Single phosphorylation of Tyr304 in the cytoplasmic tail of ephrin B2 confers high-affinity and bifunctional binding to both the SH2 domain of Grb4 and the PDZ domain of the PDZ-RGS3 protein Zhengding Su, Ping Xu and Feng Ni Biomolecular NMR and Protein Research Group, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada The B class cell-attached ephrins mediate contact-dependent cell–cell communications and transduce the contact signals to the host cells through the binding interactions of their cytoplasmic domains. Two classes of intracellular effectors of B ephrins have been identified: one contains the PSD-95/ Dlg/ZO-1 (PDZ) domain (for example PDZ-RGS3), and the second the Src homology 2 (SH2) domain (e.g. the Grb4 adaptor protein). The interaction with Grb4 requires phos- phorylation of tyrosine residues on the conserved cytoplas- mic C-terminal region of B ephrins, while binding to the PDZ domain is independent of tyrosine phosphorylation. However, the exact phosphorylation site(s) required for signaling remained obscure and it is also unknown whether the two classes of effectors can bind to B ephrins simulta- neously or if the binding of one affects the binding of the other. We report here that phosphorylation of Tyr304 in the functional C-terminal region (residues 301–333) of ephrin B2 confers high-affinity binding to the SH2 domain of the Grb4 protein. Tyrosine phosphorylation at other candi- date sites resulted in only minor change of the binding of Tyr304-phosphorylated ephrin B peptide (i.e. eph- rinB2(301–333)-pY304) with the SH2 domain. 1 H- 15 N NMR HSQC experiments show that only the eph- rinB2(301–333)-pY304 peptide forms a stable and specific binding complex with the SH2 domain of Grb4. The SH2 and PDZ domains were found to bind to the Tyr304 phosphopeptide both independently and at the same time, forming a three-component molecular complex. Taken together, our studies identify a novel SH2 domain binding motif, PHpY304EKV, on the cytoplasmic domains of B ephrins that may be essential for reverse signaling via the Grb4 adaptor protein alone or in concert with proteins containing PDZ domains. Keywords: ephrin B; Grb4; SH2; PDZ; phosphorylation. The ephrin-Eph signaling systems play critical roles in multiple cell functions including cell migration, tissue border formation in vascular development and angiogenesis, and cell–cell communications for axonal guidance and at the synaptic junction [1]. The Eph molecules resemble classical receptor tyrosine kinases (PTKs) in that they are transmem- brane proteins with kinase domains and other binding motifs projecting into the cytoplasmic space. The plasma membrane-bound ephrins, however, orchestrate cell move- ments and morphogenesis by transducing bidirectional signals into cells expressing the Eph molecules as well as cells expressing the ephrins [2–6]. The unique capacity of reverse signaling, by the B-class cell-attached ephrins in particular, is to communicate the cell contact signals to the host cells through the association of their cytoplasmic domains with intracellular effector proteins. So far, two typesofintracellulartargetsforBephrinshavebeen identified. Proteins containing PSD-95/Dlg/ZO-1 (PDZ) domains have been shown to bind to the C-termini of the B ephrins [7–10]. At least one protein containing an Src homology 2 (SH2) domain, Grb4, is recruited to the cytoplasmic tails of B ephrins upon phosphorylation [11]. Although details of these signaling events have yet to be investigated, many of the molecular interactions down- stream of the Eph and ephrin molecules appear to lead to the regulation of the cytoskeleton of the interacting cells [1,2,6]. Phosphorylation of the well-conserved cytoplasmic domains of B ephrins has been a subject of significant interest [11–16] as it is required for reverse signaling into the ephrin B-expressing cells. The short cytoplasmic tails of the B ephrins contain five tyrosine residues, all of which are located within the extreme C-terminal 33-residue region [12,13]. Three tyrosine residues are contained in a 22-residue segment CPHYEKVSGDYGHPVYIVQEMP(301–322) for ephrin B2, which was shown to be responsible for Grb4 binding and is identical in both ephrin B2 and ephrin B1 [11]. Strikingly, the Grb4 SH2 domain shows strong binding to tyrosine-phosphorylated ephrin B1, whereas one Correspondence to F. Ni, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2, Canada. Fax: + 514 4965143, Tel.: + 514 4966729, E-mail: fengni@bri.nrc.ca Abbreviations: FGF, fibroblast growth factor; HSQC, heteronuclear single quantum coherence; NOESY, nuclear Overhauser effect spectroscopy; PDZ, PSD-95/Dlg/ZO-1; SFK, Src family kinase; SH2, Src homology 2; TOCSY, total correlation spectroscopy. (Received 23 December 2003, revised 27 February 2004, accepted 9 March 2004) Eur. J. Biochem. 