Revealing mechanisms for SH2 domain mediated regulation of the protein tyrosine phosphatase SHP-2 David Barford 1 * and Benjamin G Neel 2

Minireview 249 Revealing mechanisms for SH2 domain mediated regulation of the protein tyrosine phosphatase SHP-2 David Barford 1 * and Benjamin G Neel 2 The crystal structure of the protein tyrosine phosphatase SHP-2 reveals the mechanism of auto-inhibition of phosphatase activity by its SH2 domains. Phosphotyrosine peptide stimulation of the phosphatase activity, resulting from peptide binding to the N-terminal SH2 domain, is linked to conformational changes within the protein, including an unprecedented allosteric transition of the N-terminal SH2 domain. Addresses: 1 Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Building, South Parks Road, Oxford OX1 3QU, UK and 2 Division of Hematology and Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. *Corresponding author. E-mail: davidb@biop.ox.ac.uk Structure 15 March 1998, 6:249 254 http://biomednet.com/elecref/0969212600600249 Current Biology Ltd ISSN 0969-2126 Intracellular signalling is mediated by the modulation of protein protein and protein lipid interactions associated with the assembly of multiprotein complexes [1]. Such processes function not only to alter the conformation of signalling molecules but also to recruit them to their cellular targets. An important example of this concept is the activation of cell surface receptors by extracellular ligands, including hormones, growth factors, cytokines and antigens, that results in receptor tyrosine phosphorylation by either integral or associated protein tyrosine kinases. Tyrosine phosphorylation initiates downstream signalling pathways by creating sequence-specific recognition sites for Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains and hence assists the assembly of multiprotein complexes. Signalling molecules are formed from relatively few protein families, with diversity of structure and function being generated by the shuffling of protein modules and domains endowed with specific functions: for example SH2 and SH3 domains and protein kinase and phosphatase activities [2]. In addition to recruiting proteins to activated receptors, SH2 and SH3 domains may also autoregulate enzyme catalytic activity. In the absence of a stimulatory phosphotyrosine (ptyr) signal, most SH2 domain containing enzymes, for example the protein tyrosine kinases Src, ZAP-70 and Syk, SHP protein tyrosine phosphatases (mammalian SHP-1, SHP-2 and Drosophila corkscrew), phosphatidylinositol-3-kinase and phospholipase C-γ1, are catalytically inactive as a consequence of SH2 domain mediated auto-inhibition. These proteins are reminiscent of allosterically regulated metabolic enzymes, existing in dynamic equilibrium between activated and inactive states. In this instance, the allosteric activator is a tyrosine phosphorylated receptor that stabilises the activated conformation of the enzyme. The crystal and NMR structures of SH2-domain phosphopeptide complexes have demonstrated the molecular mechanisms underlying sequence specificity for ptyr peptides [3]. More recently, the structures of Src and hematopoietic cell kinase (Hck) revealed the mechanism of regulation of the protein tyrosine kinase (PTK) domain of these enzymes by their N-terminal SH2 and SH3 domains [4,5]. Now in a recent paper, Shoelson and colleagues report the crystal structure of the SH2 domain containing protein tyrosine phosphatase (PTP), SHP-2 [6]. This work presents the first structure of an intact double SH2 domain containing protein, revealing the mechanisms of regulation of phosphatase activity by the SH2 domains. SH2 domain mediated regulation of phosphatase activity of SHP-2 contrasts strikingly with the mode of SH2 and SH3 domain mediated regulation of Src and Hck. Taken together, the SHP-2 and Src family protein kinase structures provide a framework for understanding the regulation of other SH2 domain containing proteins. Higher eukaryotes encode two SH2 domain containing PTPs, SHP-1 and SHP-2, which although highly related in structure (sharing 60% overall sequence identity) display distinct tissue distributions and biological functions [7]. Both SHP-1 and SHP-2 associate with activated receptors in vivo and in vitro via specific SH2 domain mediated recognition of ptyr residues [8,9]. SHP-1 is largely restricted to cells