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PubMed2006

Structural basis for phosphotyrosine recognition by the Src homology-2 domains of the adapter proteins SH2-B and APS.

SH2-B, APS, and Lnk constitute a family of adapter proteins that modulate signaling by protein tyrosine kinases. These adapters contain an N-terminal dimerization region, a pleckstrin homology domain, and a C-terminal Src homology-2 (SH2) domain. SH2-B is recruited via its SH2 domain to various protein tyrosine kinases, including Janus kinase-2 (Jak2) and the insulin receptor. Here, we present the crystal structure at 2.35 A resolution of the SH2 domain of SH2-B in complex with a phosphopeptide representing the SH2-B recruitment site in Jak2 (pTyr813). The structure reveals a canonical SH2 domain-phosphopeptide binding mode, but with specific recognition of a glutamate at the +1 position relative to phosphotyrosine, in addition to recognition of a hydrophobic residue at the +3 position. Biochemical studies of SH2-B and APS demonstrate that, although the SH2 domains of these two adapter proteins share 79% sequence identity, the SH2-B SH2 domain binds preferentially to Jak2, whereas the APS SH2 domain has higher affinity for the insulin receptor. This differential specificity is attributable to the difference in the oligomeric states of the two SH2 domains: monomeric for SH2-B and dimeric for APS.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Role of the proline-rich domain of dynamin-2 and its interactions with Src homology 3 domains during endocytosis of the AT1 angiotensin receptor.

In nonneural tissues, the dynamin-2 isoform participates in the formation of clathrin-coated vesicles during receptor endocytosis. In this study, the mechanism of dynamin-2 action was explored during endocytosis of the G protein-coupled AT1A angiotensin receptor expressed in Chinese hamster ovary cells. Dynamin-2 molecules with mutant pleckstrin homology domains or deleted proline-rich domains (PRD) exerted dominant negative inhibition on the endocytosis of radiolabeled angiotensin II. However, only the PRD mutation interfered with the localization of the dynamin-2 molecule to clathrin-coated pits and reduced the inhibitory effect of the GTPase-deficient K44A mutant dynamin-2. Green fluorescent protein-tagged Src homology 3 (SH3) domains of endophilin I and amphiphysin II, two major binding partners of dynamins, also inhibited AT1A receptor-mediated endocytosis of angiotensin II. These effects were partially or fully, respectively, restored by the overexpression of dynamin-2. Transient overexpression of these SH3 domains also reduced the localization of dynamin-2 to clathrin-coated pits. These data indicate that, similar to the recruitment of dynamin-1 during the recycling of synaptic vesicles, interaction of the dynamin-2 PRD with SH3 domains of proteins such as the amphiphysins and endophilins is essential for AT1A receptor endocytosis. This mechanism could be of general importance in dynamin-dependent endocytosis of other G protein-coupled receptors in nonneural tissues.

Animals↗

PubMed1996

Hydrophobic D-amino acids in the design of peptide ligands for the pp60src SH2 domain.

Src homology-2 (SH2) domains, containing approximately 100 amino acid residues, are noncatalytic motifs involved with intracellular signal transduction. These domains can be found on nonreceptor kinases, phosphatases, and in regulatory adapter proteins among others. SH2 domains bind proteins containing phosphotyrosine (pTyr) residues in a sequence specific manner. Our efforts have focused on designing peptide mimetic ligands for the SH2 domain of the nonreceptor tyrosine kinase pp60src. We employed the cocrystal structure of the 11mer Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-IIe-Tyr-Leu IC50 = 800 nM as a starting point for our design efforts. These efforts have resulted in the discovery of tripeptide ligands containing D-amino acids that are only 2-fold less potent than the 11mer.

Amino Acid Sequence↗

PubMed1999

Phosphotyrosine-containing dipeptides as high-affinity ligands for the p56lck SH2 domain.

Src homology-2 (SH2) domains are noncatalytic motifs containing approximately 100 amino acid residues that are involved in intracellular signal transduction. The phosphotyrosine-containing tetrapeptide Ac-pYEEI binds to the SH2 domain of p56lck (Lck) with an affinity of 0.1 microM. Starting from Ac-pYEEI, we have designed potent antagonists of the Lck SH2 domain which are reduced in peptidic character and in which the three carboxyl groups have been eliminated. The two C-terminal amino acids (EI) have been replaced by benzylamine derivatives and the pY + 1 glutamic acid has been substituted with leucine. The best C-terminal fragment identified, (S)-1-(4-isopropylphenyl)ethylamine, binds to the Lck SH2 domain better than the C-terminal dipeptide EI. Molecular modeling suggests that the substituents at the 4-position of the phenyl ring occupy the pY + 3 lipophilic pocket in the SH2 domain originally occupied by the isoleucine side chain. This new series of phosphotyrosine-containing dipeptides binds to the Lck SH2 domain with potencies comparable to that of tetrapeptide 1.