271, 1725–1736 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04078.x of its closest known relatives, the Nck SH2 domain, has little or no binding [11]. The phosphorylations of Tyr311 and Tyr316 were detected in both embryonic retinal tissues and 293 cells stimulated with a multimeric EphB2 [15]. In Xenopus embryos, the activated fibroblast growth factor (FGF) receptor was found to associate with and induce the phosphorylation of ephrin B1 on equivalent positions of Tyr311 and Tyr316, but not on Tyr304 [16]. The region from residues 308–316 containing two tyrosine residues was found to be critical for binding interactions with the FGF receptor. Two other tyrosine residues are at the extreme carboxyl terminus or residues YY(330–331)KV, constitu- ting a PDZ-domain binding element [8]. In neural tissues, Tyr330 was found to be the major phosphorylation site of ephrin B1 after binding to EphB2, while the phosphoryla- tion of Tyr331 could not be detected [15]. However, this phosphorylation of ephrin B1 did not significantly affect intracellular protein–protein interactions [8]. More recently, the Src family kinases (SFKs) and the PTP-BL phosphatase were proposed to be mediators of the phosphorylation state of the cytoplasmic domain of ephrin B1 whereby the SFKs may create the docking sites for Grb4 and the phosphatase may disengage Grb4 binding from B ephrins [17]. Our previous work suggested that the well-conserved 33-residue C-terminal region of residues 301–333 of ephrin B2 might encode a latent three-dimensional structure [18], which may be activated through phosphorylation for Grb4 binding. More interestingly, the 22-residue region, i.e. the ephrinB2(301–322) peptide fragment encoding the Grb4 binding site [11], appears to assume an autonomously folded structure, independently of the PDZ-binding extreme C-terminal region, i.e. residues IY330YKV [18]. As well, cellular functions ascribed to the Grb4 and PDZ-binding events can work synergistically to coordinate cell migration and morphogenesis in establishing defined patterns of cell assemblies [1,2,6]. It is therefore possible, upon phosphory- lation, for the PDZ and SH2 domains to bind simulta- neously to distinct regions of the already very short (33 residues) ephrin B cytoplasmic tails. In the current work, we first identify the phosphorylation site in ephrin B2 that creates high-affinity binding to the Grb4 SH2 domain using synthetic ephrinB2(301–333) fragments containing single or double phosphorylations at five candidate tyrosine residues, Tyr304, Tyr311, Tyr316, Tyr330 and Tyr331. We then assess whether the binding of the PDZ domain of PDZ- RGS3 affects binding of the Grb4 SH2 domain to phosphorylated peptides, through studies of ternary inter- actions among the PDZ domain, the phosphotyrosine peptide and the Grb4 SH2 domain. We also show that phosphorylations of the ephrinB2(301–333) peptide alter only the binding interactions and the three-dimensional structure of the Grb4-binding region, i.e. residues 301–322, while leaving the PDZ-binding C-terminus essentially unperturbed. Experimental procedures Construction of expression vectors for the Grb4 SH2 and RGS3 PDZ domains The DNA sequences encoding the Grb4 SH2 and the RGS3 PDZ domains were individually deduced from the amino acid sequences of murine Grb4 protein and murine PDZ- RGS3 protein as published previously [7,19] using the codon preference of Escherichia coli. The synthetic genes were amplified by PCR from six pairs of overlapping synthetic primers containing the two restriction sites of NcoI and BamHI for the SH2 domain and the two restriction sites of BamHI and EcoRI for the PDZ domain at their two ends, respectively. The double-digested DNA fragment of SH2 was subcloned into the pET3215 expression vector, which was modified from pET32 and pET15 vectors (Novagen, Madison, WI, USA), removing the original fusion carrier in the pET32 vector. In order to facilitate protein purification, a His-tag with six histidine residues was placed at the N-terminus of the SH2 domain linked with a thrombin cleavage sequence. The same insert was also subcloned into the pGFN GST fusion vector, which is a derivative of the pGEX-4T-1 expression vector (Amer- sham Biosciences, Piscataway, NJ, USA), replacing the LVPR thrombin cleavage site with the FNPR sequence [20]. The double digested DNA fragment of the PDZ domain was subcloned into the pGFN vector. All these expression constructs were confirmed by DNA sequencing and trans- formed into the E. coli BL21(DE3) expression host. Protein expression and purification The SH2 protein was expressed at 37 °C. The cells were harvested four hours after induction with isopropyl thio- b- D -galactoside at D 600 ¼ 0.8. Labeling with the 15 N isotope was accomplished using M9 media containing 1.0 gÆL )1 of [ 15 N]ammonium sulfate, 5 gÆL )1 of glucose plus a supplement of trace levels of metal ions and vitamins. Uniform 15 N/ 13 C-labeling was accomplished by substitution of unlabelled glucose with 2.0 gÆL )1 of [ 13 C 6 ]glucose. Protein purification was performed under denaturing conditions with Ni-nitriloacetic acid agarose beads (Qiagen) in the presence of 20 m M 2-mercaptoethanol at pH values of 8.0, 6.3, 5.9 and 4.5 for the binding, two washing, and eluting steps, respectively. Protein fractions were analyzed using 20% Phast gels (Amersham Biosciences). The fractions containing the SH2 domain were collected and refolded by dialyzing 2–3 times against a large volume of 50 m M sodium phosphate buffer plus 20 m M 2-mercaptoethanol (pH 6.8) at 4 °C. The pellet was removed by centrifugation and the supernatant was concentrated by ultrafiltration (Millipore, Bedford, MA, USA). The protein concentration was determined at A 280 with a calculated extinction coefficient of 12 210 M )1 Æcm )1 [21]. The expression and purification of GST-SH2 and GST- PDZ proteins were carried out with standard protocols provided by the supplier (Amersham Biosciences). Throm- bin cleavage was performed at room temperature for 2 h in 1 · NaCl/P i buffer. The concentration of the PDZ protein was determined at A 280 with a calculated extinction coefficient of 12 620 M )1 Æcm )1 [21]. Peptide synthesis and purification All the peptides, either with or without phosphotyrosine (pY), were synthesized chemically using standard Fmoc solid-phase chemistry at the Biotechnology Research Insti- tute’s Peptide Facility. The synthetic peptides were purified 1726 Z. Su et al. (Eur. J. Biochem. 