of hematopoietic origin and epithelial cells, whereas SHP-2 is expressed in all cell types. The physiological function of SHP-1 is to negatively regulate several different types of signal transduction pathways, including those associated with cytokine receptors, immune recognition receptors (such as the B and T cell antigen receptors) and certain tyrosine kinases. The precise targets of SHP-1 are controversial, but may include the receptors themselves, receptor-associated PTKs, such as Janus kinases, and/or other tyrosyl phosphorylated signalling components. In contrast to SHP-1, SHP-2 positively regulates signal transduction events from activated receptor protein

250 Structure 1998, Vol 6 No 3 tyrosine kinases (RTKs) including those for growth factors, hormones and cytokines. The precise targets of SHP-2 are also unknown, but may include daughter of sevenless (DOS)-like molecules or the adhesion molecules SIRPs (signal regulation proteins). SHP-2 performs essential roles in developmental processes: the enzyme is required for fibroblast growth factor (FGF)- induced mesoderm patterning and completion of gastrulation in Xenopus [10,11] and mouse embryos [12]. The Drosophila homologue of SHP-2, corkscrew [13], was initially found to be required for signalling from the RTK torso, which directs early embryonic head and tail development. Subsequent work has established that corkscrew is required for other RTKs-triggered pathways, including those for sevenless [14], breathless and Der [15]. Like other PTP and SH2 domain containing proteins, SHP-1 and SHP-2 are constructed on a modular basis. The proteins contain two SH2 domains at the N terminus (N-SH2 and C-SH2, where N-SH2 is closest to the N terminus) followed by a PTP domain. The C-terminal 66 amino acid residues of SHP-2 contain sites of tyrosine phosphorylation and a proline-rich domain that may be capable of binding to SH3 or WW domain containing proteins. Phosphorylation of these tyrosine residues by activated PTKs creates binding sites for the adapter molecule Grb-2, suggesting a potential role for SHP-2 as an adapter protein within certain signalling pathways. Earlier studies established that both SHP-1 and SHP-2 are essentially inactive under basal conditions, resulting from auto-inhibition of the PTP domain by the N-SH2 domain. The isolated phosphatase domain retains full catalytic activity [16] that is suppressed by addition of the SH2 domains [17]. Catalytic activity of both SHPs is increased on engagement of ptyr peptides by the SH2 domains. Singly phosphorylated peptides increase activity tenfold [9,18], whereas doubly phosphorylated peptides, which engage both SH2 domains simultaneously, cause a more dramatic 100-fold activation at greatly reduced phosphopeptide concentrations [19,20]. Specific inactivation of the ptyr recognition sites of the N-SH2 and C-SH2 domains, achieved by mutating an invariant phosphate-coordinating arginine residue (Arg βb5) in both SHP-2 [9,19] and SHP-1 [21], or truncation of either the N-SH2 or C-SH2 domains of SHP-1 [21,22], revealed that the N-SH2 domain is responsible for the inhibition of PTP activity. Engagement of ptyr peptides by this domain contributes to almost all the ptyr peptide induced enzyme activation, with virtually no contribution from the C-SH2 domain. These results probably explain why the N-SH2 domain plays a more important role than the C-SH2 domain in SHP-2-mediated FGF signalling in Xenopus [11]. Optimal signalling, however, requires both SH2 domains. An understanding of the molecular basis for the autoinhibition of the PTP domain of SHP-2 by its SH2 domains and for the stimulation of catalytic activity by ptyr peptides has been provided by the crystal structure of the enzyme determined by Shoelson and colleagues [6]. The structure corresponds to the full-length protein with a truncation of the C-terminal 66 residues. This form of SHP-2 is activated by phosphopeptides in an identical manner to the wild-type enzyme [6]. The mechanism of inhibition of the PTP domain is close to that predicted previously. However, the nature of the allosteric conformational changes which lead to activation of the PTP domain by phosphopeptide engagement of the N-SH2 domain is a surprise, and illustrates a previously unrecognised structural transition within an SH2 domain. The inactive form of SHP-2 adopts a closed conformation