Binding, Competitive↗

PubMedDate not supplied

Reexamination of the recognition preference of the specificity pocket of the Abl SH3 domain.

Src homology-3 (SH3) domains mediate important protein-protein interactions in a variety of normal and pathological cellular processes, thus providing an attractive target for the selective interference of SH3-dependent signaling events that govern these processes. Most SH3 domains recognize proline-rich peptides with low affinity and poor selectivity, and the goal to design potent and specific ligands for various SH3 domains remains elusive. Better understanding of the molecular basis for SH3 domain recognition is needed in order to design such ligands with potency and specificity. In this report, we seek to define a clear recognition preference of the specificity pocket of the Abl SH3 domain using targeted synthetic peptide libraries. High-resolution affinity panning coupled with mass spectrometric readout allows for quick identification of Trp as the preferred fourth residue in the decapeptide ligand APTWSPPPPP, which binds to Abl SH3 four times stronger than does the decapeptide containing Tyr or Phe in the fourth position. This finding is in contrast to several reports that Tyr is the only residue selected from phage displayed peptide libraries that interacts with the specificity pocket of Abl SH3. This simple, unbiased approach can fine-tune the affinity and selectivity of both natural and unnatural SH3 ligands whose consensus binding sequence has been pre-defined by combinatorial library methods.

Chromatography, Affinity↗

PubMed1996

p150TSP, a conserved nuclear phosphoprotein that contains multiple tetratricopeptide repeats and binds specifically to SH2 domains.

Src homology 2 (SH2) domains are structural modules that function in the assembly of multicomponent signaling complexes by binding to specific phosphopeptides. The tetratricopeptide repeat (TPR) is a distinct structural motif that has been suggested to mediate protein-protein interactions. Among SH2-binding phosphoproteins purified from the mouse B cell lymphoma A20, a 150-kDa species was identified and the corresponding complementary DNA (cDNA) was molecularly cloned. This protein encoded by this cDNA, which we have termed p150TSP (for TPR-containing, SH2-binding phosphoprotein), is located predominantly in the nucleus and is highly conserved in evolution. The gene encoding p150TSP (Tsp) was mapped to chromosome 7 of the mouse with gene order: centromere-Tyr-Wnt11-Tsp-Zp2. The amino-terminal two-thirds of p150TSP consist almost entirely of tandemly arranged TPR units, which mediate specific, homotypic protein interactions in transfected cells. The carboxyl-terminal third of p150TSP, which is serine- and glutamic acid-rich, is essential for SH2 binding; this interaction is dependent on serine/threonine phosphorylation but independent of tyrosine phosphorylation. The sequence and binding properties of p150TSP suggest that it may mediate interactions between TPR-containing and SH2-containing proteins.

Amino Acid Sequence↗

PubMed1998

Common in vitro substrate specificity and differential Src homology 2 domain accessibility displayed by two members of the Src family of protein-tyrosine kinases, c-Src and Hck.

Hck and Src are members of the Src family of protein- tyrosine kinases that carry out distinct and overlapping functions in vivo (Lowell, C. A., Niwa, M., Soriano, P., and Varmus, H. E. (1996) Blood 87, 1780-1792). In an attempt to understand how Hck and Src can function both independently and in concert, we have compared 1) their in vitro substrate specificity and 2) the accessibility of their Src homology 2 (SH2) domain. Using several synthetic peptides, we have demonstrated that Hck and Src recognize similar structural features in the substrate peptides, suggesting that both kinases have the intrinsic ability to carry out overlapping cellular functions by phosphorylating similar cellular proteins in vivo. Using a phosphotyrosine-containing peptide that has previously been shown to bind the SH2 domain of Src family kinases with high affinity, we found that although Src could bind to the phosphopeptide, Hck showed no interaction. The inability of Hck to bind the phosphopeptide was not a result of a stable intramolecular interaction between its SH2 domain and C-terminal regulatory phosphotyrosine residue (Tyr-520), as most Hck molecules in the purified Hck preparation were not tyrosine-phosphorylated. In contrast to intact Hck, a recombinant truncation analog of Hck was able to bind the phosphopeptide with an affinity similar to that of the Src SH2 domain, suggesting that conformational constraints are imposed on intact Hck that limit accessibility of its SH2 domain to the phosphopeptide. Furthermore, the difference in SH2 domain accessibility is a potential mechanism that enables Src and Hck to perform their respective unique functions by 1) targeting them to different subcellular compartments, whereupon they phosphorylate different cellular proteins, and/or 2) facilitating direct binding to their cellular substrates.

