Interactions between the Fyn SH3-domain and adaptor protein Cbp/PAG derived ligands, effects on kinase activity and affinity ´ Silje A Solheim1,2, Evangelia Petsalaki3, Anne J Stokka1,2, Robert B Russell3, Kjetil Tasken1,2 and Torunn Berge1,2 The Biotechnology Centre of Oslo, Norway Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo, Norway European Molecular Biology Laboratory, Heidelberg, Germany Keywords kinase activity; proline-rich motifs; protein– protein interactions; SH3 domain; tyrosine phosphorylation Correspondence T Berge, The Biotechnology Centre of Oslo, Gaustadalleen 21, N-0319 Oslo, Norway Fax: +47 2284 0501 Tel: +47 2284 0519 E-mail: torunn.berge@biotek.uio.no Website: http://www.biotek.uio.no (Received May 2008, revised 24 June 2008, accepted August 2008) doi:10.1111/j.1742-4658.2008.06626.x Csk-binding protein ⁄ phosphoprotein associated with glycosphingolipidenriched domains is a transmembrane adaptor protein primarily involved in negative regulation of T-cell activation by recruitment of C-terminal Src kinase (Csk), a protein tyrosine kinase which represses Src kinase activity through C-terminal phosphorylation Recruitment of Csk occurs via SH2domain binding to PAG pTyr317, thus, the interaction is highly dependent on phosphorylation performed by the Src family kinase Fyn, which docks onto PAG using a dual-domain binding mode involving both SH3- and SH2-domains of Fyn In this study, we investigated Fyn SH3-domain binding to 14-mer peptide ligands derived from Cbp ⁄ PAG-enriched microdomains sequence using biochemical, biophysical and computational techniques Interaction kinetics and dissociation constants for the various ligands were determined by SPR The local structural impact of ligand association has been evaluated using CD, and molecular modelling has been employed to investigate details of the interactions We show that data from these investigations correlate with functional effects of ligand binding, assessed experimentally by kinase assays using full-length PAG proteins as substrates The presented data demonstrate a potential method for modulation of Src family kinase tyrosine phosphorylation through minor changes of the substrate SH3-interacting motif Tyrosine phosphorylation is one of the key regulatory protein modifications in multicellular organisms and tightly controls and coordinates a wide range of cellular responses such as growth, metabolism, tissue repair, migration and apoptosis [1–3] Phosphorylation modulates enzymatic activity as well as creating new binding sites for the recruitment of active molecules into signalling complexes, and assists in building dynamic networks for the transduction of information from the extracellular environment to intracellular signalling pathways Accurate and specific processing of information is vital to maintaining cellular homeostasis, and errors in signal transduction pathways are linked to a range of diseases such as cancer, autoimmunity and diabetes [4,5] Tyrosine phosphorylation is a reversible modification, regulated by protein tyrosine kinases (PTKs) and phosphatases (PTPs) [6,7] PTKs of the Src kinase family employ a well-conserved modular arrangement of interaction domains in the regulation of kinase Abbreviations Cbp ⁄ PAG, Csk binding protein ⁄ phosphoprotein associated with glycosphingolipids-enriched microdomains; Csk, C-terminal Src kinase; GST, glutathione S-transferase; PPII, polyproline type II; PRD, proline-rich domain; PTK, protein tyrosine kinase; SFK, Src family kinase; SH2, Src homology 2; SH3, Src homology 3; TCR, T-cell receptor FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS 4863 Modulation of the Fyn SH3–PAG interaction S A Solheim et al activity and inhibition, as well as in the combinatorial assembly of active signalling complexes The common structure consists of an N-terminal membrane-targeting region (SH4), a Src-homology (SH3) domain and a Src-homology (SH2) domain, capable of binding to proline-rich motifs and phosphotyrosine residues, respectively These interaction domains are followed successively by a tyrosine kinase (SH1) domain [8,9] In addition, Src family kinases (SFKs) contain both a C-terminal auto-inhibitory phosphorylation site and an activating auto-phosphorylation site in the kinase domain Interactions occur frequently via individual domains; however, both domains may also cooperate to facilitate specific and stable complex formation [10– 12] In T cells, the SFKs Lck and Fyn have central roles in early signal transduction events as the most proximal signalling molecules to be activated downstream of the T-cell receptor following direct interaction between the receptor and peptide–MHC complexes on antigen presenting cells This activation leads to a cascade of tyrosine phosphorylation-dependent signalling pathways [2,3,13] Fyn associates with and phosphorylates the transmembrane adaptor protein Csk-binding protein ⁄ phosphoprotein associated with glycosphingolipid-enriched microdomains (Cbp ⁄ PAG) [14], which is localized to lipid rafts by palmitoylation anchoring [15] The adaptor functions primarily as a negative regulator of SFKs and T-cell activation via recruitment of C-terminal Src kinase (Csk) to PAG pTyr317 (human) [16–18], however, the regulatory role of PAG may be more complex as the adaptor may also act as an activating partner of Lyn, an SFK, and pSTAT3 in B-cell lymphomas, as suggested by Tauzin et al [19] The involvement of either SH3 or SH2 domains has been discussed for the association of Fyn with PAG A [20,21]; in fact, Fyn utilizes both domains in this interaction, as we have demonstrated previously [22] Binding of the Fyn SH3-domain to the first proline-rich region of PAG is essential for the initiation of PAG tyrosine phosphorylation, which occurs via a processive phosphorylation mechanism [23] This subsequently allows binding of