How and why phosphotyrosine-containing peptides bind to the SH2 and PTB domains

BACKGROUND: Specific recognition of phosphotyrosine-containing protein segments by Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains plays an important role in intracellular signal transduction. Although many SH2/PTB-domain-containing receptor-peptide complex structures have been solved, little has been done to study the problem computationally. Prediction of the binding geometry and the binding constant of any peptide-protein pair is an extremely important problem. RESULTS: A procedure to predict binding energies of phosphotyrosine-containing peptides with SH2/PTB domains was developed. The average deviation between experimentally measured binding energies and theoretical evaluations was 1.8 kcal/mol. Binding states of unphosphorylated peptides were also predicted reasonably well. Ab initio predictions of binding geometry of fully flexible peptides correctly identified conformations of two pentapeptides and a hexapeptide complexed with a v-Src SH2 domain receptor with root mean square deviations (rmsds) of 0.3 A, 1.2 A and 1.5 A, respectively. CONCLUSIONS: The binding energies of phosphotyrosine-containing complexes can be effectively predicted using the procedure developed here. It was also possible to predict the bound conformations of flexible short peptides correctly from random starting conformations.     

Exploiting the basis of proline recognition by SH3 and WW domains: design of N-substituted inhibitors

Src homology 3 (SH3) and WW protein interaction domains bind specific proline-rich sequences. However, instead of recognizing critical prolines on the basis of side chain shape or rigidity, these domains broadly accepted amide N-substituted residues. Proline is apparently specifically selected in vivo, despite low complementarity, because it is the only endogenous N-substituted amino acid. This discriminatory mechanism explains how these domains achieve specific but low-affinity recognition, a property that is necessary for transient signaling interactions. The mechanism can be exploited: screening a series of ligands in which key prolines were replaced by nonnatural N-substituted residues yielded a ligand that selectively bound the Grb2 SH3 domain with 100 times greater affinity.     

Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit

Tyrosine-phosphorylated peptides based on the regions of polyoma virus middle t antigen and the platelet-derived growth factor receptor that bind phosphoinositide 3-kinase are shown to activate this enzyme 2-3-fold in vitro. The concentrations of the peptides required to activate the enzyme are at least 10-1000-fold higher than the dissociation constants of these peptides for the individual SH2 domains of the 85-kDa subunit (KD < 100 nM). Doubly phosphorylated peptides are more effective than singly phosphorylated peptides. The results suggest that a fraction of the cellular phosphoinositide 3-kinase has SH2 domains with relatively low affinity for phosphopeptides and that binding of phosphopeptides to these enzymes causes activation. Thus, SH2 domains may be involved not only in recruiting the enzyme but also in regulating activity.     

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

The interaction of the Fyn SH3 domain with the p85 subunit of PI3-kinase is investigated using structural detail and thermodynamic data. The solution structure complex of the SH3 domain with a proline-rich peptide mimic of the binding site on the p85 subunit is described. This indicates that the peptide binds as a poly(L-proline) type II helix. Circular dichroism spectroscopic studies reveal that in the unbound state the peptide exhibits no structure. Thermodynamic data for the binding of this peptide to the SH3 domain suggest that the weak binding (approximately 31 microM) of this interaction is, in part, due to the entropically unfavorable effect of helix formation (delta S0 = -78 J.mol-1.K-1). Binding of the SH3 domain to the intact p85 subunit (minus its own SH3 domain) is tighter, and the entropic and enthalpic contributions are very different from those given by the peptide interaction (delta S0 = +252 J.mol-1.K-1; delta H0 = +44 kJ.mol-1). From these dramatically different thermodynamic measurements we are able to conclude that the interaction of the proline-rich peptide does not effectively mimic the interaction of the intact p85 subunit with the SH3 domain and suggest that other interactions could be important.     