Amino Acid Sequence↗

PubMed2000

Using a phage display library to identify basic residues in A-Raf required to mediate binding to the Src homology 2 domains of the p85 subunit of phosphatidylinositol 3'-kinase.

Src homology 2 (SH2) domains are found in a variety of cytoplasmic proteins involved in mediating signals from cell surface receptors to various intracellular pathways. They fold as modular units and are capable of recognizing and binding to short linear peptide sequences containing a phosphorylated tyrosine residue. Here we show that each of the SH2 domains of the p85 subunit of phosphatidylinositol 3-kinase selects phage displayed peptide sequences containing the core (L/I)-A-(R/K)-I-R. The serine/threonine kinase A-Raf, containing the sequence LQRIRS, is associated with the p85 protein in both quiescent and growth factor stimulated cells. This suggests that p85 and A-Raf exist in a protein complex in cells and that complex formation does not require growth factor stimulation. We also show that p85 and A-Raf can bind directly to each other in vitro and that this interaction is mediated in part by the p85 SH2 domains. Further, the p85 SH2 domains require at least one of four distinct basic-X-basic sequence motifs within A-Raf for binding. This is the first description of a phosphotyrosine-independent SH2 domain interaction that requires basic residues on the SH2 ligand.

3T3 Cells↗

PubMed2004

The C terminus of c-Src inhibits breast tumor cell growth by a kinase-independent mechanism.

Overexpression or increased activity of cellular Src (c-Src) is frequently detected in human breast cancer, implicating involvement of c-Src in the etiology of breast carcinomas. Curiously, overexpression of c-Src in tissue culture cells results in a weakly or non-transforming phenotype, indicating that it alone is not sufficient for oncogenesis. However, the protein has been demonstrated to potentiate mitogenic signals from transmembrane receptors. This report investigates the requirement for c-Src in breast cancer as a transducer and integrator of anchorage-dependent and -independent growth signals by utilizing the Src family pharmacological inhibitors, PP1 and PP2, or stable overexpression of the catalytically inactive c-Src mutant (K- c-Src). Both methods of inhibiting endogenous c-Src diminished formation of soft agar colonies and tumors in nude mice. The majority of the dominant-negative activity of K- c-Src was mapped to the Src homology 2 (SH2) domain and C-terminal half of the molecule, but not to the Unique domain, Src homology 3 (SH3) domain, or the N-terminal half of K- c-Src. Further analysis of the C terminus revealed that its ability to inhibit growth localized to the N-terminal lobe (N-lobe) of the catalytic region. These results underscore the requirement for c-Src to maintain the oncogenic phenotype of breast cancer cells and suggest that c-Src may be manipulated to inhibit cell growth by the direct disruption of its catalytic activity or the introduction of either the SH2 domain or the N-lobe of K- c-Src.

Breast Neoplasms↗

PubMed2000

NGF-Dependent neurite outgrowth in PC12 cells overexpressing the Src homology 2-domain protein shb requires activation of the Rap1 pathway.

The Src homology 2 (SH2) domain adaptor protein Shb has been shown to transmit NGF- and FGF-2-dependent differentiation signals in PC12 cells. To study if this involves signaling through the small GTPase Rap1, Rap1 activity was assessed in Shb-overexpressing PC12 cells. We demonstrate that NGF and EGF induce Rap1 activation in PC12-Shb cells, while FGF-2 fails to do so. However, PC12 cells expressing Shb with an inactivated SH2 domain do not respond to NGF stimulation with Rap1 activation. The CrkII SH2 domain interacts with Shb and a 130- to 135-kDa phosphotyrosine protein present mainly in PC12-Shb cells and these interactions may thus relate to the effect of Shb on Rap1 activation. Transient expression of RalGDS-RBD or Rap1GAP to block the Rap1 pathway reduces the NGF-dependent neurite outgrowth in PC12-Shb cells. These results suggest a role of Shb in NGF-dependent Rap1 signaling and this pathway may be of significance for neurite outgrowth under certain conditions.

Animals↗

PubMed2001

Autoinhibition of mixed lineage kinase 3 through its Src homology 3 domain.