the Fyn SH2-domain to PAG Tyr163 or pTyr181 and results in a dual-domain docking, enhancing the affinity of the Fyn–PAG interaction and rendering Fyn insensitive to negative regulation by Csk (Fig 1A) In this study, we explore further the initial step of the Fyn–PAG association; binding of the kinase SH3domain to the first proline-rich region of PAG Fulllength PAG variants, developed using a 2D peptide array approach, have previously been employed to demonstrate the functional effects of the Fyn–PAG complex in T cells [22] Using full-length proteins as templates, we created 14-mer peptide ligands (Fig 1B), one corresponding to the wild-type PAG interaction region (PRD1), and two structural variants of this sequence with low (PRD1-RLP*) and high (PRD1super) affinity for the Fyn SH3-domain, respectively PAG also has a second proline-rich region, known to interact with the SH3-domain of the related kinase Lyn [24], and a 14-mer peptide (PRD2) containing this binding site was included as a control We conducted binding assays of peptide ligands using the isolated Fyn SH3-domain (Fig 1C) as well as full-length Fyn affinity precipitated from human primary T-cell lysates Kinetics and specificity of Fyn SH3-domain interactions were determined using SPR, and CD spectroscopy was used to assess local structural changes upon SH3-domain binding The tertiary structure of the Fyn SH3-domain complexed to ligand is known both from X-ray diffraction and NMR studies [25,26], B C Fig The Fyn-PAG interaction (A) Schematic representation showing Fyn binding to the transmembrane adaptor PAG via SH3- and SH2-domain interactions (B) The 14-mer peptide ligands used in this study include the first (PRD1) and second (PRD2) proline-rich regions of PAG as well as variants of the first region (PRD1-RLP* and PRD1-super) developed using 2D peptide arrays [22] (C) Secondary structure elements of the Fyn SH3-domain The five b strands, numbered consecutively, are separated by the interaction loops (bold) making primary contacts with the ligand Trp119 (*) is a highly conserved residue among SFK SH3-domains 4864 FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS S A Solheim et al Modulation of the Fyn SH3–PAG interaction and this information was used to model the peptide ⁄ SH3-domain complexes in silico to investigate details of the ligand interactions We show that changes in affinity and secondary structure have significance for the functional effects of ligand binding, assessed experimentally by kinase assays using fulllength PAG proteins as substrates Results and Discussion Phosphorylation of the adaptor protein PAG is dependent on an initial interaction between the first prolinerich region of PAG and the SH3-domain of the kinase Fyn Our earlier investigations have highlighted the importance of the Fyn SH3-domain interaction in initiating the association with PAG, and how this affects tyrosine phosphorylation and thereby the functionality of PAG as a negative regulator of T-cell activation through recruitment of Csk, dependent on PAG pTyr317 [22] In this study, we recreated this association in vitro using isolated Fyn SH3-domains and synthetic peptide ligands containing wild-type PAG sequences as well as structural variants developed using a 2D peptide array approach, a rapid and semiquantitative approach for evaluation of amino acid substitutions of all residues in a defined region The Fyn–PAG interaction: association via the Fyn SH3-domain Several studies have focused on the identification of high-affinity ligands for SH3-domains, either through phage display or combinatorial peptide libraries [27– 29] Combined with data from NMR and X-ray crystallography [25,26,30,31], these reports have revealed in detail many of the requirements of SH3-domain ligand binding, and optimal core recognition motifs have been established for several kinase SH3-domains, such as RPLPPLP for Src, Fyn, Lyn and phosphatidylinositol 3-kinase This motif is a class I SH3 consensus motifs (RxxPxxP), characterized by an N-terminal arginine residue known to form an orientation-determining salt bridge with a key aspartate residue [27,28,32] Interestingly, the Fyn SH3 domain contains two such adjacent residues Asp99 and Asp100, which may both participate in formation of this salt bridge [33] Using 2D peptide array analyses, the minimal interaction sequence of the wild-type PAG-derived peptide PRD1 has been established to RELPRIP [22], i.e four residues in common with the phage display motif, whereas the high-affinity PRD1super peptide (Glu132Pro, Pro135Arg of PAG, human) of this study matched the minimal motif with five Fig Fyn interacts with the first proline rich region in PAG Lysates from human primary T cells were incubated with the indicated biotin-linked peptides and complexes precipitated using excess streptavidin beads Interactions were evaluated by western blot analysis using anti-Fyn IgG common residues By contrast, the low-affinity sequence PRD1-RLP*, lacked critical amino acids by three alanine substitutions of the minimal motif (Arg131, Leu133 and Pro134 of PAG, human), and PRD2 (PPPVPVK), the peptide corresponding to the second proline-rich region of PAG, had only two residues in common with the minimal core motif To analyse further the implications of PAG binding to the Fyn SH3-domain, 14-mer peptides were synthesised consisting of these sequence variants of the interaction site (PRD1, PRD1-RLP* and PRD1-super) as well as PRD2 (Fig 1B) Interaction data obtained using full-length proteins were first verified by performing biotin–streptavidin affinity precipitations on human T-cell lysates using biotin-tagged peptide ligands (Fig 2) The pull-down assays demonstrated a significant reduction in binding using the PRD1-RLP* peptide, whereas the PRD1-super peptide, developed as a high-affinity Fyn SH3 binder using the 2D