with the N-SH2 domain interacting extensively with the PTP domain, directly blocking the phosphatase catalytic site (Figure 1). The C-SH2 domain is linked to the neighbouring N-SH2 and PTP domains but does not share a significant interface with either of these two domains. Unexpectedly, the ptyr peptide binding sites of both SH2 domains are exposed to solvent and do not form part of the interdomain recognition surfaces. The architecture of the SH2 and PTP domains of SHP-2 are very similar to those of other SH2 and PTP domains determined previously [3,23,24]. The sequences of the PTP domains of the SHPs are more closely related to receptor PTPs (RPTPs) than to other cytosolic PTPs. This relationship is reflected in the three-dimensional structure of the PTP domain of SHP-2. The structure most closely resembles domain 1 of RPTPα and RPTPµ [25,26], possessing an additional twostranded β sheet which is absent from the cytosolic PTPs, PTP1B and Yersinia YopH (Figure 1). PTPs catalyse dephosphorylation reactions by means of a thiol phosphate enzyme intermediate involving an invariant cysteine residue. This catalytic cysteine residue is located at the base of a catalytic site cleft (9 Å in depth) that determines specificity for ptyr-containing peptides and precludes binding of shorter pser and pthr sidechains [27]. The cysteine residue is incorporated within a phosphate-binding cradle formed by the PTP signature motif sequence and composed of mainchain amide groups and the sidechain of an invariant arginine residue. Binding of a ptyr residue to the catalytic site is accompanied by closure of a surface, the WPD, which brings an essential catalytic aspartic acid residue within hydrogen-bonding distance of the scissile phenolic oxygen atom of the ptyr substrate, creating a catalytically competent enzyme conformation. The phenyl ring of the ptyr residue forms hydrophobic interactions with a tyrosine sidechain of the ptyr-recognition and with residues of the WPD. A fourth catalytic site, the Q, containing an invariant glutamine residue that

Minireview A dual SH2 domain phosphatase Barford and Neel 251 Figure 1 The overall structure of SHP-2 [6]. The N- SH2, C-SH2 and protein tyrosine phosphatase (PTP) domains are shown in yellow, green and cyan, respectively. The catalytic site s of the PTP domain are indicated: the PTP is in red; the ptyrrecognition is in green; the WPD is in yellow; and the Q is in white. The position of the N-SH2 domain D E, which inserts into the PTP domain catalytic site, and sidechain of the catalytic cysteine residue are also indicated together with the SH2 domain EF and BG s. (The figure was produced using MOLSCRIPT [33].) coordinates the nucleophilic water molecule during the thiolysis reaction [28], is situated adjacent to the ptyrrecognition and WPD s (Figures 1 and 2). In the inactive form of SHP-2, the D E and adjacent D and E β strands of the N-SH2 domain insert deep into the catalytic site cleft of the PTP domain forming interactions with residues of the catalytic s (i.e. the phosphatebinding cradle, the ptyr-recognition and the Q ; Figures 1 and 2). These interactions inhibit enzyme activity for two reasons. Firstly, Asp61 and Tyr62 of the D E of N-SH2 mimic the interactions of a ptyr substrate with the catalytic domain and sterically block access of phosphopeptides to the catalytic site. The sidechain of Asp61 forms water-mediated hydrogen bonds to the mainchain groups of the ptyr-binding cradle and the sidechain of the catalytic cysteine residue, whereas the sidechain of Tyr62 interacts with Tyr279 (equivalent to Tyr46 of PTP1B) within the ptyr-recognition. Secondly, the N-SH2 domain inactivates the phosphatase by restraining the WPD in the open, catalytically inactive conformation. Blockade of the catalytic site may be a common mechanism of enzymatic inhibition of PTPs. Bilwes et al., observed that the membrane proximal PTP domain of the receptor-like phosphatase, RPTPα, crystallised as a dimer whereby the catalytic site of each subunit was sterically blocked by a wedge connecting two helices, α1 and α2, within a helix-turn-helix segment from the opposite subunit of the dimer. Residues of this wedge directly interact with equivalent catalytic site residues that are part of the phosphopeptide-binding site of PTP1B and restrain the WPD in the inactive conformation [25]. These structural data have been