271) Ó FEBS 2004 by HPLC using a reverse-phase C18 semipreparative column with an acetonitrile gradient of 10–35%. The purity of the peptides was verified by a reversed-phase analytical HPLC column and the identity of the final products was verified by mass spectral analysis and NMR assignments. Fluorescence polarization measurements Fluorescein-labeled peptides were prepared through reac- tion of the ephrinB2(301–333) peptide and its variants containing a single phosphotyrosine residue (Fig. 3), or double phosphotyrosine residues with 5-iodoacetamido- fluorescein (Molecular Probes, Eugene, OR, USA). Upon completion of labeling, an excess of 2-mercaptoethanol was added to consume the excess 5-iodoacetamidofluorescein followed by the removal of small organic compounds using a C18 September-Pak column. The eluate containing fluorescein-labeled peptide was further purified by HPLC. The authenticity of fluorescein labeling was confirmed by mass spectroscopy. Fluorescence polarization was performed at 25 °Con a Perkin-Elmer fluorescence polarization instrument, the EnVision TM multilabel plate reader, which was equipped with two fluorescence polarizers. All the polarization values are expressed in mili-polarization units (mP). Fluorescence measurements were carried out with an excitation wave- length of 490 nm and an emission wavelength of 520 nm. For binding studies, each fluorescein-labeled peptide was dissolvedin50m M phosphate buffer (pH 6.8, with 20 m M 2-mercaptoethanol) to a concentration of 25 n M .The dissociation constants were obtained by fitting the binding curves using the computer program ORIGIN TM 6.0 (Nor- thampton, MA, USA) based on the following equations: L þ P () K on K off LÁP where L and P denote the ligand and protein, respectively. The fluorescence polarization (DmP) is related to the dissociation constant K d as follows: DmP ¼ C 0 ½L T ½P K d þ½P where C 0 is a constant dependent on the properties of the ligand, [L] T is the total ligand concentration, [P]isthe concentration of free SH2 protein, and K d is the equilibrium dissociation constant. Average K d values were determined from multiple independent measurements. NMR spectroscopy and resonance assignments All NMR spectra were collected at 15 °C on a Bruker Avance 500 MHz or 800 MHz spectrometer using triple- resonance probes equipped with pulse field gradients. Protein samples for NMR analysis contained % 0.5 m M of uniformly 15 N- or 15 N-/ 13 C-labeled protein. Assignments of the H/ 15 N/ 13 C NMR signals from the main-chain atoms were obtained from a combined analysis of the 1 H- 15 N HSQC, CBCA(CO)NH and HNCACB experiments [22]. Proton NMR experiments with the peptides, which included data acquisition, processing and analysis, were the same as described previously [18,23]. GST pull-down assays A GST pull-down experiment was employed to recons- titute the three-component molecular complexes formed by the ephrin B peptides, the SH2 domain and the PDZ protein. GST or GST-PDZ proteins bound to glutathione agarose beads were incubated with 5 lL of the SH2 protein in the binding buffer (50 m M sodium phosphate, pH 6.8) for 2 h at 4 °C in the absence or presence of ephrin B2 peptides in excess amount. The beads were washed extensively with the binding buffer and the samples were boiled for 10 min in the SDS/PAGE sample buffer and analyzed by SDS/PAGE Phast gel (Amersham Biosciences). Results Characterization of the expressed SH2 and PDZ protein domains The SH2 domain (Fig. 1A) of murine Grb4 was expressed using a synthetic DNA fragment subcloned for protein production in the E. coli host (Experimental procedures). The over-expressed SH2 protein was found in inclusion bodies and purified with Ni-nitiloacetic acid agarose beads under denaturing conditions. The quality of the expressed SH2 domain is improved at each step of the purification procedure (Fig. 1B). A synthetic DNA fragment encoding the PDZ domain of PDZ-RGS3 was prepared in the same way as for the SH2 domain based on the published amino acid sequence of the RGS3 protein [20]. The PDZ domain was over-expressed as a fusion to the GST carrier protein in a mostly soluble form and the intact domain can be purified after digesting the GST-PDZ fusion protein with thrombin (Fig. 1C). The expressed PDZ domain is reasonably soluble in solution and is functionally active as it has the same binding affinity to the ephrin-B2 peptide as the GST-PDZ fusion protein does (see below). The 1 H- 15 N HSQC spectrum of the SH2 domain shows a good dispersion of the 1 H- 15 N correlation peaks (Fig. 2A), indicative of a well-folded protein. Nearly complete assignments of the main chain 1 H N , 15 N, 13 C a , and 13 C b NMR signals of the SH2 domain were obtainable using triple-resonance heteronuclear NMR experiments. Figure 2B shows the differences between the 13 C a and 13 C b chemical shifts of an 15 N/ 13 C-labeled sample of the SH2 domain. The secondary structure elements were deduced from the positive or negative derivations of the 13 C a and 13 C b chemical shift differences from random coil values over a number of consecutive amino acids [24–26]. Overall, secondary structures of the Grb4 SH2 domain are very similar to these of the Src SH2 domain (Fig. 1A). However, there are some signifi- cant differences in the structural regions determining the binding specificity towards phosphopeptides. For example, the b-sheet structure after the EF3 segment in the Src SH2 does not exist in the Grb4 SH2 domain while the insertion after the BG2, BG3 and BG4 structures in the Grb4 SH2 domain forms one additional b-sheet structure (Fig. 1A and Fig. 2B). Ó FEBS 2004 Phosphorylation of Tyr304 on ephrin B2 (Eur. J. Biochem. 