Src family kinases phosphorylate the Bcr-Abl SH3-SH2 region and modulate Bcr-Abl transforming activity

Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for chronic myelogenous leukemia. Recently, we observed that inhibition of myeloid Src family kinase activity (e.g. Hck, Lyn, and Fyn) induces growth arrest and apoptosis in Bcr-Abl-transformed cells, suggesting that cell transformation by Bcr-Abl involves Src family kinases (Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J., and Smithgall, T. E. (2002) Oncogene 21, 8075-8088). Here, we report the unexpected observation that Hck, Lyn, and Fyn strongly phosphorylate the SH3-SH2 region of Bcr-Abl. Seven phosphorylation sites were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Tyr89 and Tyr134 in the Abl-derived SH3 domain; Tyr147 in the SH3-SH2 connector; and Tyr158, Tyr191, Tyr204, and Tyr234 in the SH2 domain. SH3 domain Tyr89, the most prominent phosphorylation site in vitro, was strongly phosphorylated in chronic myelogenous leukemia cells in a Src family kinase-dependent manner. Substitution of the SH3-SH2 tyrosine phosphorylation sites with phenylalanine substantially reduced Bcr-Abl-mediated transformation of TF-1 myeloid cells to cytokine independence. The positions of these tyrosines in the crystal structure of the c-Abl core and the transformation defect of the corresponding Bcr-Abl mutants together suggest that phosphorylation of the SH3-SH2 region by Src family kinases impacts Bcr-Abl protein conformation and signaling.     

Conformational basis for SH2-Tyr(P)527 binding in Src inactivation

Src protein-tyrosine kinase contains a myristoylation motif, a unique region, an Src homology (SH) 3 domain, an SH2 domain, a catalytic domain, and a C-terminal tail. The C-terminal tail contains a Tyr residue, Tyr527. Phosphorylation of Tyr527 triggers Src inactivation, caused by Tyr(P)527 binding to the SH2 domain. In this study, we demonstrated that a conformational contribution, not affinity, is the predominant force for the intramolecular SH2-Tyr(P)527 binding, and we characterized the structural basis for this conformational contribution. First, a phosphopeptide mimicking the C-terminal tail is an 80-fold weaker ligand than the optimal phosphopeptide, pYEEI, and similar to a phosphopeptide containing three Ala residues following Tyr(P) in binding to the Src SH2 domain. Second, the SH2-Tyr(P)527 binding is largely independent of the amino acid sequence surrounding Tyr(P)527, and only slightly decreased by an inactivating mutation in the SH2 domain. Furthermore, even the unphosphorylated C-terminal tail with the sequence of YEEI suppresses Src activity by binding to the SH2 domain. These experiments demonstrate that very weak affinity is sufficient for the SH2-Tyr(P)527 binding in Src inactivation. Third, the effective intramolecular SH2-Tyr(P)527 binding is attributed to a conformational contribution that requires residues Trp260 and Leu255. Although the SH3 domain is essential for Src inactivation by Tyr(P)527, it does not contribute to the SH2-Tyr(P)527 binding. These findings suggest a conformation-based Src inactivation model, which provides a unifying framework for understanding Src activation by a variety of mechanisms.     

Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.     

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.     

Tight association of GRB2 with receptor protein-tyrosine phosphatase alpha is mediated by the SH2 and C-terminal SH3 domains.

Receptor protein-tyrosine phosphatase alpha (RPTPalpha), a transmembrane member of the extensive family of protein-tyrosine phosphatases (PTPs), is constitutively phosphorylated on Tyr789, a consensus binding site for the SH2 domain of the SH3-SH2-SH3 adaptor protein GRB2. We have previously shown that GRB2 binds to P.Tyr789 in vivo and in vitro via its SH2 domain. Here, we report that not only the GRB2 SH2 domain, but also the C-terminal SH3 domain is involved in binding to RPTPalpha in vitro and in vivo. Although the N-terminal SH3 domain of GRB2 is essential for binding to the Ras guanine nucleotide exchange factor Son of Sevenless (Sos), an RPTPalpha-GRB2-Sos complex could not be detected. The inclusion of peptides encompassing an hSos1 proline-rich motif in cell lysates resulted in enhanced binding of RPTPalpha to GRB2 in vitro, suggesting that steric hindrance prohibits formation of the RPTPalpha-GRB2-Sos complex. In vitro binding experiments indicated that the binding of GRB2 to Sos/dynamin and RPTPalpha was mutually exclusive. Analysis of in vitro binding kinetics coupled with results from transient co-transfections demonstrated that RPTPalpha is tightly bound to GRB2. The site of interaction of the C-terminal SH3 domain of GRB2 with RPTPalpha was mapped using deletion mutants to an 18-residue region in the N-terminal PTP domain. Arg469, within this region, was identified as one of the residues that is involved in the interaction with the C-terminal SH3 domain of GRB2. RPTPalpha residues 469-486 are localized close to the catalytic site cleft in the structure of the N-terminal PTP-domain, suggesting that interaction with the C-terminal SH3 domain may block access to the catalytic site, thus inhibiting RPTPalpha activity.     

