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S E Shoelson

Publications and source records attributed to S E Shoelson.

At least 37 records · Page 2Linked to original sources
PubMed1997

SH2 and PTB domain interactions in tyrosine kinase signal transduction.

Proteins with SH2 or phosphotyrosine binding (PTB) domains bind activated tyrosine kinase receptors and their substrates to propagate signals into cells. Both of the domains recognize phosphotyrosine. Selectivity in these interactions is conferred by short flanking peptide motifs. Therefore, potential exists for modulating tyrosine kinase signaling pathways by the discovery of compounds that selectively bind SH2 and PTB domains. Recent advances with small peptides and nonpeptide compounds suggest that this opportunity can be realized.

Drug Design↗
PubMed1997

Cellular effects of phosphotyrosine-binding domain inhibitors on insulin receptor signaling and trafficking.

Shc and insulin receptor substrate 1 (IRS-1) are cytoplasmic substrates of tyrosine kinase receptors that engage, localize, and activate downstream SH2 enzymes. Each contains a phosphotyrosine-binding (PTB) domain that is structurally unrelated to SH2 domains. We have designed high-affinity, cellular inhibitors of the Shc PTB domain by incorporating nonnatural, phosphatase-resistant amino acids into short peptides. None of the inhibitors bind the IRS-1 PTB domain, consistent with distinct specificities for domains. The best inhibitor of the Shc domain was introduced by electroporation into Rat1 fibroblasts that express human insulin receptors. Insulin-stimulated phosphorylation of Shc was inhibited, with no effect on IRS-1, and downstream effects on mitogen-activated protein kinase and DNA synthesis were both inhibited. The PTB domain inhibitor had less influence on epidermal growth factor-induced effects and essentially no impact on serum- or phorbol ester-induced effects. The inhibitor did not affect insulin internalization and its degradation. We conclude that the PTB domain of Shc is critical for its phosphorylation by the insulin receptor, that Shc is an important mediator of insulin's mitogenic effects, and that Shc is not central to insulin receptor cycling in these cells. PTB domains can be inhibited selectively in cells and represent potential targets for drug discovery.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Tyrosine-phosphorylated Cbl binds to Crk after T cell activation.

Crk is a Src homology 2 (SH2)/Src homology 3 (SH3)-containing adapter protein that has been implicated in intracellular signaling in fibroblasts and PC12 pheochromocytoma cells. Crk has been shown to bind to a tyrosine-phosphorylated protein of 116 kDa after TCR-mediated T cell activation. Here we demonstrate that the Crk-associated p116 phosphoprotein is not the Crk-associated substrate (Cas) but, rather, is a protein product of the c-cbl proto-oncogene. Whereas Cas was not tyrosine-phosphorylated after T cell activation, Cbl became highly phosphorylated. Crk immunoprecipitates from activated T cell lysates contain tyrosine-phosphorylated Cbl. This association is mediated by the SH2 domain of Crk, as evidenced by the interaction between Cbl and the fusion protein product of a glutathione S-transferase (GST) expression construct encoding the Crk-SH2 domain in vitro. Furthermore, phosphopeptide-binding studies revealed that the GST-Crk SH2 domain binds to a tyrosine-phosphorylated peptide corresponding to amino acids 770-781 of Cbl with high affinity. Cbl is a protein tyrosine kinase (PTK) substrate that becomes phosphorylated after engagement of numerous cell surface receptors including the TCR. Data revealed by genetic studies in the nematode, Caenorhabditis elegans, implicates a Cbl-like molecule, Sli-1, as a negative regulator of the Let-23-signaling pathway. Because the signal from the Let-23 pathway affects the activation status of the Let-60 (Ras homologue in C. elegans) pathway, the activation-dependent association between Crk and Cbl may represent another TCR-generated signal leading to Ras-related pathways.

Amino Acid Sequence↗
PubMed1996

Structure of the IRS-1 PTB domain bound to the juxtamembrane region of the insulin receptor.