used to explain the signalling properties of a chimaeric molecule consisting of the extracellular domain of the epidermal growth factor (EGF) receptor and the transmembrane and cytosolic portion of the RPTP CD45 [29], although this model is unlikely to be valid for all RPTPs [26]. This chimaera restores signalling within CD45 deficient T cells only in the absence of EGF. It was proposed that in the presence of growth factor, dimerisation of the chimaera is induced with resulting inhibition of CD45 phosphatase activity [25]. Stimulation of the PTP activity of SHP-2 by association of the N-SH2 domain with a phosphopeptide results from an unexpected allosteric transition within the SH2 domain. Although the phosphopeptide-recognition site of the N-SH2 domain is distinct and non-overlapping with the site of interaction between the SH2 domain and the PTP domain, the two sites are linked via an allosteric change between two distinct SH2 domain conformations: an active (A) peptide-bound state and an inactive (I) PTP domain interaction state (Figure 3). Compared with the structures of the isolated N-SH2 domain, both in the absence of and in complex with peptide, the residues of N-SH2 that interact with the PTP domain of SHP-2 (i.e. residues of the secondary β sheet βd, βe and βf) and helix αb, have undergone a concerted conformational change of approximately 2 Å (Figure 3) [20,30]. Motion of the αb helix links the PTP-binding site of N-SH2 to a conformational change of the EF, which together with the adjacent BG forms the SH2 domain binding site for phosphopeptide residues C-terminal to the ptyr

252 Structure 1998, Vol 6 No 3 Figure 2 Mechanism of inhibition of the protein tyrosine phosphatase (PTP) domain of SHP-2 by the N-SH2 domain. (a) The structure of the catalytic site of the PTP domain of SHP-2 [6] and comparison with (b) the structure of a phosphotyrosine peptide (in yellow) bound at the catalytic site of PTP1B [27]. Residues of the N-SH2 domain D E of SHP-2 occupy a similar position to the peptide bound to the PTP1B catalytic site. A water molecule bridging the sidechains of Cys459 and Asp61 is shown as an orange sphere. The colours of the domains and catalytic site s are as described in Figure 1. residue. The two conformations of the N-SH2 domain possess two mutually exclusive properties. The I conformation is competent to bind to the PTP domain, sharing surface complementarity with the catalytic site, but contains a blocked phosphopeptide-binding site as a result of the motion of the EF towards the BG. In contrast, the phosphopeptide-binding state (A) possesses a disrupted PTP domain recognition surface. The allosteric transition from the I to the A state of the N-SH2 domain is regulated by stimulatory ptyr peptides that bind to the N-SH2 domain. A conformational transition of this nature within SH2 domains has not been previously observed. The authors suggest that the presence of an alanine residue at position αb4 of the B helix, unique to the N-SH2 domains of SHPs, facilitates motion of the αb helix relative to the central β sheet of the SH2 domain. Within over 70 other SH2 domains, more bulky valine, isoleucine or leucine residues occur at this position, which create a more rigid interface between the αb helix and the β sheet [6]. The residues involved in mediating the inhibition of the PTP domain of SHP-2 are conserved within SHP-1 and corkscrew, suggesting that these enzymes are subject to the same regulatory mechanisms. The C-SH2 domain forms few interactions with the PTP and N-SH2 domains and the peptide-binding site is remote from any interdomain interface. Moreover, the conformation of the C-SH2 domain within SHP-2 is similar to its conformation within the crystal structure of the isolated N-SH2 and C-SH2 domains with bound peptide [20]. These observations are largely consistent with data indicating that phosphopeptide binding to the C-SH2 domain alone has little to no stimulatory affect on PTP activity in SHP-2 [9,19] and SHP-1 [21]. One observation not fully explained by the crystal structure of SHP-2 is the tenfold higher activation of SHPs by doubly phosphorylated peptides relative to singly phosphorylated peptides [19]. The structure would predict that as the C-SH2 domain does not contribute to inhibition of the PTP domain, at concentrations of singly phosphorylated peptides sufficiently high to saturate