Son of sevenless binds to the SH3 domain of src-type tyrosine kinase

Measurement of serum levels of ECP has been widely used for monitoring airway inflammation in bronchial asthma and recently been applied to measure anti-inflammatory effect of theophylline. However, reduced levels of ECP in theophylline-administered patients may express not only in vivo effect of theophylline but also in vitro effect after sampling because serum ECP measures released ECP during coagulation and theophylline has been reported to inhibit eosinophil degranulation in vitro. In order to answer the question, we tested whether theophylline added to blood after sampling reduces measured levels of serum ECP. Various concentrations of theophylline were added to SST tube, to which venous blood from atopic patients was drawn. Serum was, then, obtained by centrifugation after 15 min to 6 hours of incubation at room temperature. Theophylline significantly reduced serum ECP in a concentration-dependent manner. Percent reduction of ECP levels at 1 hour of incubation were 11.9%, 18.7%, 22.8%, and 51.7% at theophylline levels of 5, 12.5, 22.5, and 120 micrograms/ml, respectively. Kinetics of serum ECP release was also inhibited in the presence of theophylline. These results suggest that in vitro effect of theophylline on serum ECP levels should be considered when data of serum ECP in patients who take theophylline are interpreted.     

Two SH2 domains of p120 Ras GTPase-activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase-activating protein

p120 GTPase-activating protein (GAP) is a negative regulator of Ras that functions at a key relay point in signal transduction pathways that control cell proliferation. Among other proteins, p120 GAP associates with p190, a GAP for the Ras-related protein, Rho. To characterize the p120.p190 interaction further, we used bacterially expressed glutathione S-transferase fusion polypeptides to map the regions of p120 necessary for its interactions with p190. Our results show that both the N-terminal and the C-terminal SH2 domains of p120 are individually capable of binding p190 expressed in a baculovirus/insect cell system. Moreover, the two SH2 domains together on one polypeptide bind synergistically to p190, and this interaction is dependent on tyrosine phosphorylation of p190. In addition, mutation of the highly conserved Arg residues in the critical FLVR sequences of both SH2 domains of full-length p120 reduces binding to tyrosine-phosphorylated p190. The dependence on p190 phosphorylation for complex formation with p120 SH2 domains observed in vitro is consistent with analysis of the native p120.p190 complexes formed in vivo. These findings suggest that SH2-phosphotyrosine interaction is one mechanism by which the cell regulates p120.p190 association and thus may be a means for coordinating the Ras- and Rho-mediated signaling pathways.     

Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain

BACKGROUND: Receptor-mediated endocytosis appears to require the GTP-binding protein dynamin, but the process by which dynamin is recruited to clathrin-coated pits remains unclear. Dynamin contains several proline-rich clusters that bind to Src homology 3 (SH3) domains, which are short modules found in many signalling proteins and which mediate protein-protein interactions. Amphiphysin, a protein that is highly expressed in the brain, interacts with dynamin in vitro, as do Grb2 and many other SH3 domain-containing proteins. In this study, we examined the role of amphiphysin in receptor-mediated endocytosis in vivo. RESULTS: To address the importance of the amphiphysin SH3 domain in dynamin recruitment, we used a transferrin and epidermal growth factor (EGF) uptake assay in COS-7 fibroblasts. Amphiphysin is present in these cells at a low level and indeed in other peripheral tissues. Confocal immunofluorescence revealed that cells transfected with the amphiphysin SH3 domain showed a potent blockade in receptor-mediated endocytosis. To test whether the cellular target of amphiphysin is dynamin, COS-7 cells were contransfected with both dynamin and the amphiphysin SH3 domain; here, transferrin uptake was efficiently rescued. Importantly, the SH3 domains of Grb2, phospholipase C gamma and spectrin all failed to exert any effect on endocytosis. The mechanism of amphiphysin action in recruiting dynamin was additionally tested in vitro: amphiphysin could associate with both dynamin and alpha-adaptin simultaneously, further supporting a role for amphiphysin in endocytosis. CONCLUSIONS: Our results suggest that the SH3 domain of amphiphysin recruits dynamin to coated pits in vivo, probably via plasma membrane adaptor complexes. We propose that amphiphysin is not only required for synaptic-vesicle endocytosis, but might also be a key player in dynamin recruitment in all cells undergoing receptor-mediated endocytosis.     