271) 1727 Identification of the binding site(s) on ephrin B2 for the SH2 domain of Grb4 Six synthetic peptides including ephrinB2(301–333) and its five individual tyrosine-phosphorylated derivatives, ephrinB2(301–333)-pY304, ephrinB2(301–333)-pY311, ephrinB2(301–333)-pY316, ephrinB2(301–333)-pY330 and ephrinB2(301–333)-pY331 (Fig. 3), were labeled with fluorescein through the Cys301 residue at their N-termini. The highly sensitive fluorescence polarization method was used to detect the binding interactions between the peptides and the Grb4 SH2 domain. The affinity of the binding interactions was evaluated by measuring the changes of fluorescence polarization of the peptides at each step of titration with the Grb4 SH2 domain, i.e. the binding isotherms (Fig. 4A). The ephrinB2(301–333)- pY304 peptide was found to have the strongest binding to the Grb4 SH2 domain while the other five peptides, ephrinB2(301–333), ephrinB2(301–333)-pY311, eph- rinB2(301–333)-pY316, ephrinB2(301–333)-pY330 and ephrinB2(301–333)-pY331, showed low or no binding affinity to the SH2 domain. The dissociation constant of the ephrinB2(301–333)-pY304/Grb4 SH2 complex was estimatedtobe0.2l M from the titration curves (Table 1). The calculated dissociation constants of the complexes between Grb4 SH2 and each of the other five peptides (i.e. ephrinB2(301–333), ephrinB2(301–333)- pY311, ephrinB2(301–333)-pY316, ephrinB2(301–333)- pY330 and ephrinB2(301–333)-pY331) were larger than 500 l M . These results show that only phosphorylated Tyr304 can result in high-affinity and specific binding of ephrinB2(301–333) to the Grb4 SH2 domain. The fluorescence binding experiments also indicate that the Cys301 residue may not be required for binding as it is labeled by the bulky fluorescein group in the peptide fragments. Fig. 1. Comparison of amino acid sequences of SH2 domains from Src and the mouse Grb4, and expression and purification of the Grb4 SH2 and RGS3 PDZ domains. (A) Secondary structural elements are from the X-ray crystallographic structure of the Src SH2 domain in complex with a pYEEI peptide [35]. The notation used for the binding pockets of the phosphotyrosine peptide is as described previously [35]. Residues determining the binding specificity are underlined. (B) SDS/PAGE of each purification step of the expressed SH2 domain of Grb4. Lane 1: the soluble part of the cell lysate; Lane 2: the insoluble part of the cell lysate; Lane 3: the Ni-nitiloacetic acid agarose beads bound with the SH2 protein at pH 8.0; Lane 4: the Ni-nitiloacetic acid agarose beads with the bound SH2 protein at pH 6.3; Lane 5: the eluted protein at pH 4.5 and Lane M: molecular mass markers (Amersham Bioscience). The sample of the Ni-nitiloacetic acid agarose beads with the bound SH2 protein at pH 5.9 was not shown here as the protein was already pure. The position of the SH2 protein is marked by the arrow. (C) SDS/PAGE patterns following each purification step of the expressed GST-PDZ. Lane 1: the soluble part of the cell lysate. The high-density band at the bottom is lysozyme, which was used to lyse the cells. Lane 2: the purified GST-PDZ fusion protein by the glutathione-agarose beads. Lane 3: The digestion of the GST-PDZ fusion protein by thrombin at room temperature for 1 h. The fusion proteins are readily and completely digested within 30 min. Lane 4: the purified PDZ protein by glutathione-agarose beads followed by ion-exchange chromatography. Lane M: molecular mass markers (Amersham Bioscience). 1728 Z. Su et al. (Eur. J. Biochem. 271) Ó FEBS 2004 We next address the questions of whether secondary tyrosine phosphorylations would affect the Grb4 SH2 domain binding to the ephrinB2(301–333)-pY304 peptides and more specifically whether the combined phosphory- lation of Tyr311 and Tyr316 would replace phosphory- lation at Tyr304. The choice of Tyr311 and Tyr316 was because these two residues were reported to be the major detectable phosphorylation sites in vivo [15] and a peptide derived from residues 301–322 of ephrinB2 or the N-terminal 22 residues of ephrinB2(301–333) was found to contain all the phosphorylation sites for high-affinity binding [11]. As shown in Table 1, the phosphorylation of Tyr311 or Tyr316 does not significantly affect the binding of the ephrinB2(301–333)-pY304 peptide to the Grb4 SH2 domain. Our data also show that the peptide with double phosphorylations at Tyr311 and Tyr316 has no significant binding to the Grb4 SH2 domain. Therefore, the high-affinity Grb4 SH2 binding of the ephrinB2(301– 333) fragment conferred by the Tyr304 phosphorylation is independent of phosphorylations at the other two tyrosine residues, Tyr311 and Tyr316. To further assess the binding specificity of the Grb4 SH2 domain, we synthesized the short phosphorylated peptide, ephrin B2(301–309) or CPHpY304EKVSG with a number of substitutions at the KV positions. The single amino acid substitutions successively transformed the pYEKV sequence of ephrin B2 to the pYEEI sequence specific for the Src SH2 domain. As shown in Fig. 4B, only the CPHpY304EKVSG peptide exhibited high-affinity binding to the Grb4 SH2 domain. The dissociation constant of the CPHpY304EKVSG/Grb4 SH2 complex was estimated to be 0.23 l M from the fluorescence titration curve, whereas all amino acid substitutions C-terminal to the pTyr304 led to dramatically reduced binding (Table 