Mixed lineage kinase 3 (MLK3) is a serine/threonine protein kinase that functions as a mitogen-activated protein kinase kinase kinase to activate the c-Jun NH(2)-terminal kinase pathway. MLK3 has also been implicated as an I kappa B kinase kinase in the activation of NF-kappa B. Amino-terminal to its catalytic domain, MLK3 contains a Src homology 3 (SH3) domain. SH3 domains harbor three highly conserved aromatic amino acids that are important for ligand binding. In this study, we mutated one of these corresponding residues within MLK3 to deliberately disrupt the function of its SH3 domain. This SH3-defective mutant of MLK3 exhibited increased catalytic activity compared with wild type MLK3 suggesting that the SH3 domain negatively regulates MLK3 activity. We report herein that the SH3 domain of MLK3 interacts with full-length MLK3, and we have mapped the site of interaction to a region between the zipper and the Cdc42/Rac interactive binding motif. Interestingly, the SH3-binding region contains not a proline-rich sequence but, rather, a single proline residue. Mutation of this sole proline abrogates SH3 binding and increases MLK3 catalytic activity. Taken together, these data demonstrate that MLK3 is autoinhibited through its SH3 domain. The critical proline residue in the SH3-binding site of MLK3 is conserved in the closely related family members, MLK1 and MLK2, suggesting a common autoinhibitory mechanism among these kinases. Our study has revealed the first example of SH3 domain-mediated autoinhibition of a serine/threonine kinase and provides insight into the regulation of the mixed lineage family of protein kinases.

Amino Acid Sequence↗

PubMed1995

Production of monoclonal antibodies to Bruton's tyrosine kinase.

Bruton's X-linked agammaglobulinemia is caused by mutations in a cytoplasmic protein tyrosine kinase termed Bruton's tyrosine kinase (BTK). The protein is expressed in all members of the B cell lineage and is critical for B cell development. The protein consists of several modules, including a pleckstrin homology domain and the Src homology domains SH1, SH2, and SH3. We report here the production of monoclonal antibodies against the pleckstrin homology domain of human BTK. The antibody was produced by immunizing mice with a FLAG-BTK fusion protein. Hybridoma supernatants were screened by ELISA using a GST-BTK fusion protein as the antigen. Selected monoclonal antibodies recognize denatured BTK on Western blots of peripheral blood mononuclear cell lysates. Mouse BTK protein is also detected. These antibodies should be useful in assessing patients with immune deficiency, as well as in studying normal B cell development.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1998

Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains.

We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX1-2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Differential activation of acute phase response factor/Stat3 and Stat1 via the cytoplasmic domain of the interleukin 6 signal transducer gp130. II. Src homology SH2 domains define the specificity of stat factor activation.

Distinct yet overlapping sets of STAT transcription factors are activated by different cytokines. One example is the differential activation of acute phase response factor (APRF, also called Stat3) and Stat1 by interleukin 6 and interferon-gamma. Interleukin 6 activates both factors while, at least in human cells, interferon-gamma recruits only Stat1. Stat1 activation by interferon-gamma is mediated through a cytosolic tyrosine motif, Y440, of the interferon-gamma receptor. In an accompanying paper (Gerhartz, C., Heesel, B., Sasse, J., Hemmann, U., Landgraf, C., Schneider-Mergener, J., Horn, F., Heinrich, P. C., and Graeve, L. (1996) J. Biol. Chem. 271, 12991-12998), we demonstrated that two tyrosine motifs within the cytoplasmic part of the interleukin 6 signal transducer gp130 specifically mediate APRF activation while two others can recruit both APRF and Stat1. By expressing a series of Stat1/APRF domain swap mutants in COS-7 cells, we now determined which domains of Stat1 and APRF are involved in the specific recognition of phosphotyrosine motifs. Our data demonstrate that the SH2 domain is the sole determinant of specific STAT factor recruitment. Furthermore, the SH2 domain of Stat1 is able to recognize two unrelated types of phosphotyrosine motifs, one represented by the interferon-gamma receptor Y440DKPH peptide, and the other by two gp130 YXPQ motifs. By molecular modeling, we propose three-dimensional model structures of the Stat1 and APRF SH2 domains which allow us to explain the different binding preferences of these factors and to predict amino acids crucial for specific peptide recognition.

Acute-Phase Proteins↗

PubMed2000

An intramolecular interaction between Src homology 3 domain and guanylate kinase-like domain required for channel clustering by postsynaptic density-95/SAP90.