Dual function C-terminal domain of dynamin-1: modulation of self-assembly by interaction of the assembly site with SH3 domains

Impairment of endocytosis by mutational targeting of dynamin-1 GTPases can result in paralysis and embryonic lethality. Dynamin-1 assembles at coated pits where it functions to cleave vesicles from donor membranes. Receptor endocytosis is modulated by SH3 (src homology 3) domain proteins, which directly bind to dynamin C-terminal proline motif sequences, affecting both the dynamin GTPase activity and its recruitment to coated pits. We have determined that dynamin-dynamin interactions, which are required for dynamin helix formation, involve these same SH3 domain-binding C-terminal proline motif sequences. Consequently, SH3 domain proteins induce the in vitro disassembly of dynamin helices. Our results therefore suggest the the dual function of the dynamin C-terminus (involving amino acids 800-840) permits direct regulation of dynamin assembly and function through interaction with SH3 domain proteins. Additionally, the N-terminal GTPase domain plays an important role in assembly. Finally, we show that the central PH (pleckstrin homology) domain exerts a strong inhibitory effect on the capacity for dynamin-1 self-assembly.     

A novel ligand for an SH3 domain of the adaptor protein Nck bears an SH2 domain and nuclear signaling motifs

Nck is a small protein composed of Src homology regions (SH) 2 and 3, paralleling the adaptors c-Crk and Grb2/Ash, but its function remains enigmatic. To clarify Nck signaling, a human brain cDNA library was searched for targets of the SH3 moiety of Nck. A novel molecule detected therefrom (referred to as Nck-, Ash- and phospholipase Cgamma-binding protein 4) contained proline-rich sequences and, through the function of one of them, interacted with the middle SH3 domain of Nck. A NAP4 fusion peptide exhibited an affinity for Nck, Ash and phospholipase Cgamma in whole cell lysates. NAP4 also had an SH2 domain, which could bind to activated EGF receptor. These intermolecular interactions imply the intricacy of Nck-mediated signaling around the receptor protein-tyrosine kinases. In addition, NAP4 bore a putative nuclear localization signal and a Q-run/P-run composite, both characteristic of nuclear proteins, and might therefore relate to the presence of Nck in the cellular nucleus.     

Solution structure of the SH3 domain from Bruton's tyrosine kinase

X-linked agammaglobulinemia (XLA) is a heritable immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk). Btk belongs to the Tec family of tyrosine kinases. Each member of the family contains five regions and mutations causing XLA have been isolated in all five regions. We have determined the solution structure of the Src homology 3 (SH3) domain of Btk using two- and three-dimensional nuclear magnetic resonance (NMR) spectroscopy on natural abundance and 15N-labeled protein material. The structure determination is complemented by investigation of backbone dynamics based on 15N NMR relaxation. The Btk SH3 forms a well-defined structure and shows the typical SH3 topology of two short antiparallel beta-sheets packed almost perpendicular to each other in a sandwich-like fold. The N- and C-termini are more flexible as are peptide fragments in the RT and n-Src loops. The studied Btk SH3 fragment adopts two slowly interconverting conformations with a relative concentration ratio of 7:1. The overall fold of the minor form is similar to that of the major form, as judged on the basis of observed NOE connectivities and small chemical shift differences. A tryptophan (W251) ring flip is the favored mechanism for interconversion, although other possibilities cannot be excluded. The side chain of Y223, which becomes autophosphorylated upon activation of Btk, is exposed within the potential SH3 ligand binding site. Finally, we compare the present Btk SH3 structure with other SH3 structures.     

Cloning of a novel human diacylglycerol kinase (DGKtheta) containing three cysteine-rich domains, a proline-rich region, and a pleckstrin homology domain with an overlapping Ras-associating domain