SUMMARY: Crystal structures of the insulin receptor substrate-1 (IRS-1) phosphotyrosine-binding (PTB) domain, alone and complexed with the juxtamembrane region of the insulin receptor, show how this domain recognizes phosphorylated "NPXY" sequence motifs. The domain is a 7-stranded beta sandwich capped by a C-terminal helix. The insulin receptor phosphopeptide fills an L-shaped cleft on the domain. The N-terminal residues of the bound peptide form an additional strand in the beta sandwich, stabilized by contacts with the C-terminal helix. These interactions explain why IRS-1 binds to the insulin receptor but not to NPXpY motifs in growth factor receptors. The PTB domains of IRS-1 and Shc share a common fold with pleckstrin homology domains. Overall, ligand binding by IRS-1 and Shc PTB domains is similar, but residues critical for phosphotyrosine recognition are not conserved.

Amino Acid Sequence↗
PubMed1996

Evidence for a role for the phosphotyrosine-binding domain of Shc in interleukin 2 signaling.

Stimulation via the T-cell growth factor interleukin 2 (IL-2) leads to tyrosine phosphorylation of Shc, the interaction of Shc with Grb2, and the Ras GTP/GDP exchange factor, mSOS. Shc also coprecipitates with the IL-2 receptor (IL-2R), and therefore, may link IL-2R to Ras activation. We have further characterized the Shc-IL-2R interaction and have made the following observations. (i) Among the two phosphotyrosine-interaction domains present in Shc, the phosphotyrosine-binding (PTB) domain, rather than its SH2 domain, interacts with the tyrosine-phosphorylated IL-2R beta chain. Moreover, the Shc-PTB domain binds a phosphopeptide derived from the IL-2R beta chain (corresponding to residues surrounding Y338, SCFTNQGpYFF) with high affinity. (ii) In vivo, mutant IL-2R beta chains lacking the acidic region of IL-2Rbeta (which contains Y338) fail to phosphorylate Shc. Furthermore, when wild type or mutant Shc proteins that lack the PTB domain were expressed in the IL-2-dependent CTLL-20 cell line, an intact Shc-PTB domain was required for Shc phosphorylation by the IL-2R, which provides further support for a Shc-PTB-IL-2R interaction in vivo. (iii) PTB and SH2 domains of Shc associate with different proteins in IL-2- and T-cell-receptor-stimulated lysates, suggesting that Shc, through the concurrent use of its two different phosphotyrosine-binding domains, could assemble multiple protein complexes. Taken together, our in vivo and in vitro observations suggest that the PTB domain of Shc interacts with Y338 of the IL-2R and provide evidence for a functional role for the Shc-PTB domain in IL-2 signaling.

Amino Acid Sequence↗
PubMed1996

Evidence for a physical association between the Shc-PTB domain and the beta c chain of the granulocyte-macrophage colony-stimulating factor receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates the growth and function of several myeloid cell types at different stages of maturation. The effects of GM-CSF are mediated through a high affinity receptor that is composed of two chains: a unique, ligand-specific alpha chain and a beta common chain (beta c) that is also a component of the receptors for interleukin 3 (IL-3) and IL-5. Beta c plays an essential role in the transduction of extra cellular signals to the nucleus through its recruitment of secondary messengers. Several downstream signaling events induced by GM-CSF stimulation have been described, including activation of tyrosine kinases and tyrosine phosphorylation of cellular proteins (including beta c) and activation of the Ras/mitogen-activated protein kinase and the JAK/STAT pathways. A region within the beta c cytoplasmic tail (amino acids 517-763) has been reported to be necessary for tyrosine phosphorylation of the adapter protein, Shc, and for the subsequent GM-CSF-induced activation of Ras. In this paper, we describe a physical association between the tyrosine phosphorylated GM-CSF receptor (GMR)-beta c chain and Shc in vivo. Using a series of cytoplasmic truncation mutants of beta c and various mutant Shc proteins, we demonstrate that the N-terminal phosphotyrosine-binding (PTB) domain of Shc binds to a short region of beta c (amino acids 549-656) that contains Tyr577. Addition of a specific phosphopeptide encoding amino acids surrounding this tyrosine inhibited the interaction between beta c and shc. Moreover, mutation of a key residue within the phosphotyrosine binding pocket of the Shc-PTB domain abrogated its association with beta c. These observations provide an explanation for the previously described requirement for Tyr577 of beta c for GM-CSF-induced tyrosine phosphorylation of Shc and have implications for Ras activation through the GM-CSF, IL-3, and IL-5 receptors.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Stimulation through the T cell receptor induces Cbl association with Crk proteins and the guanine nucleotide exchange protein C3G.

