Rotational coupling of the transmembrane and kinase domains of the Neu receptor tyrosine kinase

Ligand binding to receptor tyrosine kinases (RTKs) regulates receptor dimerization and activation of the kinase domain. To examine the role of the transmembrane domain in regulation of RTK activation, we have exploited a simplified transmembrane motif, [VVVEVVV](n), previously shown to activate the Neu receptor. Here we demonstrate rotational linkage of the transmembrane domain with the kinase domain, as evidenced by a periodic activation of Neu as the dimerization motif is shifted across the transmembrane domain. These results indicate that activation requires a specific orientation of the kinase domains with respect to each other. Results obtained with platelet-derived growth factor receptor-beta suggest that this rotational linkage of the transmembrane domain to the kinase domain may be a general feature of RTKs. These observations suggest that activating mutations in RTK transmembrane and juxtamembrane domains will be limited to those residues that position the kinase domains in an allowed rotational conformation.     

Molecular dynamics (MD) investigations of preformed structures of the transmembrane domain of the oncogenic Neu receptor dimer in a DMPC bilayer

The critical Val/Glu mutation in the membrane spanning domain of the rat Neu receptor confers the ability for ligand-independent signaling and leads to increased dimerization and transforming ability. There is evidence that the two transmembrane interacting helices play a role in receptor activation by imposing orientation constraints to the intracellular tyrosine kinase domains. By using MD simulations we have attempted to discriminate between correct and improper helix-helix packing by examining the structural and energetic properties of preformed left-handed and right-handed structures in a fully hydrated DMPC bilayer. The best energetic balance between the residues at the helix-helix interface and the residues exposed to the lipids is obtained for helices in symmetrical left-handed interactions packed together via Glu side chain/Ala backbone interhelical hydrogen bonds. Analyses demonstrate the importance of the ATVEG motif in helix-helix packing and point to additional contacting residues necessary for association. Our findings, all consistent with experimental data, suggest that a symmetrical left-handed structure of the helices could be the transmembrane domain configuration that promotes receptor activation and transformation. The present study may provide further insight into signal transduction mechanisms of the ErbB/Neu receptors.     

Structural and Functional Properties of Platelet-Derived Growth Factor and Stem Cell Factor Receptors

The receptors for platelet-derived growth factor (PDGF) and stem cell factor (SCF) are members of the type III class of PTK receptors, which are characterized by five Ig-like domains extracellularly and a split kinase domain intracellularly. The receptors are activated by ligand-induced dimerization, leading to autophosphorylation on specific tyrosine residues. Thereby the kinase activities of the receptors are activated and docking sites for downstream SH2 domain signal transduction molecules are created; activation of these pathways promotes cell growth, survival, and migration. These receptors mediate important signals during the embryonal development, and control tissue homeostasis in the adult. Their overactivity is seen in malignancies and other diseases involving excessive cell proliferation, such as atherosclerosis and fibrotic diseases. In cancer, mutations of PDGF and SCF receptors—including gene fusions, point mutations, and amplifications—drive subpopulations of certain malignancies, such as gastrointestinal stromal tumors, chronic myelomonocytic leukemia, hypereosinophilic syndrome, glioblastoma, acute myeloid leukemia, mastocytosis, and melanoma.The type III tyrosine kinase receptor family consists of platelet-derived growth factor (PDGF) receptor α and β, stem cell factor (SCF) receptor (Kit), colony-stimulating factor-1 (CSF-1) receptor, and Flt-3 (Blume-Jensen and Hunter 2001). Members of this receptor family are characterized by five Ig-like domains in their extracellular part, a single transmembrane domain, and an intracellular part consisting of a rather well-conserved juxtamembrane domain, a tyrosine kinase domain with a characteristic inserted sequence without homology with kinases, and a less well-conserved carboxy-terminal tail. The ligands for these receptors are all dimeric molecules, and on binding they induce receptor dimerization. Although the overall mechanisms for the activation of the type III tyrosine kinase receptors and the signaling pathways they induce are similar, the receptors are expressed on different cell types and thus have different functions in vivo.Here we will describe the structural and functional properties of the PDGF receptors and Kit.     