peptide array approach, displayed an  2.5-fold increase in Fyn binding relative to the wild-type (PRD1) peptide Interaction with the first proline-rich sequence was exclusive as no association was observed in pull-down assays performed with the peptide PRD2 containing the second PAG proline-rich domain Conformation of peptide ligands in solution Polyproline sequences are known to adopt a distinct secondary structure in aqueous solutions due to the unique conformational properties of the cyclic imino nature of proline residue [34,35] This structure, a polyproline type II helix (PPII), has a specific geometry exploited in SH3-domain ligand binding that enables direct interactions with the hydrophobic binding grooves of this domain Using CD spectroscopy, we investigated the extent of PPII helix conformation of the peptide ligands PRD1, PRD1-RLP*, FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS 4865 Modulation of the Fyn SH3–PAG interaction S A Solheim et al 50 A 75 kDa PAG –50 20 40 60 10 80 100 120 s –100 B PRD1 PRD1-RLP* PRD1-super PRD2 –250 –300 200 210 230 220 240 Wavelength (nm) 250 260 Fig Far-UV CD spectroscopy of the PAG-derived peptide ligands Spectra were recorded in 10 mM Hepes (pH 7.4), 150 mM NaCl at a sample concentration of 90 lM Time wt PAG PAG RLP* super PAG 250 –200 20 300 –150 Relative intensity (%) [θ] (deg·cm2·dmol–1) (x105) 100 kDa pPAG 200 150 100 50 PRD1-super and PRD2 (Fig 3) In water, typical CD spectra of the PPII helix show a negative band at 200– 205 nm and a positive band at 217–225 nm [36] Although the positive band was absent from our samples, all four peptides displayed strong negative bands at  201 nm, indicating that they are not completely unstructured in aqueous buffer The negative maxima, believed to arise from the extent of PPII conformation, was found to correspond with the number of proline residues in each peptide sequence (PRD2 > PRD1 = PRD1-super > PRD1-RLP*) The absence of a characteristic positive shoulder from all spectra was thought to arise from the presence of other secondary structure elements in the samples [34,35] Time (min) Fig Kinetics of PAG phosphorylation by wild-type Fyn In vitro pulse-chase assays were performed using active Fyn kinase and various full-length PAG constructs incubated on ice in the presence of 80 nM [32P]ATP[cP] for 10 Addition of 0.5 mM ATP initiated the chase reaction, aliquots were withdrawn at specified time points and reactions stopped by mixing with SDS ⁄ PAGE sample buffer and boiling Time points were resolved on SDS ⁄ PAGE gels and detected using autoradiography (A) Phosphorylation of wildtype PAG using active Fyn kinase illustrating the autoradiography gels used for quantification of phosphorylation levels Coomassie stain shows equal loading of PAG in each sample (B) Tyrosine phosphorylation effects of the PAG–Fyn SH3 interaction modulation Phosphorylation levels were quantified as pixel intensity of the highest molecular species relative to phosphorylated wild-type (wt) PAG at Error bars show SEM over three repeat experiments Effects of PAG–Fyn SH3 interaction modulation The functional effects of modulating the affinity of the PAG–Fyn SH3 interaction were examined in an in vitro kinase assay using full-length, recombinant PAG protein and active Fyn kinase In T cells, PAG has a dual role as both a ligand and a substrate for Fyn, and with nine potential tyrosine phosphorylation sites the adaptor is an excellent subject for quantitative analysis of phosphorylation effects mediated via SH3domain interactions In a pulse-chase assay, active Fyn and recombinant PAG were incubated on ice with a low concentration of radioactive ATP After 10 min, a large excess of ATP was added and the phosphorylation process was continued with aliquots withdrawn at the indicated time points as illustrated for wild-type PAG in Fig 4A Phosphorylated PAG species appeared as distinct bands, with the slower migrating species corresponding to more heavily phosphorylated protein 4866 accumulating over time Quantitative analyses of PAG phosphorylation of the three variants were performed over a time course of by measuring pixel intensity of the highest band at each time point relative to fully phosphorylated wild-type PAG (Fig 4B) The wild-type PAG protein was rapidly phosphorylated by Fyn, reaching the highest molecular mass species after  The full-length high affinity SH3-binder, superPAG, was phosphorylated in a similar fashion; however, the accumulation of protein tyrosine phosphorylation appeared more pronounced (approximately twofold) The rapid phosphorylation progress and the appearance of discrete bands, as illustrated in Fig 4A, are typical of a processive phosphorylation process [22,23,37], governed by an initial interaction between the Fyn SH3-domain and PAG This binding mode is indispensable for rapid and efficient PAG phosphorylation, as full-length PAG-RLP* with a FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS S A Solheim et al Modulation of the Fyn SH3–PAG interaction greatly reduced Fyn SH3-domain affinity showed a significantly delayed tyrosine phosphorylation Phosphorylation of the PAG-RLP* protein most likely follows a distributive course caused by random collisions between the substrate and kinase Efficient phosphorylation of PAG-RLP* consequently occurs only after phosphorylation of Tyr168 and Tyr181, which according to our interaction model [22] allows Fyn to dock onto its substrate via SH2-domain binding, thus switching to tyrosine phosphorylation in a processive manner Again, the interaction was deemed specific for the first proline-rich PAG sequence, as all substrate molecules included in the kinase assays contained an unaltered second proline-rich region (amino acids 252– 265 of PAG, human) which did not appear to affect tyrosine phosphorylation by Fyn in vitro Structural impact of Fyn