We and others have recently identified Cbl, the protein product of the c-cbl protooncogene, as an early tyrosine kinase substrate upon T cell activation and have shown that Cbl forms in vivo complexes with Src family tyrosine kinases, Grb2 adaptor protein, and the p85 subunit of PI-3 kinase. Here we show that Cbl associates with all three forms of the human Crk protein, predominantly CrkL, following T cell receptor activation of Jurkat T cells. Association between Cbl and Crk proteins was confirmed in normal human peripheral blood-derived T cells. In vitro, Cbl was able to interact with the Crk SH2 domain but not the SH3 domain. A phosphopeptide corresponding to a potential Crk SH2 domain-binding motif in Cbl (pYDVP) specifically inhibited binding between Cbl and Crk SH2 domain. Anti-Cbl antibody completely immunodepleted the CrkL-associated 120kDa phosphotyrosyl polypeptide, suggesting that the recently described p130cas-related Crk-associated p116 of T cells may be Cbl. Consistent with this possibility, the 4F4 antibody used to characterize the p116 polypeptide cross-reacted with Cbl protein when it was resolved on one- or two-dimensional gels. CrkL was constitutively associated with a substantial amount of the guanine nucleotide exchange protein C3G, and a fraction of the C3G protein was coimmunoprecipitated with Cbl in activated Jurkat T cells. These results suggest the possibility that Cbl may participate in a signaling pathway that regulates guanine nucleotide exchange on small G-proteins in T cells.

Adaptor Proteins, Signal Transducing↗
PubMed1996

4'-O-[2-(2-fluoromalonyl)]-L-tyrosine: a phosphotyrosyl mimic for the preparation of signal transduction inhibitory peptides.

Development of phosphotyrosyl (pTyr) mimetics which are stable to protein-tyrosine phosphatases (PTPs), yet can retain biological potency when incorporated into peptides, is an active area of drug development. Since a majority of pTyr mimetics derive their "phosphofunctionality" from phosphorus-containing moieties, such as phosphonates, evolution of new inhibitors and modes of prodrug derivatization have been restricted to chemistries appropriate for phosphorus-containing moieties. A new, nonphosphorus-containing pTyr mimetic has recently been reported, L-O-(2-malonyl)tyrosine (OMT,5), which can be incorporated into peptides that exhibit good PTP and Src homology 2 (SH2) domain inhibitory potency. For phosphonate-based pTyr mimetics such as phosphonomethyl phenylalanine (Pmp,2) introduction of fluorines alpha to the phosphorus has provided higher affinity pTyr mimetics. This strategy has now been applied to OMT, and herein is reported 4'-O-[2-(2-fluoromalonyl)]-L-tyrosine (FOMT,6) a new fluorine-containing nonphosphorus pTyr mimetic. Incorporation of FOMT into appropriate peptides results in good inhibition of both PTP and SH2 domains. In an assay measuring the inhibition of PTP 1B-mediated dephosphorylation of phosphorylated insulin receptor, the peptide Ac-D-A-D-E-X-L-amide exhibited a 10-fold enhancement in inhibitory potency for X = FOMT (19) (IC(50) = 10 microM) relative to the unfluorinated peptide, X = OMT (18) (IC(50) = 10 microM. Molecular modeling indicated that this increased affinity may be attributable to new hydrogen-bonding interactions between the fluorine and the enzyme catalytic site, and not due to lowering of pKa values. In a competition binding assay using the p85 PI 3-kinase C-terminal SH2 domain GST fusion construct, the inhibitory peptide, Ac-D-X-V-P-M-L-amide, showed no enhancement of inhibitory potency for X = FOMT (22) (IC(50) = 18 microM) relative to the unfluorinated peptide, X = OMT (21) (IC(50) = 14 microM). The use of FOMT would therefore appear to have particular potential for the development of PTP inhibitors.

