed by kinase and in efforts to understand mechanistically the dissociation of Lck (Shin et al., 1991; Pitcher et al., rearrangements that lead to activation (Schindler et al., 1999).The structure of the complex rationalizes this 1999; Sicheri et al., 1997; Williams et al., 1997; Xu et al., observation, as Ser 408 is exposed and accessible for 1997, 1999).Crystallographic analysis of numerous phosphorylation, but the mechanism by which this tyrosine and serine/threonine kinases in both active phosphorylation promotes dissociation of the complex and inactive conformations has revealed a diverse set of remains obscure.Although the sequences required for structural deformations that lead to loss of catalytic co-receptor interaction are found only in Lck, the function in inactive kinases (Huse and Kuriyan, 2002). unique regions of other Src family members may These deformations involve key elements of the active mediate distinct binding interactions.In Fyn, for site, including the C helix (which contains a critical example, this region may associate with the cytoplasmic glutamic acid residue, Glu 310 in Src), the relative chains of the T-cell receptor via its amino-terminal orientations of the N- and C-lobes, the G-loop, and the region (Timson Gauen et al., 1992), but this interac- activation loop itself.Not surprisingly, the active site tion is weak and not well characterized.Additionally, conformations in active kinases are strikingly similar, as studies of chimeric Src/Yes proteins show that the they must be competent to catalyse the phosphotransfer Yes unique region cannot substitute for the corres- reaction.Comparison of inactive kinases shows ponding region of Src, supporting the notion of substantial conformational variability, as distinct reg- distinct interactions and function for this domain ulatory mechanisms involving phosphorylation, interdo- (Summy et al., 2003). main, and protein–protein interactions impinge upon these plastic elements of the catalytic core to control kinase activity in particular pathways.In Src, the SH3 and SH2 domains and phosphorylation of the activation loop and C-terminal tail comprise the key regulatory elements (Sicheri et al., 1997; Williams et al., 1997; Xu et al., 1997, 1999; Schindler et al., 1999).

Structure of the unique domain of Lck The most N-terminal segment of the Src family architecture, the unique domain, has been the last to yield to structural analysis, perhaps in part because this region in isolation may lack a deﬁned structure in many cases.The function of this domain is unclear for most members of the family, but in Lck it has long been known to mediate association with the cytoplasmic tails of T-cell coreceptors CD4 and CD8a (Rudd et al., Figure 4 Structures of the Lck unique region in complex with 1988; Veillette et al., 1988; Shaw et al., 1989). The CD4 and CD8a cytoplasmic tails.The three-dimensional NMR Lck/coreceptor interactions require conserved cysteine structures of the Lck unique domain in complex with the motifs: a CxCP motif in CD4 and CD8a and a CxxC cytoplasmic tails of T-cell coreceptors CD4 (left) and CD8a (right) are shown in a ribbon representation.Lck is shown in dark blue motif in the Lck unique domain (Shaw et al., 1990; and the cytoplasmic tails of CD4 and CD8a are shown in gray.The Turner et al., 1990). These four conserved cysteine bound Zn2 þ ion is indicated as a blue sphere.Portions of the Lck residues (two from Lck and two from either of the unique domain and coreceptor tails that are unstructured and not coreceptors) coordinate a Zn2 þ ion that is critical for included in the complex are represented by dotted lines.The Zn 2 þ - complex formation (Huse et al., 1998; Lin et al., 1998). binding core of the complexes is formed by two cysteine residues in Lck (Cys 20 and Cys 23) and two in either of the coreceptors Recent solution NMR analysis of the Lck unique region (cysteines 420 and 422 in CD4, 194, and 196 in CD8a), small but and the CD4 and CD8a cytoplasmic tails has demon- signiﬁcant hydrophobic cores also help stabilize each complex.The strated that each of these sequences are unstructured in interaction between Lck and CD4 is regulated; phosphorylation of isolation, but that they fold together to form stable, serine 408 in CD4 promotes coreceptor dissociation and internalization.The dileucine internalization motif in CD4 (leucines 413 ternary Lck : Zn : CD4 and Lck : Zn : CD8a complexes in and 414) that is required for recognition by the clathrin adaptor 2 þ the presence of Zn (Kim et al., 2003). The elucidation complex is buried by the interaction with Lck, explaining the need of the three-dimensional structures of these complexes for dissociation of Lck prior to receptor internalization