SH3-domain ligand binding Far-UV CD was employed to characterize local structural changes in the Fyn SH3-domain following binding of PAG-derived peptide ligands CD spectra of the Fyn SH3-domain alone (Fig 5A) showed a minimum at A B Fig Far-UV CD spectroscopy of the Fyn SH3-domain interaction with PAG derived ligands (A) Far-UV CD spectra of the Fyn SH3-domain, the PRD1 peptide and the SH3 domain-peptide complex measured in 10 mM Hepes (pH 7.4), 150 mM NaCl (B) Spectra of SH3-domain ligand mixtures at a ligand concentration range of 10–90 lM Spectra of free peptide were subtracted from those of the mixture (C) Difference spectra of the SH3-ligand mixtures [data presented in B with spectra of the free SH3-domain subtracted (a)b)] C FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS 4867 Modulation of the Fyn SH3–PAG interaction S A Solheim et al 207 nm and two maxima at 221 and 236 nm, comparable with previous reports on SFK SH3-domains [38,39] Addition of peptide containing the wild-type binding site (PRD1) resulted in a positive shift of the maxima and an increase in positive ellipticity over the 220– 240 nm region, whereas the minimum was slightly blueshifted with an increase in negative ellipticity The latter observation may indicate an increase in PPII conformation caused by stabilization of the proline-containing peptide upon binding to the SH3-domain [34] Ligand binding over a concentration range of 10– 90 lm showed that the wild-type sequence and the high-affinity SH3-binder, PRD1-super, induced a local concentration-dependent structural change over the same region (220–240 nm, Fig 5B) Spectra for both peptides were found to approach a saturation maximum Increasing concentrations of the low-affinity SH3-binder, PRD1-RLP*, did not affect secondary structure, as estimated by CD measurements; neither did the control peptide PRD2 containing the second proline-rich region The largest difference, Dh, in the 215–250 nm region was plotted for the highest peptide concentrations (90 lm, Fig 5C) This clearly revealed that the relative local structural change was significantly greater for the high-affinity binder, showing a broad positive band with an ellipticity maximum centred at 225 nm Positive ellipticity in this region has been attributed to interactions with clustered aromatic amino acids [39,40], which may involve tyrosine residues located in the interaction loops of the SH3domain Other parts of the differential spectrum were not significantly influenced by ligand binding to the SH3-domain for either peptide (data not shown) Determination of affinity and kinetic constants Binding affinities and dissociation constants for ligands derived from the first proline-rich domain of PAG were analysed using SPR To this end, we immobilized Fyn SH3-fusion proteins to surfaces of biosensor chips, as described above, and assayed for binding by passing the peptides PRD1, PRD1-RLP* and PRD1super over immobilized proteins Thus, a single chip was used for all peptides, minimizing effects of variations in ligand concentration Binding profiles (Fig 6A) obtained this way corresponded well with A B C D Fig SPR measurements of the interaction between immobilized Fyn SH3-domains and peptides PRD1, PRD1-RLP* and PRD1-super (A) All three peptides were injected over the sensor chip at 100 lM to reveal differences in binding (B–D) Peptides (B, PRD1; C, PRD1-RLP*; D, PRD1-super) were injected over the sensor chip for kinetic analysis of the SH3-domain interaction Ligand binding curves for peptide concentrations of 3.1, 12.5, 50 and 200 lM are shown Binding was measured in resonance units (RU), where RU corresponds to the binding of pgỈmm)2 4868 FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS S A Solheim et al Modulation of the Fyn SH3–PAG interaction Table Kinetic constants for PAG-derived ligands and Fyn SH3-domain interactionsa ka1 (M)1Ỉs)1) PRD1 PRD1-super kd1 (s)1) ka2 (s)1) kd2 (s)1) KD (M) 7.7 · 104 6.8 · 104 0.41 0.1 2.5 · 10)4 6.9 · 10)4 0.01 0.003 5.2 · 10)6 1.2 · 10)6 a Apparent association (ka) and dissociation (kd) rate constants and affinity constants (KD) were calculated from three independent experiments Numbers in the table are shown for one representative experiment No kinetic data could be obtained for the Fyn SH3 ⁄ PRD1-RLP* peptide interaction SD < 1% for all constants, apart from PRD1 ka2 and kd2 where SD < 5% results from pull-down assays using biotinylated peptides Approximately twice as much PRD1-super peptide as the PRD1 peptide was found to bind to immobilized SH3-domains over multiple experiments, while virtually no binding to the immobilised proteins was observed for the PRD1-RLP* peptide To determine association and dissociation rate constants for the peptides, concentration series were injected over the sensor chips (Fig 6B–D, representative curves from a single run are shown) Kinetic data from the binding curves were evaluated using a twostate conformational change model, which was found to provide a better fit than the : Langmuir model based on analyses of residuals and v2 values Our choice of model is backed by findings using CD spectroscopy (Fig 5), which indicated that ligand binding induced a structural change in the Fyn SH3-domain A summary of kinetic constants is given in Table Dissociation constants showed that the PRD1-super peptide bound to the Fyn SH3-domain with approximately fourfold higher affinity than the wild-type interaction sequence KD estimated for the wild-type PRD1 peptide was in the low micromolar range ( 4–7 lm) in repeat experiments, of the same order of magnitude as previous studies on SFK SH3-domains using similar technology [28,39,41–43] Molecular modelling of the Fyn SH3-domain and PAG-derived peptides The interaction pockets of the SH3-domain are made by loops linking the individual b strands together (the RT loop, the n-Src