Amino Acid Sequence↗
PubMed1996

Spatial constraints on the recognition of phosphoproteins by the tandem SH2 domains of the phosphatase SH-PTP2.

The domain organization of many signalling proteins facilitates a segregation of binding, catalytic and regulatory functions. The mammalian SH2 domain protein tyrosine phosphatases (PTPs) contain tandem SH2 domains and a single carboxy-terminal catalytic domain. SH-PTP1 (PTP1C, HCP) and SH-PTP2 (Syp, PTP2C, PTP1D) function downstream from tyrosine kinase-linked insulin, growth factor, cytokine and antigen receptors. As well as directing subcellular localization by binding to receptors and their substrates, the two SH2 domains of these PTPs function together to regulate catalysis. Here we report the structure of the tandem SH2 domains of SH-PTP2 in complex with monophosphopeptides. A fixed relative orientation of the two domains, stabilized by a disulphide bond and a small hydrophobic patch within the interface, separates the peptide binding sites by approximately 40 A. The defined orientation of the SH2 domains in the structure, and data showing that peptide orientation and spacing between binding sites is critical for enzymatic activation, suggest that spatial constraints are important in this multidomain protein-protein interaction.

Amino Acid Sequence↗
PubMed1996

Crystal structure of the PI 3-kinase p85 amino-terminal SH2 domain and its phosphopeptide complexes.

Crystal structures of the amino-terminal SH2 domain of the p85alpha subunit of phosphatidylinositol (PI) 3-kinase, alone and in complex with phosphopeptides bearing pTyr-Met/Val-Xaa-Met motifs, show that phosphopeptides bind in the two-pronged manner seen in high-affinity Lck and Src SH2 complexes, with conserved interactions between the domain and the peptide segment from phosphotyrosine to Met+3. Peptide binding requires the rearrangement of a tyrosyl side chain in the BG loop to create the hydrophobic Met+3 binding pocket. The structures suggest a mechanism for the biological specificity exhibited by PI 3-kinase in its interactions with phosphoprotein partners.

Amino Acid Sequence↗
PubMed1996

Structural basis for IL-4 receptor phosphopeptide recognition by the IRS-1 PTB domain.

We present the NMR structure of the PTB domain of insulin receptor substrate-1 (IRS-1) complexed to a tyrosine-phosphorylated peptide derived from the IL-4 receptor. Despite the lack of sequence homology and different binding specificity, the overall fold of the protein is similar to that of the Shc PTB domain and closely resembles that of PH domains. However, the PTB domain of IRS-1 is smaller than that of Shc (110 versus 170 residues) and binds to phosphopeptides in a distinct manner. We explain the phosphopeptide binding specificity based on the structure of the complex and results of site-directed mutagenesis experiments.

Amino Acid Sequence↗
PubMed1995

Intermolecular phosphorylation between insulin holoreceptors does not stimulate substrate kinase activity.