Oncogene Structure and regulation of Src kinases TJ Boggon and MJ Eck 7922 The autoinhibited conformation of Src autoinhibited conformation has important implications for controlling the activation of Src kinases. et al Crystal structures of both Src (Williams .,. 1997; Xu Why is this assembled conformation of Src catalyti- et al., 1997, 1999) and Hck (Sicheri et al., 1997; cally compromised? The position of the SH2 and SH3 Schindler et al., 1999) in the autoinhibited, tail- domains does not sterically occlude the catalytic cleft. phosphorylated state show that the SH3 and SH2 Rather, these domains lock the kinase domain in an domains turn inward and make intramolecular interac- inactive conformation (Figure 5).The principle distor- tions that lock the catalytic domain in an inactive tions of the active site that render it inactive are (i) the conformation.The SH3 and SH2 domains pack against displacement of the C helix, which removes the the catalytic domain on the side opposite the catalytic catalytically important Glu 310 from the active site cleft.The SH3 domain packs against the N lobe of the cleft, (ii) the activation loop adopts an a-helical kinase, while the SH2 domain packs against the C lobe. conformation, which precludes binding of peptide This ‘assembled’ conformation is stabilized by intramo- substrates and also sequesters Tyr 416, making it lecular interactions of the SH3 and SH2 domains with inaccessible for phosphorylation, and (iii) the relative ﬂexible polypeptide segments; the SH3 domain binds the orientation of the N and C lobes is constrained in an linker segment that connects the SH2 and kinase orientation that may not be optimal for catalysis domains, and the SH2 domain binds the phosphorylated (Sicheri et al., 1997; Williams et al., 1997; Xu et al., C-terminal tail (Figure 3, left panel).Importantly, the 1997, 1999; Schindler et al., 1999). intramolecular sequences coordinated by the SH3 and The precise structural mechanism by which the SH3 SH2 domains are not ‘high-afﬁnity’ binding sites.The and SH2 domain assembly promotes displacement of linker segment or Src does not contain the PxxP motif, the C helix is not completely clear.However, one can but the linker of both Src and Hck does adopt a PPII gain insight into this question by considering the C helix helical conformation in association with their respective to have two preferred conformations, ‘active’ and SH3 domains.Similarly, the C-terminal tail bears a ‘displaced’, which may differ little in intrinsic stability. phosphorylated tyrosine, but has a glycine, rather than Thus, the switch from active to displaced (or vice versa) the preferred isoleucine, at the pY þ 3 position.As could be effected by factors that either stabilized the discussed below, the fact that these targeting domains displaced position or destabilized the active conforma- use their ligand-binding surfaces to maintain the tion.The SH3 and SH2 domains may in part promote

Figure 5 Detailed schematic of the active site in inactive vs active Src kinases.The active site of a Src kinase in an inactive conformation (left panel, Src) and in the active conformation (right panel, Lck).Portions of the N lobe are shown in blue, the activation loop in green and the catalytic loop in red.Changes in the positioning of Glu-310 and the C helix between the active and inactive conformations are evident.In the inactive state, the C helix is rotated outward, and Glu 310 is coordinated by Arg 409 and Tyr 382.Leu 407 and ﬂanking residues in the activation loop also help to ﬁx the C-helix in the inactive position.Phosphorylation of Tyr 416 restructures the activation loop, allowing the C helix to rotate inward and Glu 310 to form the requisite interaction with Lys 295 (which in turn coordinates the phosphates of the bound ATP, not shown).Note that in the active state Arg 409 forms a salt bridge with the phosphorylated Tyr 416 rather than with Glu 310; this electrostatic switch is one component of the activation mechanism.Figure adapted from Xu et al.(1999)