loop, and the 310 helix as indicated in Fig 1C), flanked by strands b4 and b5 The variable loops n-Src and RT are principal determinants for ligand recognition, orientation, and specificity of this domain, with residues Tyr91 and Tyr137 forming interaction pocket 1; Tyr93, Tyr137 and Trp119 forming pocket 2, and the valley between n-Src and RT loops included by Trp119 and Tyr132 making up the third interaction pocket [31,44] Molecular modelling of the ligand ⁄ SH3-domain interactions were performed using the modeller software as described above Three available structures (1fyn [25], 1azg [26] and 1a0n [26]) showing the Fyn SH3 domain binding to peptides were superposed to establish the positioning of the SH3 binding PxxP motif and the alignment of our peptides to a molecular model (Fig 7) To model our peptides binding to the SH3 domain, we selected the NMR structure 1azg of the Fyn SH3 domain complexed to a proline-rich peptide P2L (PPRPLPVAPGSSKT) corresponding to residues 91–104 of the p85 subunit [26] P2L contains the class I SH3 consensus motif RxxPxxP, sharing sequence properties with both PRD1 and PRD1-super The first four residues of each peptide (CHQS) were common to all models, thus, all peptide sequences were numbered starting with the first residue of the model sequence, arginine for PRD1 and PRD1-super and alanine for PRD1-RLP*, respectively Alignment of the PRD1 peptide containing the wildtype PAG interaction sequence binding to Fyn SH3 (Fig 7B), predicted both hydrophobic and electrostatic interactions involved in formation of the complex The model showed peptide residues Pro4 and Pro7 fitting well into binding grooves and created by the n-Src loop and the 310 helix, stabilized by stacking interactions of the pyrrolidine ring of Pro7 with the aromatic side chain of Tyr137 Modelling of the PRD1-super peptide, which included two amino acid substitutions (Glu2Pro and Pro8Arg), again showed main interactions via the core residues for binding (Arg1, Pro4 and Pro7) The proline substitution (Pro2) positioned directly after the arginine residue occupied pocket and was stacked against the aromatic residue Trp119 Previous reports have suggested that pocket 3, binding arginine via acidic side chains, could favourably accommodate binding of a proline residue in this way [45], and it is likely that the proline substitution provides a major contribution to the increased affinity for the PRD1-super peptide Advantages of the Arg8 introduction were less obvious as alignment of flanking residues correlated with greater flexibility in positioning; however, further stabilization of the complex could be provided by H-bond interactions via water molecules and other amino acids not evaluated in this model Impact of the various amino acid substitutions was demonstrated by superposing the variant peptides PRD1-RLP* (green) and PRD1-super (blue) onto the wild-type PRD1 peptide (beige) (Fig 7C) The modelled structures showed that alanine substitution of FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS 4869 Modulation of the Fyn SH3–PAG interaction S A Solheim et al Fig Molecular models showing the Fyn SH3-domain complexed to PAG-derived peptides (A) Superposition of the three available PDB structures for the Fyn SH3 domain complexed to ligand peptides (1azg (yellow) [26], 1fyn (green) [25] and 1a0n (red) [26]) (B) Models of the Fyn SH3domain in complex with PRD1 and PRD1-super peptides based on the NMR structure 1azg [26] The peptide is displayed as sticks; the SH3-domain is shown as ribbons, while selected amino acids predicted to be essential for interaction with the ligand are shown as spheres (C) Superposition of the PRD1-RLP* (green) and PRD1super (blue) peptides on the PRD1 (beige) peptide complexed to the Fyn SH3-domain Arg1 appeared to reduce interactions with acidic residues of pocket (e.g residues surrounding Trp119) The substitution removes the arginine residue of the RxxPxxP motif, thus abrogating the formation of the salt bridge with Asp99, which defines the orientation of the peptide ligand [33] Alanine substitution of Pro4 eliminated its stacking interactions with the central tryptophan residue Trp119, believed to extensively reduce the ability of the PRD1-RLP* ligand to form a stable complex with the Fyn SH3-domain The overlay structures emphasised the significance of the PRD1-super Pro2 substitution, revealing a good fit adjacent to Arg1 where interactions with aromatic and acidic residues such as Trp119, Asp99 and Asp118 of the SH3-domain were likely It is highly probable that the predicted interactions outlined above result in the local structural changes in the Fyn SH3-domain as observed by CD measurements, and that these changes affect binding affinity as evaluated by SPR 4870 Conclusions We have described in detail the interactions of a natural Fyn SH3-domain ligand extracted from the adaptor protein PAG and low- and high-affinity variants of this sequence developed using a 2D array approach Interaction kinetics and local structural impact of these ligands binding to the Fyn SH3-domain were analysed using SPR and CD, respectively, and the results of these investigations related to functional effects of ligand binding, assessed experimentally by kinase assays using full-length PAG proteins containing the modified binding motifs as substrates In summary, our data demonstrate that substrate phosphorylation may be modified through minor changes in the SH3-domain interacting motif Optimal binding sequence motifs have previously been found using random or biased library approaches, performed with several cycles of selection for optimalization [27–29,46] In this study, we used a FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS S A Solheim et al natural ligand as a point of reference for the development of high- and low-affinity ligands using a 2D peptide array