1). Two-dimensional NMR spectroscopy was employed to investigate the specificity of binding of phosphorylated ephrinB2(301–333) peptides to the Grb4 SH2 domain. The 1 H- 15 N HSQC spectrum of the Grb4 SH2 domain (Fig. 2A) Fig. 2. Characterization of the Grb4 SH2 by heteronuclear NMR. (A) The 1 H- 15 N HSQC spectrum of the SH2 domain of Grb4 with the assignment of the amide 1 H- 15 N correlations to specific residues. The measurement was performed at 15 °C and pH 6.8. Sequence specific assignments of the backbone 15 Nand 13 C resonances were achieved using triple-resonance NMR experiments with a 15 N/ 13 C-labeled SH2 sample. (B) Secondary structure of the Grb4 SH2 protein deduced from the differences of the 13 C a and 13 C b chemical shifts of the 15 N/ 13 C-labeled SH2 domain. Ó FEBS 2004 Phosphorylation of Tyr304 on ephrin B2 (Eur. J. Biochem. 271) 1729 responded to the addition of the ephrinB2 peptide phos- phorylated at Tyr304, or ephrinB2(301–333)-pY304 (Fig. 5A). Almost twice the numbers of HSQC peaks were found at lower peptide concentrations and most of the peaks redistributed at a molar ratio of 2 : 1 for the peptide- SH2 concentrations (Fig. 5A, red spectrum). More signifi- cantly, in a stepwise titration experiment, the HSQC peaks did not undergo gradual shifting with increased concentra- tions of the peptide (data not shown), indicating that the free and peptide-bound SH2 must exchange slowly as observed for other SH2 domains. In contrast, the nonphos- phorylated peptide, ephrinB2(301–333), did not induce any significant changes to the HSQC peaks up to a molar concentration ratio of 2 : 1 (Fig. 5B, red spectrum). As well, no significant changes in the HSQC peaks were observed in the presence of two other single tyrosine-phosphorylated peptides, ephrinB2(301–333)-pY311 and ephrinB2(301– 333)-pY316, similar to that of the unphosphorylated ephrinB2(301–333) (spectra not shown). Taken together, both the NMR and fluorescence polarization experiments Fig. 4. The effects of tyrosine phosphorylation on ephrin B2 binding to the SH2 domain of Grb4 analyzed by fluorescence polarization. (A) Fluorescence binding curves were collected for the unmodified peptide ephrinB2(301–333) (j) and the three singly phosphorylated peptides, ephrinB2(301–333)-pY304 (d), ephrinB2(301–333)-pY311 (m) and ephrinB2(301–333)-pY316 (.). Data for two other singly phosphorylated peptides including ephrinB2(301–333)-pY330 and ephrinB2(301–333)-pY331 are not shown, only their experimentally determined binding constants are listed in Table 1 for comparison. (B) Binding curves were collected for YEKV-pY304 (d), YEKI- pY304 (j), YEKI-pY304 (m) and YEEI-pY304 (r)(Fig.3). Fig. 3. Schematic representation of peptides for fluorescence polarization assays. The 33-residue peptide, ephrinB2(301–333), is derived from the extreme C-terminal sequence of the cytoplasmic domain of ephrin B2. The phosphorylated tyrosine residues are labeled with pY at the five individual sites, Tyr304, Tyr311, Tyr316, Tyr330 or Tyr331, for the five tyrosine-phosphorylated peptides, ephrinB2(301–333)-pY304, eph- rinB2(301–333)-pY311, ephrinB2(301–333)-pY316, ephrinB2(301–333)-pY330 and ephrinB2(301–333)-pY331. The consensus binding sequence of ephrin B2 for the Grb4 SH2 is minimized to a nine-residue segment, ephrinB2(301–309) or CPHpYEKVSG (referred to as YEKV-pY304). Three variants of the short peptide are generated to determine the binding specificity of the Grb4 SH2 in relation to the closest consensus binding sequence of the Src SH2 domain (i.e. pYEEI). Table 1. The effect of tyrosine phosphorylations on the binding affinity of ephrinB2(301–333) to the SH2 domain of Grb4 and to the PDZ domain of PDZ-RGS3. In the combination experiments, the two C terminal tyrosine residues were ignored due to their distant positions. ND, not determined. pY position K d (SH2) (l M ) K d (PDZ) (l M ) EphrinB2(301–333) series WT >500 3.0 ± 0.17 pY304 0.21 ± 0.05 3.1 ± 0.18 pY311 and pY316 >500 ND pY311 or pY316 >500 2.9 ± 0.16 pY304 and pY311 0.21 ± 0.06 ND pY304 and pY316 0.22 ± 0.05 ND pY330 or pY331 >500 2.8 ± 0.16 EphrinB2(301–309) series YEKV-pY304 0.23 ± 0.05 – YEEV-pY304 >500 – YEKI-pY304 >500 – YEEI-pY04 >500 – 1730 Z. Su et al. (Eur. J. Biochem. 271) Ó FEBS 2004 show that the phosphorylation of Tyr304 confers high- affinity and specific binding of ephrinB2(301–333) to the Grb4 SH2 domain. The ephrinB2(301–333)-pY304 peptide can bind to the Grb4 SH2 and RGS3 PDZ domains simultaneously It has been shown that the cytoplasmic tail of B-class ephrins constitutes a PDZ domain binding motif, YYKV, whose binding to PDZ domains is phosphorylation-inde- pendent [8]. Figure 6A shows that the GST-PDZ fusion protein binds to the fluorescein-labeled ephrinB2(301–333) peptide and this binding has an affinity of K d ¼ 3.0 l M (Table 1). The binding affinities of the GST-PDZ fusion protein to two single tyrosine phosphorylated peptides at the extreme C-terminus (i.e. the PDZ binding motif), i.e. ephrinB2(301–333)-pY330 and ephrinB2(301–333)-pY331, are essentially the same as that of the ephrinB2(301–333) peptide (Table 1). Therefore, the fluorescein-labeled eph- rinB2(301–333) peptides with single tyrosine phosphoryla- tion at Tyr304, Tyr311, Tyr316, Tyr330 or Tyr331 had essentially the same binding affinities to the GST-PDZ protein in agreement with previous observations [8]. Binding experiments with the isolated PDZ domain showed similar binding affinities to the ephrin B peptides as that of the GST-PDZ protein. Therefore, the GST-PDZ fusion protein was used for all other binding experiments including the GST pull-down experiments (see below). On the other hand, the SH2 domain of Grb4 binds to the cytoplasmic tail of ephrin B2 in a phosphorylation-depend- ent manner. It is therefore interesting to know whether Grb4 and PDZ-RGS3 can bind to the cytoplasmic tail of ephrin B2 simultaneously or whether Grb4 binding can affect or even exclude the binding of the PDZ-RGS3 Fig. 5. 