Oncogene Structure and regulation of Src kinases TJ Boggon and MJ Eck 7923 the displaced conformation by pinning the linker order to maintain the inactive conformation (Young segment against the N lobe of the kinase domain. et al., 2001). Molecular dynamics studies of the Tryptophan 260 lies at the junction between the linker assembled Src kinase reveal tight correlation in the and the N lobe, and packs against the carboxy-terminal movements of the N lobe and SH3 domain, consistent end of the C helix in the assembled conformation, where with the intimate association observed in crystal it helps to stabilize the displaced, inactive position of the structure (Young et al., 2001). Furthermore, these C helix.In support of this idea, in the structure of the studies indicate that these correlated motions extend active Lck kinase domain, this residue adopts an from the SH3 domain into the SH2 domain, suggesting alternate conformation and does not contact the C that conformational coupling between these domains is helix (Yamaguchi and Hendrickson, 1996).Also, required for effective autoinhibition.This prediction has mutagenesis studies support the notion that Trp 260 been experimentally confirmed, glycine mutations in the helps to communicate the position of the SH2 and SH3 short turn connecting the SH3 and SH2 domain domains to the active site (LaFevre-Bernt et al., 1998). deregulate Src (Young et al., 2001). Additional residues in the linker may also participate In summary, in an assembled state, Src kinases are (Gonfloni et al., 1997). In particular, Leu 255 inserts catalytically compromised by the displacement of the C into a hydrophobic pocket on the back of the N lobe helix.The SH3 and SH2 domains are turned inward, and may stabilize the displaced conformation (Gonfloni their recognition surfaces sequestered by weak intramo- et al., 1999). lecular interactions.Additionally, the helical conforma- The SH2 and SH3 domains also communicate to the tion of the activation loop precludes binding of peptide C helix via the activation loop.In the assembled substrates and also protects Tyr 416 from phosphoryla- conformation, the activation loop forms a short helix, tion.The interdependencies in the positions and con- which is propped between the N and C lobes (Schindler formations of the SH3 and SH2 domains, the kinase et al., 1999; Xu et al., 1999). In this conformation, Tyr lobes, the activation loop, and the C helix suggest that 416 is buried in the cleft between the lobes of the kinase, maintenance of the autoinhibited conformation relies on where it is not accessible for phosphorylation.Further- this set of cooperative, self-consistent interactions.The more, the more N-terminal portion of the activation autoinhibited Src kinase is a precariously set mousetrap, loop, including Leu 407, creates a steric barrier that small perturbations in key interdomain interactions can blocks the active position of the C helix (Figure 5). be sufficient to spring the kinase into its active Release of the SH3/SH2 clamp is likely to induce conformation.Indeed, the oncogenic v-Src protein lacks disordering of the helical activation loop and removal of the autoinhibitory phosphorylation site in the the blockage created by Leu 407.Subsequent phosphor- C-terminal tail (Takeya and Hanafusa, 1983; Cooper ylation of Tyr 416 creates an electrostatic switch that et al., 1986). Additionally, mutations in the RT loop further promotes the active position of the C helix and of the SH3 domain that perturb the SH3-kinase induces a new conformation in the activation loop interaction (Kato et al., 1986) or mutations in the SH2 (Figure 5).The activation loop conformation in the domain that block binding of the phosphorylated tail active Lck kinase structure closely resembles that in the are sufficient to activate Src. insulin receptor tyrosine kinase (IRTK) (Yamaguchi and Hendrickson, 1996).In IRTK, the phosphorylated activation loop forms a b-sheet interaction with a bound substrate peptide (Hubbard, 1997).By analogy with Insights into Src activation IRTK, the phosphorylated and subsequently reorga- nized activation loop in Src kinases is thought to form a Why such a complex, multicomponent