approach This is a simple and rapid method for analysis of individual amino acid substitutions, and the high-affinity SH3-binder, PRD1-super, developed using this strategy was found to have a dissociation constant in the low micromolar range, comparable to that of the phage-display library peptide VSL12 (1.2 versus 0.6 lm) [46] In vivo manipulation of protein interaction domains in this way could provide valuable insight into the functional consequences of localized protein–protein interactions As an example, the high-affinity, fulllength PAG (superPAG) construct has been used in T cells to reveal that the negative regulatory potential of PAG is enhanced by this modification, consistent with a higher degree of PAG phosphorylation and concomitant Csk recruitment [22] On a similar note, is the kinase targeting strategy used by viruses such as HIV and Herpesvirus saimiri which encode accessory proteins containing proline-rich domains [47–49] These proteins, Nef and Tip, respectively, interact with kinase SH3-domains with high affinity to modify the behaviour of virus-infected cells, and have been shown to control both pathogenicity and T-cell proliferation This demonstrates the potential of manipulating signalling pathways in vivo by creating high-affinity ligands to compete with natural SH3-protein interactions Experimental procedures Modulation of the Fyn SH3–PAG interaction Biotin-peptide pull-down assay Human peripheral blood T cells were purified from normal donors by negative selection as described [18] and lysed in ice-cold lysis buffer (50 mm Tris pH 7.4, 100 mm NaCl, mm NaF, 10 mm NaPPi, mm EDTA, 1% Triton X-100, 50 mm n-b-octyl-d-glucoside, mm Na3VO4 and mm phenylmethylsulfonyl fluoride) After 30 pre-clear at °C using streptavidin beads (Invitrogen, Carlsbad, CA, USA), T-cell lysates were incubated with different biotinylated peptides for h at °C with rotation followed by 30 incubation with streptavidin beads at room temperature Biotin alone was used as a control for non-specific binding Samples were washed extensively in ice-cold lysis buffer prior to western blot analysis using anti-Fyn IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) Protein expression and purification Recombinant proteins were expressed and purified as described previously [22] For thrombin cleavage of the GST-tag, washed glutathione–Sepharose beads bound to GST–Fyn SH3-domain were incubated with human thrombin (Sigma, St Louis, MO, USA) in 50 mm Tris (pH 8.0), 150 mm NaCl, 0.1% b-mercaptoethanol and 2.5 mm CaCl2 at °C overnight The supernatant containing the Fyn SH3domain was purified further by gel filtration over a Superdex 75 column (GE Healthcare Europe GmbH, Uppsala, Sweden) using 10 mm Hepes (pH 7.4), 150 mm NaCl, and concentrated using a Vivaspin spin column (Sartorius Biolab, Auckland, New Zealand) Recombinant proteins were stored at )80 °C in 10 mm Hepes (pH 7.4), 150 mm NaCl Peptide synthesis Peptides PRD1 (CHQSRELPRIPPES), PRD1-RLP* (CHQSAEAARIPPES), PRD1-super (CHQSRPLPRIPRES) and PRD2 (EEEAPPPVPVKLLD) were synthesized with or without biotin-tags coupled to the N-terminus in-house or purchased from AnaSpec Inc (San Jose, CA, USA) Purity was analysed by HPLC and mass spectroscopy All peptides were lyophilized prior to re-suspension in NaCl ⁄ Pi, and peptide concentration was determined using a Biochrom 30 amino acid analyser (Biochrom, Cambridge, UK) Expression plasmids of glutathione S-transferase -tagged proteins The constructs expressing different variants of glutathione S-transferase (GST)-PAGDTM have been described previously [22] A construct expressing the SH3-domain of Fyn was created by sub-cloning amino acids 83-537 into the BamHI ⁄ NotI sites of the pGEX-4T-2 vector and introducing a stop codon at amino acid residue 142 In vitro kinase assay Pulse-chase experiments using full-length, active Fyn kinase (Upstate, Charlottesville, VA, USA) were performed as described elsewhere [37] and protein phosphorylation quantified using imagequant (GE Healthcare Europe) Circular dichroism CD spectroscopy was performed on a nitrogen-flushed JASCO spectropolarimeter J810 (Jasco, Tokyo, Japan) equipped with a circulating water bath Samples were analysed at 20 °C using a quartz cuvette (Hellma GmbH & co, Mullheim, Germany) with a pathlength of 0.1 cm CD specă tra were recorded five times at 50 nmỈmin)1 with a width of nm over a wavelength range of 190–260 nm Samples were analysed in 10 mm Hepes, pH 7.4, 150 mm NaCl Total buffer absorbance was analysed and was found to be satisfactory for the range of wavelengths used in the measurements The scans were averaged and a sample-free buffer spectrum was subtracted Data smoothing was FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS 4871 Modulation of the Fyn SH3–PAG interaction S A Solheim et al performed using an inverse square algorithm in sigmaplot 8.0 (SPSS, Chicago, IL, USA) as described previously [50] Surface plasmon resonance Measurements were performed on a BIAcore T100 instrument (BIAcore Life Sciences ⁄ GE Healthcare Europe) GST–Fyn SH3 fusion protein or GST protein alone (diluted in 10 mm sodium acetate, pH 4.5) were immobilized on CM5 sensor chips (BIAcore) via cross-linking of free amine groups to the N-hydroxysuccinimide ⁄ 1-ethyl-3[3-dimethylaminopropyl] carbodiimide hydrochloride activated flow-cell surfaces to a final response of 8500 RU (1 RU = pgỈmm)2), followed by blocking of free succinimide ester groups with m ethanolamine GST–protein was immobilised as a reference surface to the same molar extent The chip was treated with 10 mm dithiothreitol in running buffer (10 mm Hepes, pH 7.4, 150 mm NaCl, 0.05% P20, mm EDTA, 0.005% SDS) to remove GST dimer products After extensive washing of the surface with running buffer, peptide binding was assessed by injecting the indicated concentrations in running buffer over the