Substrate conformational restriction and CD45-catalyzed dephosphorylation of tail tyrosine-phosphorylated Src protein

Hydrolysis of the tail phosphotyrosine in Src family members is catalyzed by the protein-tyrosine phosphatase CD45, activating Src family-related signaling pathways. Using purified recombinant phospho-Src (P-Src) (amino acid residues 83-533) and purified recombinant CD45 catalytic (cytoplasmic) domain (amino acid residues 565-1268), we have analyzed the kinetic behavior of dephosphorylation. A time course of phosphatase activity showed the presence of a burst phase. By varying the concentration of P-Src, it was shown that the amplitude of this burst phase increased linearly with respect to P-Src concentration. Approximately 2% of P-Src was shown to be rapidly dephosphorylated followed by a slower linear phase. A P-Src protein substrate containing a functional point mutation in the Src homology domain 2 (SH2) led to more rapid dephosphorylation catalyzed by CD45, and this reaction showed only a single linear kinetic phase. These results were interpreted in terms of a model in which P-Src exists in a relatively slow dynamic equilibrium between "closed" and "open" conformational forms. Combined mutations in the SH2 and SH3 domain or the addition of an SH3 domain ligand peptide enhanced the accessibility of P-Src to CD45 by biasing P-Src to a more open form. Consistent with this model, a phosphotyrosine peptide that behaved as an SH2 domain binding ligand showed approximately 100-fold greater affinity for unphosphorylated Src versus P-Src. Surprisingly, P-Src possessing combined SH3 and SH2 functional inactivating point mutations was dephosphorylated by CD45 more slowly compared with P-Src completely lacking SH3 and SH2 domains. Additional data suggest that the SH3 and SH2 domains can inhibit accessibility of the P-Src tail to CD45 by interactions other than direct phosphotyrosine binding by the SH2 domain. Taken together, these results suggest how activation of Src family member signaling pathways by CD45 may be influenced by the presence or absence of ligand interactions remote from the tail.     

Development of Grb2 SH2 Domain Signaling Antagonists: A Potential New Class of Antiproliferative Agents

Aberrant signaling through protein-tyrosine kinase (PTK)-dependent pathways is associated with several proliferative diseases. Accordingly, PTK inhibitors are being developed as new approaches for the treatment of certain cancers. Growth factor receptor bound protein 2 (Grb2) is an important downstream mediator of PTK signaling that serves obligatory roles in many pathogenic processes. One of the primary functions of Grb2 is to bind to specific phosphotyrosyl (pTyr)-containing sequences through its Src homology 2 (SH2) domain. Agents that bind to the Grb2 SH2 domain and prevent its normal function could disrupt associated PTK signaling and serve as alternatives to kinase-directed inhibitors. Starting from the X-ray crystal structure of a lead peptide bound to the Grb2 SH2 domain, this review will summarize important contributions to these efforts. The presentation will be thematically arranged according to the region of peptide modified, proceeding from the N-terminus to the C-terminus, with a special section devoted to aspects of conformational constraint.     

Defining the specificity space of the human SRC homology 2 domain

Src homology 2 (SH2) domains are the largest family of interaction modules encoded by the human genome to recognize tyrosine-phosphorylated sequences and thereby play pivotal roles in transducing and controlling cellular signals emanating from protein-tyrosine kinases. Different SH2 domains select for distinct phosphopeptides, and the function of a given SH2 domain is often dictated by the specific motifs that it recognizes. Therefore, deciphering the phosphotyrosyl peptide motif recognized by an SH2 domain is the key to understanding its cellular function. Here we cloned all 120 SH2 domains identified in the human genome and determined the phosphotyrosyl peptide binding properties of 76 SH2 domains by screening an oriented peptide array library. Of these 76, we defined the selectivity for 43 SH2 domains and refined the binding motifs for another 33 SH2 domains. We identified a number of novel binding motifs, which are exemplified by the BRDG1 SH2 domain that selects specifically for a bulky, hydrophobic residue at P + 4 relative to the Tyr(P) residue. Based on the oriented peptide array library data, we developed scoring matrix-assisted ligand identification (or SMALI), a Web-based program for predicting binding partners for SH2-containing proteins. When applied to SH2D1A/SAP (SLAM-associated protein), a protein whose mutation or deletion underlies the X-linked lymphoproliferative syndrome, SMALI not only recapitulated known interactions but also identified a number of novel interacting proteins for this disease-associated protein. SMALI also identified a number of potential interactors for BRDG1, a protein whose function is largely unknown. Peptide in-solution binding analysis demonstrated that a SMALI score correlates well with the binding energy of a peptide to a given SH2 domain. The definition of the specificity space of the human SH2 domain provides both the necessary molecular basis and a platform for future exploration of the functions for SH2-containing proteins in cells.     