mechanism of considerable portion of the binding surface for substrate autoinhibition? The architecture of the assembled Src peptides; thus, autoinhibited Src is unable to bind kinase appears to have evolved to create an intrinsic peptide substrate. coupling of activation of Src with targeting to its Finally, the restriction of interdomain mobility caused appropriate cellular substrates.Src kinases are known to by the SH3/SH2 clamp may also directly inhibit the be activated by binding of cognate ligands to their SH2 kinase, both because the relative orientation of the N and/or SH3 domains (Brown and Cooper, 1996).Src is and C lobes induced by the clamp do not appear to be both recruited to, and activated by, the PDGF receptor ideal for catalysis and because greater flexibility may be via interaction of its SH2 domain with the tyrosine- required for efficient catalysis and for turnover of the phosphorylated receptor (Kypta et al., 1990; Alonso nucleotide substrate.The effectiveness of the clamp et al., 1995). A number of Src substrates contain binding appears to depend upon a rigid coupling between the motifs for both the SH3 and SH2 domains of Src; focal SH3 and SH2 domains (Young et al., 2001). Although adhesion kinase (Fak) is one example (Thomas et al., NMR and crystallographic analysis of SH3–SH2 frag- 1998; Sieg et al., 1999). The closely spaced proline and ments of Src kinases indicated that there is no defined phosphotyrosine motifs in Fak have been shown to bind orientation between the two domains when they are simultaneously to an SH3/SH2 fragment of Fyn, ‘excised’ from the intact kinase (Eck et al., 1994; Arold although the tandem interaction does not appear to et al., 2001), computational and biochemical analyses confer an avidity effect (Arold et al., 2001). Addition- show that, in the context of the intact kinase, a relatively ally, Src kinases can be activated by displacement of rigid coupling between these two domains is required in their SH3 domain, while the SH2 domain remains

Oncogene Structure and regulation of Src kinases TJ Boggon and MJ Eck 7924 engaged with the C-terminal tail.This effect has been SLAM cytoplasmic tail with its phosphopeptide-binding demonstrated in vitro by activation of Hck with the HIV site and simultaneously bind the Fyn SH3 domain via a protein Nef (Moarefi et al., 1997). Nef contains a PxxP distinct binding surface (Chan et al., 2003). SAP is also motif within a loop that is preconfigured in a PPII an unusual SH2 domain in that it binds SLAM in a conformation and confers tight binding to the Hck SH3 ‘three-pronged’ fashion which tolerates, but does not domain (Lee et al., 1995). require, phosphorylation of SLAM (Poy et al., 1999). More recently, Fyn has been discovered to be Binding of SAP to the Fyn SH3 domain is expected to recruited to SLAM-family receptors and activated by disrupt the autoinhibitory interaction of the SH3 interactions of its SH3 domain with the adapter protein domain, and therefore to unleash the catalytic activity SAP (SLAM-associated protein) (Latour et al., 2001) of Fyn (Chan et al., 2003; Latour et al., 2003). The (Figure 6).SLAM receptors are present on T and NK activation of Fyn by SAP and SLAM is critical for cells, as well as B cells and other antigen-presenting cells, proper immune modulation; inherited mutations in where they make homotypic ‘self-ligand’ interactions SAP which abolish or weaken binding to SLAM are that are critical for a proper immune response (Engel the basis of X-linked lymphoproliferative syndrome et al., 2003). SAP is an unusual adapter protein in that it (Engel et al., 2003). consists of a single SH2 domain, but it can nevertheless The SLAM/SAP/Fyn interaction also highlights function as an adapter protein because it can bind the significant gaps in our structural understanding of Src