flow-cell surface at a sample rate of lLỈmin)1 Kinetic analysis was performed using the BIAcore T100 evaluation software Curve fitting was executed by the software program (global fitting algorithm) which also performed calculations of dissociation constants (KD) Molecular modelling of the protein–peptide complexes Modelling of the protein–peptide complexes was performed using modeller [51] and visualized using pymol (http:// www.pymol.org) The Protein Data Bank [52] entry of the Fyn tyrosine kinase SH3-domain bound to a proline-rich peptide 3BP-2 (PPAYPPPPPVP, PDB ID: 1fyn [25]) and P2L (PPRPLPVAPGSSKT, PDB ID: 1azg and 1a0n [26]) were used to establish the positioning of the PxxP core motif of the peptides and 1azg was selected as a template to create models for binding of the PRD1, PRD1-super, and PRD1-RLP* peptides In aligning the peptides of this study to that in the 1azg, it was assumed that the core SH3-domain binding motif (PxxP) would overlay with the equivalent position as described in the original study [26] Acknowledgements This work was supported by Norwegian Research Council FUGE Career Fellowship (159306 to TB), by EU Grant 3D repertoire LSHG-CT-2005-212028 (to RR), and by grants from the Norwegian Functional Genomics Programme, the Norwegian Research Council, the Norwegian Cancer Society, and the European Union (grant no 037189, thera-cAMP) (to 4872 KT) We thank Gladys M Tjørhom, Jorun Solheim, and Ola Blingsmo for excellent technical assistance, Dr Per E Kristiansen for use of the circular dichroism equipment, and Dr Matthew Betts for helpful discussion References Pawson T (2004) Specificity in signal transduction: from phosphotyrosine–SH2 domain interactions to complex cellular systems Cell 116, 191–203 Hubbard SR & Till JH (2000) Protein tyrosine kinase structure and function Annu Rev Biochem 69, 373–398 Palacios EH & Weiss A (2004) Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation Oncogene 23, 7990–8000 Gschwind A, Fischer OM & Ullrich A (2004) The discovery of receptor tyrosine kinases: targets for cancer therapy Nat Rev Cancer 4, 361–370 Hermiston ML, Xu Z & Weiss A (2003) CD45: a critical regulator of signaling thresholds in immune cells Annu Rev Immunol 21, 107–137 Hunter T (1995) Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling Cell 80, 225–236 Pao LI, Badour K, Siminovitch KA & Neel BG (2007) Nonreceptor protein-tyrosine phosphatases in immune cell signaling Annu Rev Immunol 25, 473–523 Boggon TJ & Eck MJ (2004) Structure and regulation of Src family kinases Oncogene 23, 7918–7927 Xu W, Harrison SC & Eck MJ (1997) Three-dimensional structure of the tyrosine kinase c-Src Nature 385, 595–602 10 Guappone AC & Flynn DC (1997) The integrity of the SH3 binding motif of AFAP-110 is required to facilitate tyrosine phosphorylation by, and stable complex formation with, Src Mol Cell Biochem 175, 243–252 11 Nakamoto T, Sakai R, Ozawa K, Yazaki Y & Hirai H (1996) Direct binding of C-terminal region of p130Cas to SH2 and SH3 domains of Src kinase J Biol Chem 271, 8959–8965 12 Richard S, Yu D, Blumer KJ, Hausladen D, Olszowy MW, Connelly PA & Shaw AS (1995) Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1 Mol Cell Biol 15, 186–197 13 Straus DB & Weiss A (1992) Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor Cell 70, 585–593 14 Yasuda K, Nagafuku M, Shima T, Okada M, Yagi T, Yamada T, Minaki Y, Kato A, Tani-Ichi S, Hamaoka T et al (2002) Cutting edge: Fyn is essential for tyrosine phosphorylation of Csk-binding protein ⁄ phospho- FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS S A Solheim et al 15 16 17 18 19 20 21 22 23 24 25 protein associated with glycolipid-enriched microdomains in lipid rafts in resting T cells J Immunol 169, 2813–2817 Posevitz-Fejfar A, Smida M, Kliche S, Hartig R, Schraven B & Lindquist JA (2008) A displaced PAG enhances proximal signaling and SDF-1-induced T cell migration Eur J Immunol 38, 250–259 Brdicka T, Pavlistova D, Leo A, Bruyns E, Korinek V, Angelisova P, Scherer J, Shevchenko A, Hilgert I, Cerny J et al (2000) Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation J Exp Med 191, 1591–1604 Kawabuchi M, Satomi Y, Takao T, Shimonishi Y, Nada S, Nagai K, Tarakhovsky A & Okada M (2000) Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases Nature 404, 999–1003 Torgersen KM, Vang T, Abrahamsen H, Yaqub S, Horejsi V, Schraven B, Rolstad B, Mustelin T & Tasken K (2001) Release from tonic inhibition of T cell activation through transient displacement of C-terminal Src kinase (Csk) from lipid rafts J Biol Chem 276, 29313–29318 Tauzin S, Ding H, Khatib K, Ahmad I, Burdevet D, van Echten-Deckert G, Lindquist JA, Schraven B, Din NU, Borisch B et al (2008) Oncogenic association of the Cbp ⁄ PAG adaptor protein with the Lyn tyrosine kinase in human B-NHL rafts Blood 111, 2310–2320 Smida M, Posevitz-Fejfar A, Horejsi V, Schraven B & Lindquist JA (2007) A novel negative regulatory function of PAG: blocking Ras activation Blood 110, 596– 615 Davidson D, Schraven B & Veillette A (2007) PAGassociated FynT regulates calcium signaling and promotes anergy in T lymphocytes Mol Cell Biol 27, 1960–1973 Solheim SA, Torgersen KM, Tasken K & Berge T (2008) Regulation of FynT function by dual-domain docking on PAG ⁄ Cbp J Biol Chem 283, 2773–2783 Pellicena P & Miller WT (2001) Processive phosphorylation of p130Cas by Src depends on SH3-polyproline interactions J Biol Chem 276, 28190–28196 Ingley E, Schneider JR, Payne CJ, McCarthy DJ, Harder KW, Hibbs ML & Klinken SP (2006) Csk-binding protein mediates sequential enzymatic down-regulation and degradation of Lyn in erythropoietinstimulated cells J Biol Chem 281, 31920–31929 Musacchio A, Saraste M & Wilmanns M (1994) Highresolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides Nat Struct Biol 1, 546–551 Modulation of the Fyn SH3–PAG interaction 26 Renzoni DA, Pugh