Diacylglycerol kinase (DGK) attenuates levels of second messenger diacylglycerol in cells and produces another (putative) messenger, phosphatidic acid. We have previously purified a 110-kDa DGK from rat brain (Kato, M., and Takenawa, T. (1990) J. Biol. Chem. 265, 794-800). Here we report the cDNA cloning from human brain and retina cDNA libraries. The cDNA encodes a novel DGK isotype, termed DGKtheta, of 941 amino acids with an apparent molecular mass of 110 kDa. DGKtheta contains a C-terminal putative catalytic domain, which is present in all eukaryotic DGKs. In contrast to other DGK isotypes, DGKtheta contains three cysteine-rich domains instead of two. The third cysteine-rich domain is most homologous to the second one in other DGK isotypes. This particular sequence homology extends C-terminally beyond the typical cysteine/histidine core structure and is DGK-specific. DGKtheta furthermore contains various domains for protein-protein interaction, such as a proline- and glycine-rich domain with a putative SH3 domain-binding site and a pleckstrin homology domain with an overlapping Ras-associating domain. DGKtheta is expressed in the brain and, to a lesser extent, in the small intestine, duodenum, and liver. In situ hybridization of DGKtheta mRNA in adult rat brain reveals high expression in the cerebellar cortex and hippocampus. DGKtheta activity in COS cell lysates is optimal toward diacylglycerols containing an unsaturated fatty acid at the sn-2 position.     

Screening for high-affinity ligands to the Src SH2 domain using capillary isoelectric focusing-electrospray ionization ion trap mass spectrometry

A solution-based microscale approach for determination of high-affinity noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray ionization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentrated up to 2 orders of magnitude and separated on the basis of their isoelectric points. The complexes are then dissociated in the mass spectrometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information for the ligands involved in tight complex formation may be obtained using the MSn capabilities of the ion trap. The methodology can potentially be used to screen rapidly combinatorial mixtures of compounds for high-affinity ligands.     

Cloning of a novel signal-transducing adaptor molecule containing an SH3 domain and ITAM

We molecularly cloned a cDNA coding for a novel phosphotyrosine molecule with a 70 kDa molecular mass, named STAM (signal transducing adaptor molecule), which is tyrosine-phosphorylated rapidly after stimulation with various cytokines such as IL-2, IL-3, IL-4, IL-7, GM-CSF, EGF and PDGF. STAM contains an SH3 (Src-homology 3) domain and the ITAM (immunoreceptor tyrosine-based activation motif), suggesting that STAM acts as an adaptor molecule involved in signal transducing pathways from the cytokine receptors.     

Growth factor receptor-bound protein 2 SH2/SH3 domain binding to CD28 and its role in co-signaling

The co-stimulatory antigen CD28 has been shown to bind to several intracellular proteins including phosphatidylinositol 3-kinase, growth factor receptor-bound protein 2 (Grb2), and ITK. Paradoxically, Grb2 and phosphatidylinositol 3-kinase binding has been mapped to a similar pYMNM motif within the CD28 cytoplasmic tail. Given the importance of CD28 co-signaling to T cell function, questions exist regarding the mechanism by which Grb2 binds to CD28, and whether the interaction plays a role in co-stimulation. To biochemically characterize Grb2/CD28 binding, we initially utilized glutathione S-transferase-Grb2 fusion proteins carrying inactivating mutations within the SH2 and SH3 domains of Grb2, and assessed their ability to bind to CD28. In vitro binding experiments indicated that the Grb2 SH2 domain is critical for the association, while the SH3 domain plays an additional role in facilitating optimal binding. Enhanced binding via the SH3 domains was not observed when the C-terminal PXXP motif within CD28 was disrupted, thereby indicating that both SH2 and SH3 domains contribute to CD28 binding. Mutations that alter Grb2 binding were found to block the CD28-dependent interleukin-2 production. Further, tyrosine phosphorylation of Vav and the costimulation-dependent activation of Jun N-terminal kinase was blocked in cells defective in CD28/Grb2 binding. These results provide evidence for an alternate CD28-mediated signaling process involving Grb2 binding to the co-receptor.     