Figure 6 Insights into the activation of Src kinases: recruitment and activation of Fyn by SLAM/CD150 coreceptors in immune cells. In FynT, as in all Src kinases, activation is thought to be accomplished by release of the inhibitory intramolecular interactions of the SH2 and SH3 domains, accompanied by autophosphorylation of the activation loop.FynT is a lymphocyte-speciﬁc isoform of Fyn, and is brought to the cytoplasmic tail of SLAM via interaction of its SH3 domain with the adapter protein SAP.SAP is an SH2-only protein that can simultaneously bind SLAM and the FynT SH3 domain.The structure of the entire complex has not been elucidated, but the crystal structure of a SLAM/SAP/Fyn-SH3 complex is known (inset).In the assembled conformation of FynT (left), the SAP- binding site on the SH3 domain is occluded by intramolecular interactions; recruitment by SAP is expected to activate the kinase by displacement of the SH3 domain (right).The active Fyn kinase phosphorylates indicated tyrosines in the SLAM tail.The FynT SH2 domain may also bind one of these sites, further stabilizing the active signaling complex.Also, SAP may interact directly with the catalytic domain of FynT.The ribbon diagram of the SLAM/SAP/FynSH3 complex was drawn from the coordinates of PDB deposition 1M27

Oncogene Structure and regulation of Src kinases TJ Boggon and MJ Eck 7925 kinase activation (Figure 6).Although the active similar to that of Src kinases (Nagar et al., 2003), despite conformation of the Lck kinase domain is known, the fact that Abl lacks the autoinhibitory tail phosphor- relatively little is known about how full-length Src ylation site that is crucial for Src inhibition.This kinases may assemble in active signaling complexes.Are similarity was expected, and reinforced by mutagenesis there precisely defined interdomain or interprotein studies based upon the Src structures and sequence interactions in the active state akin to those observed comparisons, but it remained unclear how Abl was in autoinhibited Src? How do Src kinases recognize their maintained in an inactive conformation in the absence protein substrates? Do the SH2 and SH3 domains of the ‘latch’ provided by the SH2–tail interaction in Src ‘direct’ substrate to the kinase domain in a precise kinases.Structure/function studies of Abl by Superti- manner, or do they simply co-localize the kinase with Furga and colleagues defined an N-terminal ‘cap’ region substrate? To date, no Src kinase has been crystallized in in Abl that appeared to stabilize its autoinhibited complex with a peptide/protein substrate.In the case of conformation (Pluk et al., 2002). The crystal structure the SAP/Fyn interaction, SAP may bind to the Fyn of Abl confirmed this prediction, and quite unexpectedly kinase domain in addition to the SH3 domain (Chan revealed that the N-terminal myristoyl group contri- et al., 2003). Additionally, phosphorylation of addi- butes to autoinhibition by inserting into a hydrophobic tional tyrosines in SLAM is required for stable pocket within the C lobe of the kinase domain.This association with Fyn, suggesting that the Fyn SH2 deep hydrophobic pocket appears to be unique to Abl, domain may bind SLAM following initial recruitment and insertion of the acyl chain induces a conformational and phosphorylation of SLAM by Fyn (Latour et al., rearrangement on the back of the C lobe that creates the 2003).These additional interactions hint at the existence docking site for the SH2 domain (Nagar et al., 2003). of a conformationally defined, active signaling complex Thus, the myristoylated N-terminus functionally re- (Figure 6), but further structural analysis is needed to places the SH2–tail interaction in Abl, and, as in Src, address these questions. disruption of this latch leads to oncogenic activation Also, it is unclear whether the unique domain of some (Azam et al., 2003; Hantschel et al., 2003; Harrison, Src kinases may participate in catalytic regulation.The 2003; Nagar et al., 2003). In the oncogenic BCR-Abl available structures of Src and Hck lack the unique (the chimeric product of the translocated Philadelphia regions.One clue that the unique region of Src may serve chromosome in chronic myelogenous leukemia), the a regulatory function is the presence of serine phosphor- N-terminal myristoylation site and cap regions are ylation sites (Gould et al., 1985; Chackalaparampil and truncated and replaced with BCR-derived sequences. Shalloway, 1988; Shalloway et al., 1992). In Lck serines Also, in parallel with Src kinases, appropriate targeting 42 and 59 are phosphorylated by protein kinase C, and interactions are thought to disrupt the assembled state these phosphorylations affect the function and cata- and induce kinase activation (Nagar et al., 2003). lytic activity of the kinase (Watts et al., 1993; Winkler The