Members of the postsynaptic density-95 (PSD-95)/SAP90 family of membrane-associated guanylate kinase (MAGUK) proteins function as multimodular scaffolds that organize protein-signaling complexes at neuronal synapses. MAGUK proteins contain PDZ, Src homology 3 (SH3), and guanylate kinase (GK)-like domains, all of which can function as sites for specific protein-protein interactions. We report here a direct protein-protein interaction between the SH3 domain and the GK region in the PSD-95 family of MAGUKs. The SH3 domain of the PSD-95 family appears to have an atypical binding specificity, because the classical SH3 binding (-P-X-X-P-) motif is absent from the GK domain. Although SH3-GK binding can occur in either an intramolecular or intermolecular manner, the intramolecular mode is preferred, possibly because of additional tertiary interactions available when the SH3 and GK domains are adjacent in the same polypeptide. Mutations disrupting the intramolecular SH3-GK interaction do not interfere with PSD-95 association with the K(+) channel Kv1.4 or with the GK domain-binding protein GKAP. The same mutations, however, inhibit the clustering of Kv1.4 by PSD-95, suggesting that the intramolecular SH3-GK interaction may modulate the clustering activity of PSD-95.

Animals↗

PubMed2001

A transcription function for the T cell-specific adapter (TSAd) protein in T cells: critical role of the TSAd Src homology 2 domain.

T cell-specific adapter (TSAd) protein is an Src homology 2 (SH2) domain-containing adapter molecule implicated in T cell receptor for antigen (TCR)-mediated interleukin 2 (IL-2) secretion in T cells. Here, we demonstrate that a substantial fraction of TSAd is found in the T cell nucleus. Nuclear import of TSAd is an active process that depends on TSAd SH2 domain recognition of a phosphotyrosine-containing ligand. Importantly, we show that TSAd can act as a potent transcriptional activator in T cells. Furthermore, the TSAd SH2 domain appears to be essential for this transcription-activating function independent of its role in nuclear import. Biochemical analyses suggest that a single TSAd SH2 domain ligand of 95-100 kD may be involved in these processes. Consistent with a role as a transcription activator, cotransfection of TSAd with an IL-2 promoter-reporter gene construct results in a considerable upregulation of IL-2 promoter activity. Further, we show that this augmentation requires a functional TSAd SH2 domain. However, TSAd does not appear to modulate the activity of the major recognized IL-2 gene transcription factors, nuclear factor kappaB (NF-kappaB), nuclear factor of activated T cells (NFAT), or activator protein 1 (AP-1). These findings point to the function of TSAd as a novel transcription-regulatory protein in T cells and illustrate the importance of the TSAd SH2 domain in this role.

Active Transport, Cell Nucleus↗

PubMed1994

Interaction of Grb2 via its Src homology 3 domains with synaptic proteins including synapsin I.

Grb2 is a 25-kDa adaptor protein composed of a Src homology 2 (SH2) domain and two flanking Src homology 3 (SH3) domains. One function of Grb2 is to couple tyrosine-phosphorylated proteins (through its SH2 domain) to downstream effectors (through its SH3 domains). Using an overlay assay, we have identified four major Grb2-binding proteins in synaptic fractions. These proteins interact with wild-type Grb2 but not with Grb2 containing point mutations in each of its two SH3 domains corresponding to the loss of function mutants in the Caenorhabditis elegans Grb2 homologue sem-5. Two of the proteins, mSos and dynamin, were previously shown to bind Grb2. The third protein of 145 kDa is brain specific and to our knowledge has not been previously described. The fourth protein is synapsin I. Dynamin is required for synaptic vesicle endocytosis and synapsin I is thought to mediate the interaction of synaptic vesicles with the presynaptic cytomatrix. These data suggest that Grb2, or other proteins containing SH3 domains, may play a role in the regulation of the exo/endocytotic cycle of synaptic vesicles and therefore of neurotransmitter release.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The Src homology-2 domains (SH2 domains) of the protein tyrosine kinase p56lck: structure, mechanism and drug design.

Src homology 2 (SH2) domains are found in many intercellular signal-transduction proteins which bind phosphotyrosine containing polypeptide sequences with high affinity and specificity and are considered potential targets for drug discovery. The protein p56lck is a member of the family of Src tyrosine kinase. The SH2 domain is thought to be responsible for the recruitment and regulation of p56lck kinase activity. There have been enormous efforts in the development of SH2 domain inhibitors for diseases such as cancer, osteoporosis and other diseases. This review focuses on current understanding of SH2 domain structure, mechanism and drug discovery with an emphasis on p56lck SH2 domain. A potential impact of the accumulated crystallographic effort on the development of methods for structure-based drug design is briefly addressed.

Amino Acid Sequence↗