1 H- 15 N-HSQC spectra of the SH2 domain of Grb4 titrated by ephrinB2(301–333). ThespectraofthefreeGrb4-SH2domain (black) were overlaid on those in the presence of ephrinB(301–333) peptides (red). (A) Titration with ephrinB2(301–333)-pY304. (B) Titration with ephrinB2(301–333). The protein concentration was 0.5 m M and the peptide concentration was 1 m M . Ó FEBS 2004 Phosphorylation of Tyr304 on ephrin B2 (Eur. J. Biochem. 271) 1731 protein. We set out to address this question by use of the SH2 domain of Grb4 and the PDZ domain of the RGS3 protein. We titrated the SH2 protein into the fluorescein- labeled ephrinB2(301–333)-pY304 after saturation by the GST-PDZ fusion protein. Consequently, the fluorescence polarization increased further from that elicited by PDZ binding with increasing concentrations of the SH2 protein. It was found that after normalization, the polarization changes had similar trends as the titration experiments in the absence of the GST-PDZ protein (Fig. 6B). Therefore, it appears that binding of the PDZ domain to the ephrinB2(301–333)-pY304 peptide has no impact on the binding of the Grb4 SH2 domain. Formation of three-component complexes among the SH2 domain, the PDZ domain and ephrinB2(301–333) peptides shown by GST pull-down experiments In vitro GST pull-down experiments were also carried out to verify the ternary interactions among the Grb4 SH2 and RGS3 PDZ domains and the ephrinB2(301–333) peptides. First, the Grb4 SH2 domain-binding peptide, eph- rinB2(301–333)-pY304, was used for the formation of the three-component complex with the Grb4 SH2 and RGS3 PDZ domains. The purified GST-PDZ protein, which was bound to the glutathione-agarose beads, was mixed with the Grb4 SH2 domain and the ephrinB2(301–333)-pY304 peptide. The beads were washed extensively to remove the unbound proteins. The proteins bound to the bead were visualized by use of SDS/PAGE (Fig. 7). As indicated by this kind of pull-down data, both the Grb4 SH2 and the RGS3 PDZ domains showed strong binding to the ephrinB2(301–333)-pY304 peptide in a three-component molecular complex. Control experiments showed the lack of binding of the Grb4 SH2 domain to the GST-PDZ protein nor to the GST carrier protein. Second, pull-down experi- ments were also carried out for the other five peptides including ephrinB2(301–333), ephrinB2(301–333)-pY311, ephrinB2(301–333)-pY316, ephrinB2(301–333)-pY330 and ephrinB2(301–333)-pY331. None of these peptides can link the Grb4 SH2 and the RGS3 PDZ domains to form a ternary complex (Fig. 7). Effects of tyrosine phosphorylation on the conformation of ephrinB2(301–333) The unphosphorylated peptide, ephrinB2(301–333), was shown to form a well-folded b-hairpin structure for the putative SH2-binding region of residue 301–322 along with a very flexible C-terminal tail from residues 323–333 [18]. Phosphorylation at Tyr304, Tyr311 or Tyr316 does not affect the conformational characteristics of these PDZ- binding C-terminal tail residues. For example, the typical NOE contact between the aCH proton of Ala327 and the NH proton of Ile329, and the consecutive H N -H N NOEs from Lys329 to Tyr331, existed in all the peptides independent of phosphorylation (Fig. 8A,B). Thus, the conformation of the PDZ binding domain of eph- rinB2(301–333) is essentially independent of tyrosine phosphorylation within the SH2-binding element of eph- rinB2(301–333). On the other hand, tyrosine phosphorylation appears to have a profound effect on the b-hairpin conformation of the SH2-binding region. First, all three kinds of single phos- phorylation at the Tyr304, Tyr311 or Tyr316 site caused perturbation to the loop region in the b-hairpin structure as the unique NOEs detected previously [18] completely disappeared in the NOESY spectrum of the phosphorylated ephrinB2(301–333) peptides, as shown in Fig. 8A for ephrinB2(301–333)-pY304. Second, the consecutive back- bone H N -H N NOEs from residues Tyr316 to Gln319 were observed for ephrinB2(301–333)-pY304 and ephrinB2(301– 333)-pY316 (Fig. 8B) but not for ephrinB2(301–333)- pY311. Third, there is a dramatic reduction of NOE contacts among the sidechains of aromatic residues pur- porting to side-chain packing interactions of a b-hairpin structure (Fig. 8C). Many previously identified long-range NOEs such as those between Val318 and His303, Gln319 and His303, Lys306 and Tyr316 were absent in the phosphorylated peptides as shown in Fig. 8C for eph- rinB2(301–333)-pY304. Interestingly, the long-range NOE contacts involving residues Lys306 and Tyr316, Glu305 and Fig. 6. Effect of PDZ binding on the interactions of the Grb4-SH2 domain with the ephrinB2(301–333)-pY304 peptide. (A) The PDZ- binding curves are for the fluorescein-labeled peptides including eph- rinB2(301–333) (j), ephrinB2(301–333)-pY304 (d), ephrinB2(301– 333)-pY311 (m), ephrinB2(301–333)-pY316 (.). Corresponding data for the phosphorylated peptides at Tyr330 and Tyr331 are listed in Table 1. (B) Binding experiments performed with the fluorescein- labeled ephrinB2(301–333)-pY304 peptide in the absence of the GST- PDZ protein (d)orwith75l M of the GST-PDZ protein (s). 