We photocoupled benzoylphenylalanineB25, B29 epsilon-biotin insulin (BBpa-insulin) to native insulin receptors to obtain a uniform receptor population with covalently bound, non-dissociable ligand. We employed BBpa-insulin-bound and autophosphorylated (activated) receptor to phosphorylate substrate insulin receptor under conditions where the substrate receptor never interacts with insulin. The substrate receptor becomes phosphorylated in this inter-receptor fashion and reaches a phosphorylation state 50% of the maximal obtainable by autophosphorylation. However, this phosphorylation does not activate the substrate receptor to any measurable degree. We conclude that intermolecular phosphorylation of the insulin holoreceptors is unlikely to be of physiological significance.

3T3 Cells↗
PubMed1995

Possible v-Crk-induced transformation through activation of Src kinases.

p47gag-crk (v-Crk) encoded by avian sarcoma virus CT10, causes an elevation of tyrosine phosphorylation of several cellular proteins. The lack of a protein-tyrosine kinase domain in v-Crk suggests its co-operation with cellular protein-tyrosine kinase activity. We have shown that suppression of a certain fraction of c-Src activity by Csk may require the binding of Csk to tyrosine-phosphorylated paxillin. In this study, we detected co-immunoprecipitation of tyrosine-phosphorylated paxillin with v-Crk in CT10-transformed chicken embryo fibroblasts (CEF), and demonstrated that v-Crk binding to paxillin can inhibit Csk binding to paxillin. A phosphotyrosine peptide, which can inhibit v-Crk binding to paxillin, did not inhibit Csk binding to paxillin, suggesting that v-Crk and Csk bind to different tyrosine-phosphorylated sites in paxillin. We also found that the kinase activity of the endogenous c-Src in CEF is elevated severalfold after CT10-transformation. We therefore suggest that the competitive binding of overexpressed v-Crk affects an efficient interaction of Csk with tyrosine-phosphorylated paxillin in CT10-transformed CEF. This would result in a failure in the suppression of the kinase activities of a population of c-Src and other Src family protein-tyrosine kinases as well, and these kinases may then contribute to the phosphorylation of cellular proteins in CT10-transformed CEF.

Amino Acid Sequence↗
PubMed1995

PTB domains of IRS-1 and Shc have distinct but overlapping binding specificities.

PTB domains are non-Src homology 2 (SH2) phosphotyrosine binding domains originally described in the receptor tyrosine kinase substrate, Shc. By serial truncation, we show that a 174-residue region of Shc p52 (33-206) has full PTB activity. We also show that a 173-residue region of insulin receptor substrate-1 (IRS-1; residues 144-316) has related PTB activity. In vitro both domains bind directly to activated insulin receptors. Binding is abrogated by substitution of Tyr-960 and selectively inhibited by phosphopeptides containing NPXY sequences. Phosphopeptide assays developed to compare PTB domain specificities show that the Shc PTB domain binds with highest affinity to psi XN beta 1 beta 2 pY motifs derived from middle T (mT), TrkA, ErbB4, or epidermal growth factor receptors (psi = hydrophobic, beta = beta-turn forming); the IRS-1 PTB domain does not bind with this motif. In contrast, both the Shc and IRS-1 PTB domains bind psi psi psi XXN beta 1 beta 2pY sequences derived from insulin and interleukin 4 receptors, although specificities vary in detail. Shc and IRS-1 are phosphorylated by distinct but overlapping sets of receptor-linked tyrosine kinases. These differences may be accounted for by the inherent specificities of their respective PTB domains.

3T3 Cells↗
PubMed1995

L-O-(2-malonyl)tyrosine: a new phosphotyrosyl mimetic for the preparation of Src homology 2 domain inhibitory peptides.