Csk homologous kinase (CHK) and ErbB-2 interactions are directly coupled with CHK negative growth regulatory function in breast cancer

Our previous studies demonstrated that Csk homologous kinase (CHK) acts as a negative growth regulator of human breast cancer through inhibition of ErbB-2/neu-mediated Src family kinase activity (Bougeret, C., Jiang, S., Keydar, I., and Avraham, H. (2001) J. Biol. Chem. 276, 33711-33720. The interaction between the CHK SH2 domain and Tyr(P)(1248) of the ErbB-2 receptor has been shown to be specific and critical for CHK function. In this report, we investigated whether the interaction of the CHK SH2 domain and ErbB-2 is directly related to the inhibition of heregulin-stimulated Src kinase activity. We constructed three CHK SH2 domain binding mutants: G129R (enhanced binding), R147K (inhibited binding), and R147A (disrupted binding). NMR spectra for the domains of each construct were used to evaluate their interaction with a Tyr(P)(1248)-containing ErbB-2 peptide. G129R showed enhanced binding to ErbB-2, whereas binding was completely disrupted by R147A. The enhanced binding mutant showed chemical shift changes at the same residues as wild-type CHK, indicating that this mutant has the same binding characteristics as the wild-type protein. Furthermore, inhibition of heregulin-stimulated Src kinase activity was markedly diminished by R147A, whereas G129R-mediated inhibition was stronger as compared with wild-type CHK. These results indicate that the specific interaction of CHK and ErbB-2 via the SH2 domain of CHK is directly related to the growth inhibitory effects of CHK. These new CHK high affinity binding constructs may serve as good candidates for inhibition of the ErbB-2/Src transduction pathway in gene therapy studies in breast cancer.     

Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: insights from NMR mapping studies and site-directed mutagenesis.

Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the Src homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphorylates the C-terminal tail of Src-family members, resulting in downregulation of the Src family kinase activity. The Src family kinases share 37 % homology with Csk but, unlike Src-family kinases, the catalytic domain of Csk alone is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolecular interaction were studied by nuclear magnetic resonance (NMR) and site-directed mutagenesis techniques. Chemical shift perturbation of backbone nuclei (H' and (15)N) has been used to map the Csk catalytic domain binding site on the Csk-SH3. The experimentally determined interaction surface includes three structural elements: the N-terminal tail, a small part of the RT-loop, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed that mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinase activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a major role in the activation of the Csk catalytic domain.     

Purification and spectroscopic characterization of the human protein tyrosine kinase-6 SH3 domain

The human protein tyrosine kinase-6 (PTK6) polypeptide that is deduced from the cDNA sequence contains a Src homology (SH) 3 domain, SH2 domain, and catalytic domain of tyrosine kinase. We initiated biochemical and NMR characterization of PTK6 SH3 domain in order to correlate the structural role of the PTK6 using circular dichroism and heteronuclear NMR techniques. The circular dichroism data suggested that the secondary structural elements of the SH3 domain are mainly composed of beta-sheet conformations. It is most stable when the pH is neutral based on the pH titration data. In addition, a number of cross peaks at the low-field area of the proton chemical shift of the NMR spectra indicated that the PTK6 SH3 domain retains a unique and folded conformation at the neutral pH condition. For other pH conditions, the SH3 domain became unstable and aggregated during NMR measurements, indicating that the structural stability is very sensitive to pH environments. Both the NMR and circular dichroism data indicate that the PTK6 SH3 domain experiences a conformational instability, even in an aqueous solution.     