1732 Z. Su et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Fig. 7. Formation of a three-component complex through GST pull-down. Lane M: molecular mass markers. The molecular sizes of each band are 97.0, 66.0, 45.0, 30.0, 20.1 and 14.4 kDa from the top to the bottom. Lane 1: the three-component complex of GST-PDZ, ephrinB2(301–333)- pY304 and the SH2 protein. Lane 2: sample prepared similarly as that in Lane 1 except for the absence of ephrinB2(301–333)-pY304. Lane 3: sample prepared the similarly as that in Lane 2 except the GST was used instead of GST-PDZ. Lane 4–8: samples prepared the same as that in Lane 1 except that the peptides are ephrinB2(301–333), ephrinB2(301–333)-pY311, ephrinB2(301–333)-pY316, ephrinB2(301–333)-pY330 and ephrinB2(301–333)-pY331, respectively. Fig. 8. Homonuclear NOESY spectra of the ephrinB(301–333)-pY304 peptide. (A) NH-aH region of the NOESY spectrum (red) with a mixing time of 200 ms in H 2 O overlaid with a TOCSY spectrum (dark). The cross indicates the missing medium-range NOEs in ephrinB(301–333)-pY304 compared with those in the unphosphorylated ephrinB(301–333) [18]. (B) The NH-NH NOE connectivities for residues Tyr316-Gln319 and Lys329-Tyr331. (C) The aromatic region of the NOESY spectrum of the ephrinB(301–333)-pY304 peptide. The NOE experiments were carried out with a mixing time of 200 ms in H 2 O at pH 6.8 and 288 K. Some key medium- and long-range NOEs are still present, but many long-range NOEs are absent compared to the unphosphorylated ephrinB2(301–333) peptide [18]. Interestingly, the number of side-chain NOE contacts in the two other phosphorylated peptides, ephrinB2(301–333)-pY311 and ephrinB2(301–333)-pY316, was further reduced and almost all NOEs have disappeared. Ó FEBS 2004 Phosphorylation of Tyr304 on ephrin B2 (Eur. J. Biochem. 271) 1733 Tyr316 remained in ephrinB2(301–333)-pY304 but not in the two other phosphorylated peptides. Figure 9 shows a structural model of ephrinB2(301–333)- pY304, generated from the NOE data of Fig. 8. Compared to the solution conformation of the nonphosphorylated peptide [18], phosphorylation at Tyr304 dramatically increased the flexibility of the b-hairpin structure and essentially unfolded it by phosphorylation at Tyr311 or Tyr316. Instead, residues around Tyr316 were found to form a helical structure with the ephrinB2(301–333)-pY304 peptide (Fig. 9). Phosphorylation of Tyr316 destroys even this helical structure as typical NOEs determining the short helix were not observable in the ephrinB2(301–333)-pY316 peptide. In contrast, the short helix found for the PDZ- binding motif [18] remains unperturbed by any of the phosphorylations. Taken together, these results indicate that phosphorylated ephrinB2(301–333) peptides become much more flexible especially in the N-terminal region around residue Tyr304 or the PHY304EKV sequence region. This kind of flexible conformations may be more favorable for Grb4 SH2 domain binding, as almost all SH2- binding phosphotyrosine peptides appear to assume exten- ded conformations in the binding site of the complex [27]. Discussion Using fluorescence polarization and NMR spectroscopy, we have identified a sequence segment around Tyr304 or PHpY304EKV on a C-terminal 33-residue peptide of the ephrin B2 cytoplasmic region as a high-affinity binding site for the Grb4 SH2 domain, in which tyrosine phosphory- lation is critical. We also show that phosphorylation of two distant tyrosine residues, i.e. Tyr311 or Tyr316, did not affect the binding of the pTyr304 motif to the Grb4 SH2 domain. Some other SH2 domains, for instance, p85N-SH2 [28], were found to have higher affinity for doubly tyrosine- phosphorylated peptides. Therefore, our results indicate that the phosphorylations of Tyr311 and Tyr316 identified in vivo may have unknown alternative functions other than physical interactions with the Grb4 protein. Earlier work already provided some evidence for low levels of in vivo phosphorylation at an equivalent position of residue Tyr304 in the Xenopus ephrin B1 [16]. Other studies implicate Tyr311 and/or Tyr316 as the major phosphorylation sites [15,16]. However, it is not known whether these two phosphorylated tyrosine residues contribute to high-affinity binding of the ephrin B cytoplasmic domain to the Grb4 adaptor protein [11]. The equivalent Tyr304 residue of Xenopus ephrin B1 was shown to be essential for binding to Grb4 in a recent work employing truncations and residue substitutions of ephrin B1 expressed in Xenopus oocytes cells [29]. This latter work also did not find evidence for the involvement of phosphorylated Tyr311 or Tyr316 in ephrin B1 binding to the Grb4 protein, in sharp contrast to a previous claim otherwise [30]. It is now clear that phos- phorylated Tyr304 alone determines the direct binding to Grb4 while sequence elements surrounding the Tyr311 site are important for the interactions of ephrin B with tyrosine kinases or phosphatases [29]. Structurally, the Grb4 SH2 domain binding motif on B ephrins, pYEKV, is somewhat different from other SH2 binding sequences [31,32], for example, pYEEI, pYDNV, pYTDM and pYTDL for Src family SH2 domains; pYENP, pYTEV and pYMDL for the Abl SH2 domain; pYDHP, pYKFL and pYNR for the CrK SH2 domain; pYDEP, pYDED and pYDEV for the Nck SH2 domain; pYLNV, pYLNV, pYIN and pYMN for the Sem5 SH2 domain; pYMXM, pYVXM, pYIXM and pYEXM for the N-terminal SH2 domain of p85; pYXXM for the C-terminal SH2 domain of p85; pYVIP, pYILI and pYILV for the C-terminal SH2 domain of PLC-r; pYELE, pYIDI and pYVDV for the N-terminal SH2 domain of PLC-r; and pYIXV, pYVXI, pYVXL and pYVXP for the N-terminal SH2 domain of SHPTP2. The different SH2 domain