ability of Src to be ‘turned on by touch’ is et al., 1993; Kesavan et al., 2002). Additionally, in the indispensable for regulating and maintaining fidelity in Src-family cousin Abl, the N-terminal ‘cap’ its signaling pathways.Diverse multidomain signaling region contributes to the autoinhibited conformation proteins share this property, in spite of the fact that their (see below). domain architectures are unrelated to that of Src.For example, in the protein tyrosine phosphatase SHP-2, which contains two adjacent SH2 domains followed by a the catalytic phosphatase domain, the N-terminal SH2 Src: turned on by touch domain binds back to the PTPase domain and sterically occludes the active site (Hof et al., 1998). The SH2 The structural investigations of Src kinases over the past domain uses a surface that does not overlap with its dozen years have provided insights that go well beyond phosphopeptide-binding groove.The surface–surface Src itself.Most obviously, they helped to elucidate the interaction with the phosphatase domain is nevertheless general mechanisms of recognition by SH2 and SH3 disrupted by binding of cognate phosphoproteins to the domains, which are near-ubiquitous in eukaryotic signal SH2 domains due to a conformational change within the transduction.Additionally, the SH3–SH2-kinase assem- N-terminal SH2 domain that disrupts its PTPase- blage has been reused in evolution with only minor binding surface.As with Src and Abl, mutations in modifications.The Abelson kinase (Abl), and likely Tec SHP-2 that uncouple targeting and catalytic activation family kinases as well, adopt an assembled conforma- (without directly compromising either) result in disease tion very similar to that of Src kinases (Nagar et al., (Tartaglia et al., 2001; Neel et al., 2003). 2003).More generally, the underlying principle revealed A feature that is implicit in the design of these by the structural organization of Src – the use of protein proteins, but perhaps not immediately obvious, is an interaction domains to regulate catalytic activity in a enhancement of specificity that arises from the use of manner that inextricably couples targeting with catalytic targeting domains for autoinhibition.This property is activation – applies quite frequently, if not universally, readily explained in autoinhibited Src – the SH3 and SH2 in modular signaling proteins (Pawson and Nash, 2003). domains have intramolecular binding partners of modest Recent structural analysis of Abl in an autoinhibited affinity, thus they only bind and become activated by conformation reveals that its SH3, SH2, and kinase specific high-affinity binding partners that can outcom- domains adopt an assembled conformation extremely pete these intramolecular interactions.More generally, in

Oncogene Structure and regulation of Src kinases TJ Boggon and MJ Eck 7926 any ‘allosteric’ system, a portion of energy of ligand assembled with each other, but are greatly displaced binding is used to induce conformational change, and is from the autoinhibitory position on the back of the therefore ‘lost’ as intrinsic binding energy.Regulatory kinase domain.Essentially, the clamp is intact, but intramolecular interactions effectively ‘raise the bar’ for released from the kinase domain, because the C-terminal specific binding.Thus, Src is only turned on by the touch tail is unphosphorylated and does not bind the SH2 of its intended, specific partners. domain.In this structure, the kinase domain adopts a One might expect release of the SH3/SH2 clamp alone conformation very similar to that in the active Lck to be sufficient to activate Src, absent Tyr 416 kinase domain, despite the fact that Tyr 416 is not phosphorylation, because Tyr 416 is thought to be phosphorylated (D Fabbro and S Cowan-Jacob, perso- autophosphorylated.Strikingly, recent crystallographic nal communication).Thus, this structure provides an analysis of an SH3-SH2-kinase fragment of Src in an important snapshot of the Src activation pathway, and unphosphorylated state bears out this prediction.This it provides direct support for the notion that Src structure reveals a heretofore unseen conformation in kinases have evolved to intrinsically couple activation which the SH3, SH2, and linker segments remain with targeting.

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