the N-SH2 domain the activity of SHPs should be equivalent to that stimulated by doubly phosphorylated peptides. One explanation as to why this is not the case may be that inhibition of the PTP domain occurs at the peptide concentrations required to saturate the N-SH2 domain. However, this would not explain the findings that both SHP-2 with mutations in the C-SH2 domain Arg βb5 residue [9], and SHP-1 lacking the entire C-SH2 domain [21], were stimulated to a lower extent by certain singly phosphorylated peptides than the wild-type enzyme. An alternative explanation, that peptide engagement to the C-SH2 domain provides some stimulatory response, therefore seems likely. Consistent with this idea are the properties of mutant SHP-2, with N-SH2 domain Arg βb5 substitutions, that can be activated by ptyr peptides [9,19]. It is possible that the C-terminal 66 residues, missing from the SHP-2 crystal structure, play a role in the C-SH2 domain mediated regulation of PTP activity. A recent report demonstrated that a characteristic of tandem SH2 domains is an increased selectivity for phosphopeptides compared with single SH2 domains. Moreover, the relative arrangement of SH2 domains within tandem repeats can force spatial restraints on the recognition of phosphopeptides [31]. Thus, the C-SH2 domain within SHPs probably performs numerous functions. Firstly, this domain allows recruitment of SHP-2 to ptyr sites within activated receptors and, as a result of cooperativity effects, increases the effective binding affinity of the

Minireview A dual SH2 domain phosphatase Barford and Neel 253 Figure 3 Stereo view showing the structural transition between the I and A states of the N-SH2 domain of SHP-2. In particular the figure shows changes in the βd, βe and βf strands, αb helix and EF. The view of SHP-2 is similar to that in Figure 1 with the N-SH2 domain (I state) in yellow. The structure of the isolated N- SH2 domain with bound peptide (representing the A state) [30] is in red superimposed onto the I state of the N-SH2 domain [6]. The A state of N-SH2 has a disrupted PTPrecognition surface whereas the I state of N- SH2 has a blocked phosphopeptide-binding site as a result of motion of the EF. The PTP domain is shown in blue, the C-SH2 domain in green and catalytic domain s are in purple. The structure of the platelet-derived growth factor (PDGF)-1009 peptide bound to the N-SH2 domain A state is shown in balland-stick representation. Cys 459 PTP PTP WPD Structure ptyr D E EF Q BG αb N-SH2 (I) N-SH2 (A) C-SH2 Cys 459 PTP PTP WPD ptyr D E EF Q BG αb N-SH2 (I) N-SH2 (A) C-SH2 N-SH2 domain for ptyr residues, hence compensating for the energetic costs of conformational changes and disruption of protein protein interactions. Secondly, tandem SH2 domains provide increased specificity in signal transduction pathways [20,31]. The structural data of Hof et al., [6] also provide insights into possible models for the in vivo properties of SHPs. For example, it is likely that engagement of ptyr sequences by the C-SH2 domain alone could recruit SHPs to cell surface receptors without concomitant phosphatase activation. Regulation of phosphatase activity would depend on the state of phosphorylation of tyrosine residues within sequences recognised by the N-SH2 domain. In addition, C-SH2 domain directed recruitment of SHP-2 could allow SHP-2 to act as an adapter molecule, independently of its phosphatase activity. Similarly to the role of the SH2 domains of SHPs, the N-terminal SH3 and SH2 domains of Src family PTKs autoregulate protein kinase activity. The auto-inhibition of these kinases is relieved by engagement of peptide sequences of activating proteins to the respective binding sites on the SH3 and SH2 domains [32]. A distinct means of activation results from the dephosphorylation of the C- terminal ptyr residue (ptyr527). Comparison of SHP-2 and Src family PTK structures reveals striking and intriguing differences in the molecular mechanisms underlying the regulation of these proteins. Within the inactive Src PTK structures [4,5], the SH2 and SH3 domains are sequestered by intramolecular interactions on the opposite side of the kinase domain to the catalytic site; the SH2 domain interacts with ptyr527 and the SH3 domain interacts with a peptide sequence linking the SH2 and protein kinase domains. 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