Solution structure and peptide binding of the SH3 domain from human Fyn

BACKGROUND: The Src family of tyrosine kinases is involved in the propagation of intracellular signals from many transmembrane receptors. Each member of the family contains two domains that regulate interactions with other molecules, one of which is the Src homology 3 (SH3) domain. Although structures have previously been determined for SH3 domains, and ideas about peptide-binding modes have been proposed, their physiological role is still unclear. RESULTS: We have determined the solution structure of the SH3 domain from the Src family tyrosine kinase Fyn in two forms: unbound and complexed with a peptide corresponding to a putative ligand sequence from phosphatidylinositol 3' kinase. Fyn SH3 shows the typical SH3 topology of two perpendicular three-stranded beta sheets and a single turn of 3(10) helix. The interaction of SH3 with three potential ligand peptides was investigated, demonstrating that they all bind to the same site on the molecule. A previous model for ligand binding to SH3 domains predicts binding in one of two orientations (class I or II), each characterized by a consensus sequence. The ligand with the closest match to the class I consensus sequence bound with highest affinity and in the predicted orientation. CONCLUSIONS: The Fyn SH3 domain has a well-defined structure in solution. The relative binding affinities of the three ligand peptides and their orientation within the Fyn SH3 complex were consistent with recently proposed models for the binding of 'consensus' polyproline sequences. Although the affinities of consensus and non-consensus peptides are different, the degree of difference is not very large, suggesting that SH3 domains bind to polyproline peptides in a promiscuous manner.     

p62, a phosphotyrosine-independent ligand of the SH2 domain of p56lck, belongs to a new class of ubiquitin-binding proteins

p62 is a novel cellular protein which was initially identified as a phosphotyrosine-independent ligand of the SH2 domain of p56(lck). In the yeast two-hybrid system, p62 specifically interacted with ubiquitin in vivo. Furthermore, p62 bound to ubiquitin-conjugated Sepharose beads in vitro and was efficiently competed by soluble ubiquitin. The interaction was independent of ATP hydrolysis, and its dissociation did not require a reducing agent. Thus, p62 binds to ubiquitin noncovalently. Further analysis showed that the C-terminal 80 amino acids of p62 were indispensable for its interaction with ubiquitin. However, p62 has homology neither with ubiquitin C-terminal hydrolases nor with the S5a subunit of the 26 S proteasome complex, the only proteins known to bind to ubiquitin noncovalently. These results suggest that p62 belongs to a new class of ubiquitin-binding proteins and that p62 affects signal transduction at least partly through ubiquitination-mediated protein degradation.     

Conformational analysis of cyclic hexapeptides designed as constrained ligands for the SH2 domain of the p85 subunit of phosphatidylinositol-3-OH kinase

The structures of the cyclic hexapeptide cyclo(-Gly-Tyr-Val-Pro-Met-Leu-) (1) and its phosphotyrosyl (pTyr) derivative cyclo[-Gly-Tyr(PO3H2)-Val-Pro-Met-Leu-] (2), designed as constrained models of a sequence that interacts with the src homology 2 (SH2) region of the p85 subunit of phosphatidylinositol-3-OH kinase (PI-3 kinase), were studied in methanol/water solutions by 500 MHz nmr spectroscopy. Compound 1 was found to exist as a 2:1 mixture of isomers about the Val-Pro bond (trans and cis prolyl) between 292-330 K in 75% CD3O(D,H)/(D,H)2O solutions. A third species of undetermined structure (ca. 5%) was also observed. Compound 2, a model of phosphorylated peptide ligand that binds to the PI-3 kinase SH2 domain, exhibited similar conformational isomerism. When either compound was dissolved in pure solvent [i.e., 100% CD3O(H,D) or (H,D)2O] the ratio of cis to trans isomers was ca 1:1. A battery of one- and two-dimensional nmr experiments at different temperatures and solvent compositions allowed a complete assignment of both the cis and trans forms of 1 and indicated the trans compound to be the major isomer. The spectral properties of the phophorylated derivative 2 paralleled those of 1, indicating like conformations for the two compounds. Analysis of rotating frame Overhauser spectroscopy data, coupling constants, amide proton temperature dependence, and amide proton exchange rates generated a set of constraints that were employed in energy minimization and molecular dynamics calculations using the CHARMM force field. The trans isomer exists with the tyrosine and C-terminal Tyr(+3) (Met) residues at opposite corners of the 18-membered ring separated by a distance of 16-18 A, in contrast with the cis isomer where the side chains of these residues are much closer in space (7-14 A). It was previously shown that the pTyr and the third amino acid C-terminal to this residue are the critical recognition elements for pTyr-peptide binding to the PI-3 kinase SH2 domain. Such cyclic structures may offer appropriate scaffolding for positioning important amino acid side chains of pTyr-containing peptides as a means of increasing their binding affinities to SH2 domains, and in turn provide a conceptual approach toward the design of SH2 domain directed peptidomimetics.