Structural basis of the auto-inhibition mechanism of nonreceptor tyrosine kinase PTK6

Protein tyrosine kinase 6 (PTK6) is composed of SH3, SH2, and Kinase domains, with a linker region (Linker) between the SH2 and Kinase domains. Here, we report the structural basis of the SH3-Linker interaction that results in auto-inhibition of PTK6. The solution structures of the SH3 domain and SH3/Linker complex were determined by NMR spectroscopy. The structure of the SH3 domain forms a conventional β-barrel with two β-sheets comprised of five β-strands. However, the molecular topology and charge distribution of PTK6-SH3 slightly differs from that of the other SH3 domains. The structure of the N-terminal Linker within the complex showed that the proline-rich region (P175-P187) of the Linker forms a compact hairpin structure through hydrophobic interactions. The structure of the SH3/Linker complex revealed intra-molecular interaction between the amino acid pairs R22/E190, W44/W184, N65/P177, and Y66/P179. Mutations in PTK6 at R22, W44, N65, and Y66 residues in the SH3 domain increased catalytic activity compared with wild-type protein, implying that specific interactions between hydrophobic residues in the proline-rich linker region and hydrophobic residues in the SH3 domain are mainly responsible for down-regulating the catalytic activity of PTK6.     

Structural basis for dimerization of the Grb10 Src homology 2 domain. Implications for ligand specificity

Grb7, Grb10, and Grb14 are members of a distinct family of adapter proteins that interact with various receptor tyrosine kinases upon receptor activation. Proteins in this family contain several modular signaling domains including a pleckstrin homology (PH) domain, a BPS (between PH and SH2) domain, and a C-terminal Src homology 2 (SH2) domain. Although SH2 domains are typically monomeric, we show that the Grb10 SH2 domain and also full-length Grb10 gamma are dimeric in solution under physiologic conditions. The crystal structure of the Grb10 SH2 domain at 1.65-A resolution reveals a non-covalent dimer whose interface comprises residues within and flanking the C-terminal alpha helix, which are conserved in the Grb7/Grb10/Grb14 family but not in other SH2 domains. Val-522 in the BG loop (BG3) and Asp-500 in the EF loop (EF1) are positioned to interfere with the binding of the P+3 residue of a phosphopeptide ligand. These structural features of the Grb10 SH2 domain will favor binding of dimeric, turn-containing phosphotyrosine sequences, such as the phosphorylated activation loops in the two beta subunits of the insulin and insulin-like growth factor-1 receptors. Moreover, the structure suggests the mechanism by which the Grb7 SH2 domain binds selectively to pTyr-1139 (pYVNQ) in Her2, which along with Grb7 is co-amplified in human breast cancers.     

Molecular rulers: an assessment of distance and spatial relationships of Src tyrosine kinase Sh2 and active site regions

The three-dimensional structures of the inactive conformations of Hck and Src, members of the Src protein-tyrosine kinase family, have recently been described. In both cases, the catalytic domain lies on the opposite face of the enzyme from the SH2 and SH3 domains. The active conformation of these enzymes has not yet been described. Given the known role of the SH2 and SH3 domains in promoting substrate binding, enzyme activation likely reorients the relative spatial arrangement between the SH2/SH3 domains and the active site region. We describe herein a series of "molecular rulers" and their use in assessing the topological and spatial relationships of the SH2 and active site regions of the Src protein-tyrosine kinase. These synthetic compounds contain sequences that are active site-directed (-Glu-Glu-Ile-Ile-(F(5))Phe-, where (F(5))Phe is pentafluorophenylalanine) and SH2-directed (-Tyr(P)-Glu-Glu-Ile-Glu-), separated by a sequence of variable length. The most potent bivalent compound, acetyl-Glu-Glu-Leu-Leu-(F(5))Phe-(GABA)(3)-Tyr(P)-Glu-Glu-Ile-Glu-amide (where GABA is gamma-aminobutyric acid), displays a >120-fold enhancement in inhibitory potency relative to the simple monovalent active site-directed species, acetyl-Glu-Glu-Leu-Leu-(F(5))Phe-amide. The short linker length (3 GABA residues) between the active site- and SH2-directed peptide fragments suggests that the corresponding domains on the Src kinase can assume a nearly contiguous spatial arrangement in the active form of the enzyme.     

Phosphorylation-dependent interactions between ADAM15 cytoplasmic domain and Src family protein-tyrosine kinases.