binding motifs provide diver- sity to signal transduction through a wide range of SH2 domain-containing proteins [31]. Through the alignment of the Grb4 and Src SH2 domains (Fig. 1A), it is seen that the phosphotyrosine binding pocket of the Grb4 SH2 domain is almost identical to that of the Src SH2 domain, being composed of residues in the bA2, bB5, bD3, bD4, bD6andbD¢1 structural elements. On the other hand, the local environment of the Grb4 SH2 domain binding pocket is different from that in the Src SH2 domain, which is composed of residues at the bD5, bE4, EF1, EF3, BG2, BG3 and BG4 sites. This particular Fig. 9. Model of the flexible structure of ephrinB2(301–333)-pY304 in solution. A cluster of solution conformations was generated by use of NOE data shown in Fig. 7, specifying the secondary structure elements. Residues for the Grb4 SH2 domain binding, i.e. PHpY304EKV, are colored in blue, while the tail residues IY330YKV for PDZ domain binding are colored in red. This representation of the peptide confor- mation was prepared using INSIGHT II (Tripos, San Diego, CA, USA). 1734 Z. Su et al. (Eur. J. Biochem. 271) Ó FEBS 2004 [...]... domain -binding and RGS3 PDZ domain -binding to phosphorylated peptides are almost entirely independent of one another (Table 1) These observations demonstrate that the flexibly linked SH2 -binding and PDZ -binding sequences in the ephrin B peptide (Fig 9) can accept the simultaneous docking of both the Grb4 SH2 and RGS3 PDZ domains It is possible that simultaneous binding to the cytoplasmic domain of B ephrins may... pocket of Grb4 SH2 are significantly different from those in the Src SH2 domain The b-sheet after the EF3 segment in the Src SH2 domain appears to be shifted to the residues behind the BG segments in the Grb4 SH2 domain All these structural variations may determine the binding specificity of the Grb4 SH2 domain for the pYEKV motif in B ephrins (Fig 2B and Table 1) The phosphorylation of Tyr311 or 316... may also be able to coordinate the two different types of reverse signaling events mediated by the Grb4 and PDZ- RGS3 proteins Activation of reverse signaling through B ephrins leads to clustering and tyrosine phosphorylation of the cytoplasmic tail of B ephrins, and a concomitant recruitment of Grb4 and its SH3 -binding protein partners Subsequently, regulation of the cell cytoskeleton occurs through... localization and/ or alteration of tyrosine -phosphorylation levels of other proteins [11] In vivo and cell-based experiments showed that ephrin B2 or B1 lacking the cytoplasmic tails were no longer able to exert reverse signaling [14,35] Taken together, our findings strongly suggest that the high-affinity binding between phosphotyrosine304 in ephrinB2 and the Grb4 SH2 domain is one critical step in the reverse... specifying the hairpin structure It is likely that the phosphorylation of Tyr311 or Tyr316 has significant contributions to the exposure and positioning of the Tyr304 residue for binding to tyrosine kinases and phosphatases On the other hand, tyrosine phosphorylations do not affect the conformation of the PDZ binding motif at the C-terminus (Fig 9) Therefore, the 33-residue functional tail of ephrin B2 appears... capable of accommodating two relatively independent structural subunits carrying binding motifs for different docking proteins Indeed, in vitro GST pull-down experiments showed that a three-component complex can be formed among the Grb4 SH2 domain, the Tyr304- phosphorylated ephrineB2(301–333) peptide and the GST -PDZ protein from PDZ- RGS3 Fluorescence -binding experiments revealed that Grb4 SH2 domain -binding. .. Phosphorylation of Tyr304 on ephrin B2 (Eur J Biochem 271) 1735 pocket in the Grb4 SH2 domain has reduced negative charges and increased hydrophobicity, which may be related to the difference between the Grb4 SH2 domain binding motif, pYEKV, identified in this study and the Src SH2 binding motif, pYEEI reported previously [33,34] Moreover, the secondary structures around the pTyr binding pocket of Grb4. .. significant in uence on the solution conformation of the ephrin B cytoplasmic tail at least within the ephrinB2(301–333) fragment The unphosphorylated ephrin B peptide adopts a well-folded b-hairpin structure for the Grb4 SH2 domainbinding region [18] The phosphorylation of either Tyr311 or Tyr316 largely exposes Tyr304 from a folded b-hairpin as shown by the disappearance of long-range NOE contacts specifying... Axon guidance: the cytoplasmic tail Curr Opin Cell Biol 14, 221–229 7 Lu, Q., Sun, E.E., Klein, R.S & Flanagan, J.G (2001) Ephrin- B reverse signaling is mediated by a novel PDZ- RGS protein and selectively inhibits G protein- coupled chemoattraction Cell 105, 69–79 8 Lin, D., Gish, G.D., Songyang, Z & Pawson, T (1999) The carboxyl terminus of B class ephrins constitutes a PDZ domain binding motif J Biol... reverse signaling of the Eph /ephrin B system, that orchestrate the assemblage of Eph/ephrincontaining cells and tissues into defined functional aggregates and compartments Acknowledgements We thank Drs Dmitri Tolkachev and Surajit Bhattacharjya for their valuable discussion and Patrice Bouchard and Betty Zhu for technical assistance We also thank Andy Ng and Evelyne Copeland for critical reading of the manuscript . Single phosphorylation of Tyr304 in the cytoplasmic tail of ephrin B2 confers high-affinity and bifunctional binding to both the SH2 domain of Grb4 and. question by use of the SH2 domain of Grb4 and the PDZ domain of the RGS3 protein. We titrated the SH2 protein into the fluorescein- labeled ephrinB2(301–333)-pY304