DJ, Siligardi G, Das P, Morton CJ, Rossi C, Waterfield MD, Campbell ID & Ladbury JE (1996) Structural and thermodynamic characterization of the interaction of the SH3 domain from Fyn with the proline-rich binding site on the p85 subunit of PI3-kinase Biochemistry, 35, 15646–15653 27 Rickles RJ, Botfield MC, Weng Z, Taylor JA, Green OM, Brugge JS & Zoller MJ (1994) Identification of Src, Fyn, Lyn, PI3K and Abl SH3 domain ligands using phage display libraries EMBO J, 13, 5598–5604 28 Weng Z, Rickles RJ, Feng S, Richard S, Shaw AS, Schreiber SL & Brugge JS (1995) Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions Mol Cell Biol 15, 5627–5634 29 Sparks AB, Quilliam LA, Thorn JM, Der CJ & Kay BK (1994) Identification and characterization of Src SH3 ligands from phage-displayed random peptide libraries J Biol Chem 269, 23853–23856 30 Morton CJ, Pugh DJ, Brown EL, Kahmann JD, Renzoni DA & Campbell ID (1996) Solution structure and peptide binding of the SH3 domain from human Fyn Structure 4, 705–714 31 Feng S, Kasahara C, Rickles RJ & Schreiber SL (1995) Specific interactions outside the proline-rich core of two classes of Src homology ligands Proc Natl Acad Sci USA 92, 12408–12415 32 Kay BK, Williamson MP & Sudol M (2000) The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains FASEB J 14, 231–241 33 Shelton H & Harris M (2008) Hepatitis C virus NS5A protein binds the SH3 domain of the Fyn tyrosine kinase with high affinity: mutagenic analysis of residues within the SH3 domain that contribute to the interaction Virol J 5, 24 34 Rath A, Davidson AR & Deber CM (2005) The structure of ‘unstructured’ regions in peptides and proteins: role of the polyproline II helix in protein folding and recognition Biopolymers 80, 179–185 35 Li SS (2005) Specificity and versatility of SH3 and other proline-recognition domains: structural basis and implications for cellular signal transduction Biochem J 390, 641–653 36 Drake AF, Siligardi G & Gibbons WA (1988) Reassessment of the electronic circular dichroism criteria for random coil conformations of poly(l-lysine) and the implications for protein folding and denaturation studies Biophys Chem 31, 143–146 37 Scott MP & Miller WT (2000) A peptide model system for processive phosphorylation by Src family kinases Biochemistry 39, 14531–14537 38 Viguera AR, Arrondo JL, Musacchio A, Saraste M & Serrano L (1994) Characterization of the interaction of FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS 4873 Modulation of the Fyn SH3–PAG interaction 39 40 41 42 43 44 S A Solheim et al natural proline-rich peptides with five different SH3 domains Biochemistry 33, 10925–10933 Okishio N, Tanaka T, Fukuda R & Nagai M (2003) Differential ligand recognition by the Src and phosphatidylinositol 3-kinase Src homology domains: circular dichroism and ultraviolet resonance Raman studies Biochemistry 42, 208–216 Bousquet JA, Garbay C, Roques BP & Mely Y (2000) Circular dichroic investigation of the native and nonnative conformational states of the growth factor receptor-binding protein N-terminal src homology domain 3: effect of binding to a proline-rich peptide from guanine nucleotide exchange factor Biochemistry 39, 7722–7735 Kang H, Freund C, Duke-Cohan JS, Musacchio A, Wagner G & Rudd CE (2000) SH3 domain recognition of a proline-independent tyrosine-based RKxxYxxY motif in immune cell adaptor SKAP55 EMBO J 19, 2889–2899 Bhaskar K, Yen SH & Lee G (2005) Disease-related modifications in tau affect the interaction between Fyn and Tau J Biol Chem 280, 35119–35125 Lim CS, Seet BT, Ingham RJ, Gish G, Matskova L, Winberg G, Ernberg I & Pawson T (2007) The K15 protein of Kaposi’s sarcoma-associated herpesvirus recruits the endocytic regulator intersectin through a selective SH3 domain interaction Biochemistry 46, 9874–9885 Feng S, Chen JK, Yu H, Simon JA & Schreiber SL (1994) Two binding orientations for peptides to the Src SH3 domain: development of a general model for SH3ligand interactions Science 266, 1241–1247 4874 45 Alexandropoulos K, Cheng G & Baltimore D (1995) Proline-rich sequences that bind to Src homology domains with individual specificities Proc Natl Acad Sci USA 92, 3110–3114 46 Rickles RJ, Botfield MC, Zhou XM, Henry PA, Brugge JS & Zoller MJ (1995) Phage display selection of ligand residues important for Src homology domain binding specificity Proc Natl Acad Sci USA 92, 10909–10913 47 Saksela K, Cheng G & Baltimore D (1995) Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4 EMBO J 14, 484–491 48 Briggs SD, Lerner EC & Smithgall TE (2000) Affinity of Src family kinase SH3 domains for HIV Nef in vitro does not predict kinase activation by Nef in vivo Biochemistry 39, 489–495 49 Bauer F, Hofinger E, Hoffmann S, Rosch P, Schweimer K & Sticht H (2004) Characterization of Lck-binding elements in the herpesviral regulatory Tip protein Biochemistry 43, 14932–14939 50 Greenfield NJ (2006) Using circular dichroism spectra to estimate protein secondary structure Nat Protoc 1, 2876–2890 51 Sali A & Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints J Mol Biol 234, 779–815 52 Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN & Bourne PE (2000) The Protein Data Bank Nucleic Acids Res 28, 235–242 FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS ... arrays [22] (C) Secondary structure elements of the Fyn SH3-domain The five b strands, numbered consecutively, are separated by the interaction loops (bold) making primary contacts with the ligand... dependent on an initial interaction between the first prolinerich region of PAG and the SH3-domain of the kinase Fyn Our earlier investigations have highlighted the importance of the Fyn SH3-domain. .. This subsequently allows binding of the Fyn SH2-domain to PAG Tyr163 or pTyr181 and results in a dual-domain docking, enhancing the affinity of the Fyn? ??PAG interaction and rendering Fyn insensitive