Inhibition of Src homology 2 (SH2) domain-binding interactions affords one potential means of modulating protein-tyrosine kinase-dependent signaling. Small phosphotyrosyl (pTyr)-containing peptides are able to bind to SH2 domains and compete with larger pTyr peptides or native pTyr-containing protein ligands. Such pTyr-containing peptides are limited in their utility as SH2 domain inhibitors in vivo due to their hydrolytic lability to protein-tyrosine phosphatases (PTPs) and the poor cellular penetration of the ionized phosphate moiety. An important aspect of SH2 domain inhibitor design is the creation of pTyr mimetics which are stable to PTPs and have reasonable bioavailability. To date, most PTP-resistant pTyr mimetics which bind to SH2 domains are phosphonates such as (phosphonomethyl)phenylalanine (Pmp, 2), [(monofluorophosphono)methyl]phenylalanine (FPmp, 3) or [(difluorophosphono)methyl]-phenylalanine (F2Pmp, 4). Herein we report the incorporation of a new non-phosphorus-containing pTyr mimetic, L-O-(2-malonyl)tyrosine (L-OMT, 5), into SH2 domain inhibitory peptides using the protected analogue L-N alpha-Fmoc-O'-(O",O"-di-tert-butyl-2-malonyl)tyrosine (6) and solid-phase peptide synthesis techniques. Five OMT-containing peptides were prepared against the following SH2 domains: the PI-3 kinase C-terminal p85 SH2 domain (Ac-D-(L-OMT)-V-P-M-L-amide, 10, IC50 = 14.2 microM), the Src SH2 domain (Ac-Q-(L-OMT)-E-E-I-P-amide, 11, IC50 = 25 microM, and Ac-Q-(L-OMT)-(L-OMT)-E-I-P-amide, 14, IC50 = 23 microM), the Grb2 SH2 domain (Ac-N-(L-OMT)-V-N-I-E-amide, 12, IC50 = 120 microM), and the N-terminal SH-PTP2 SH2 domain (Ac-L-N-(L-OMT)-I-D-L-D-L-V-amide, 13, IC50 = 22.0 microM). These results show that peptides 10, 11, 13, and 14 have reasonable affinity for their respective SH2 domains, with the IC50 value for the SH-PTP2 SH2 domain-directed peptide 13 being equivalent to that previously observed for the corresponding F2Pmp-containing peptide. OMT may afford a new structural starting point for the development of novel and useful SH2 domain inhibitors.

Amino Acid Sequence↗
PubMed1995

T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/phospholipase C-gamma 1-binding phosphotyrosyl protein pp36/38.

Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as zeta and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors. pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor. The tyrosine-phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-gamma 1 and Grb2, providing one mechanism to recruit downstream effectors to the cell membrane. Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent manner. Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85 alpha cDNA into Jurkat cells followed by anti-hemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38. This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity. Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI-3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells.

Adaptor Proteins, Signal Transducing↗
PubMed1995

The SH3 domain-binding T cell tyrosyl phosphoprotein p120. Demonstration of its identity with the c-cbl protooncogene product and in vivo complexes with Fyn, Grb2, and phosphatidylinositol 3-kinase.

Previously, we have identified p120 as a Fyn/Lck SH3 and SH2 domain-binding protein that is tyrosine phosphorylated rapidly after T cell receptor triggering. Here, we used direct protein purification, amino acid sequence analysis, reactivity with antibodies, and two-dimensional gel analyses to identify p120 as the human c-cbl protooncogene product. We demonstrate in vivo complexes of p120cbl with Fyn tyrosine kinase, the adaptor protein Grb2, and the p85 subunit of phosphatidylinositol (PI) 3-kinase. The association of p120cbl with Fyn and the p85 subunit of PI 3-kinase (together with PI 3-kinase activity) was markedly increased by T cell activation, consistent with in vitro binding of p120cbl to their SH2 as well as SH3 domains. In contrast, a large fraction of p120cbl was associated with Grb2 prior to activation, and this association did not change upon T cell activation. In vitro, p120cbl interacted with Grb2 exclusively through its SH3 domains. These results demonstrate a novel Grb2-p120cbl signaling complex in T cells, distinct from the previously analyzed Grb2-Sos complex. The association of p120cbl with ubiquitous signaling proteins strongly suggests a general signal transducing function for this enigmatic protooncogene with established leukemogenic potential but unknown physiological function.

Adaptor Proteins, Signal Transducing↗