The adamalysins (ADAMs) are transmembrane glycoproteins involved in cell adhesion and proteolytic ectodomain processing of cytokines and adhesion molecules. Many ADAM cytoplasmic domains are proline-rich and have potential phosphorylation sites. We show here that the cytoplasmic domain of ADAM15, metargidin, can interact specifically with Src family protein-tyrosine kinases (PTKs) and the adaptor protein Grb2 in hematopoietic cells (Jurkat, THP-1, U937, and K562 cell lines). Src homology 3 domains from several Src family PTKs including Lck, Fyn, Abl, and Src associate with ADAM15 in vitro. Dephosphorylation of cell extracts resulted in decreased association of ADAM15 with Src family PTK SH3 domains, indicating that phosphorylation influences ADAM15 interactions with its binding partners. This was confirmed in vitro for Hck, Lck, and Grb2, which showed enhanced association with tyrosine-phosphorylated glutathione S-transferase-ADAM15 cytoplasmic domain compared with unphosphorylated protein. In contrast, binding of MAD2 to ADAM15 was slightly reduced by phosphorylation of the ADAM. Immunoprecipitation of ADAM15 from Jurkat cells confirmed the association with Lck in vivo, and upon PMA stimulation, the phosphorylation level of ADAM15 was increased. Cotransfection of ADAM15 and Hck showed Hck-dependent phosphorylation of ADAM15 in vivo. Hck, and to a lesser extent Lck, phosphorylated the ADAM15 cytoplasmic domain in vitro in immune complex kinase assays. Binding of ADAM15 cytoplasmic domain to Hck and Lck was also shown by Far Western analysis. In contrast to Hck, Lck activity was not required for binding to ADAM15, as shown by treatment of cells with PP1. Deletion and point mutation analysis of the ADAM15 cytoplasmic domain confirmed the importance of the proline-rich motifs for Grb2 and Lck binding and indicated the regulatory nature of Tyr(715) and Tyr(735). These data demonstrate selective, phosphorylation-dependent interactions of ADAM15 with Src family PTKs and Grb2, which highlight the potential for integration of ADAM functions and cellular signaling.     

Directed discovery of bivalent peptide ligands to an SH3 domain

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G(4)-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the beta-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67(phox) SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G(4)-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.     

Solution structure of the human Grb14-SH2 domain and comparison with the structures of the human Grb7-SH2/erbB2 peptide complex and human Grb10-SH2 domain

Grb14 is an adapter protein that is known to be overexpressed in estrogen receptor positive breast cancers, and in a number of prostate cancer cell lines. Grb14 has been demonstrated to bind to a number of activated receptor tyrosine kinases (RTKs) and to modulate signals transduced through these receptors. The RTKs to which Grb14 binds include the insulin receptor (IR), the fibroblast growth factor receptor (FGFR), the platelet-derived growth factor receptor (PDGFR), and the tunica endothelial kinase (Tek/Tie2) receptor. Grb14 has been shown to bind to these activated RTKs through its Src homology 2 (SH2) domain, with the exception of the insulin receptor, where the primary binding interaction is via a small domain adjacent to the SH2 domain (the BPS or PIR domain). Grb14 is a member of the Grb7 family of proteins, which also includes Grb7 and Grb10. We have solved the solution structure of the human Grb14-SH2 domain and compared it with the recently determined Grb7-SH2 and Grb10-SH2 domain structures.     

Backbone nuclear relaxation characteristics and calorimetric investigation of the human Grb7-SH2/erbB2 peptide complex

Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20%–30% of breast cancers. Grb7 binds to erbB2 and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In a prior study, we reported the solution structure of the Grb7-SH2/erbB2 peptide complex. In this study, T₁, T₂, and steady-state NOE measurements were performed on the Grb7-SH2 domain, and the backbone relaxation behavior of the domain is discussed with respect to the potential function of an insert region present in all three members of this protein family. Isothermal titration calorimetry (ITC) studies were completed measuring the thermodynamic parameters of the binding of a 10-residue phosphorylated peptide representative of erbB2 to the SH2 domain. These measurements are compared to calorimetric studies performed on other SH2 domain/phosphorylated peptide complexes available in the literature.     

Solution structure of the human Grb7-SH2 domain/erbB2 peptide complex and structural basis for Grb7 binding to ErbB2

The solution structure of the hGrb7-SH2 domain in complex with a ten amino acid phosphorylated peptide ligand representative of the erbB2 receptor tyrosine kinase (pY1139) is presented as determined by nuclear magnetic resonance methods. The hGrb7-SH2 domain structure reveals the Src homology 2 domain topology consisting of a central -sheet capped at each end by an -helix. The presence of a four residue insertion in the region between -strand E and the EF loop and resulting influences on the SH2 domain/peptide complex structure are discussed. The binding conformation of the erbB2 peptide is in a -turn similar to that found in phosphorylated tyrosine peptides bound to the Grb2-SH2 domain. To our knowledge this is only the second example of an SH2 domain binding its naturally occurring ligands in a turn, instead of extended, conformation. Close contacts between residues responsible for binding specificity in hGrb7-SH2 and the erbB2 peptide are characterized and the potential effect of mutation of these residues on the hGrb7-SH2 domain structure is discussed.