Oncogenic receptor tyrosine kinase in leukemia.

Growth, survival and differentiation of hematopoietic cells are regulated by the interaction between hematopoietic growth factors and their receptors. While the defect in this interaction results in an insufficient hematopoiesis, the aberrantly elevated activation leads to the transformation of hematopoietic cells. The constitutive active mutations of receptor tyrosine kinase, such as c-Kit platelet-derived growth factor receptor (PDGFR) or fins-like tyrosine kinase 3 (Flt3), play a major role in the development of hematopoietic neoplasia. The constitutive activation is provoked by several mechanisms, such as making fusion genes by chromosomal translocations, or various mutations involving regulatory regions of the receptor. The chromosomal translocation brings the receptor intracytoplasmic domain juxtaposed to an unrelated molecule which has dimerization or multimerization motif, resulting in the constitutive dimerization of the receptor. The missense, insertion or deletion mutations in the regulatory regions, such as juxtamembrane domain, activation loop and extracellular domain, cause constitutive activation by releasing the respective auto-inhibitory functions of each regulatory region. Constitutive active receptors generate different signals quantitatively and qualitatively from wild type receptor, which mediate the oncogenic phenotype. Given the frequent involvement of constitutive active receptor tyrosine kinase in hematopoietic malignancies, targeted inhibitions of active tyrosine kinase and downstream aberrant signaling are rapidly developing novel therapeutic modality with much promise.     

The single transmembrane domains of ErbB receptors self-associate in cell membranes.

Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) alpha-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM alpha-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia coli cell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM equivalent to ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups.     

Studies of Receptor Tyrosine Kinase Transmembrane Domain Interactions: The EmEx-FRET Method

The energetics of transmembrane (TM) helix dimerization in membranes and the thermodynamic principles behind receptor tyrosine kinase (RTK) TM domain interactions during signal transduction can be studied using Förster resonance energy transfer (FRET). For instance, FRET studies have yielded the stabilities of wild-type fibroblast growth factor receptor 3 (FGFR3) TM domains and two FGFR3 pathogenic mutants, Ala391Glu and Gly380Arg, in the native bilayer environment. To further our understanding of the molecular mechanisms of deregulated FGFR3 signaling underlying different pathologies, we determined the effect of the Gly382Asp FGFR3 mutation, identified in a multiple myeloma cell line, on the energetics of FGFR3 TM domain dimerization. We measured dimerization energetics using a novel FRET acquisition and processing method, termed “emission-excitation FRET (EmEx-FRET),” which improves the precision of thermodynamic measurements of TM helix association. The EmEx-FRET method, verified here by analyzing previously published data for wild-type FGFR3 TM domain, should have broad utility in studies of protein interactions, particularly in cases when the concentrations of fluorophore-tagged molecules cannot be controlled.     

二聚化:受体酪氨酸激酶活化的重要机制

受体酪氨酸激酶家族是一类具有内源性蛋白酪氨酸激酶活性的生长因子受体。它们具有相似的分子结构 ,其配体介导的受体活化主要是通过二聚化的机制来实现的。配体介导同源或异源的受体二聚化 ,不同的配体以不同的机制介导受体的二聚化。本文介绍了受体酪氨酸激酶家族不同亚类受体在其配体介导下二聚化的机制 ,并着重介绍了表皮生长因子受体家族各成员间的异二聚化及其引起的胞内信号转导途径的多样化     

An extended surface of binding to Trk tyrosine kinase receptors in NGF and BDNF allows the engineering of a multifunctional pan-neurotrophin.

Neurotrophin-mediated cell survival and differentiation of vertebrate neurons is caused by ligand-specific binding to the Trk family of tyrosine kinase receptors. However, sites in the neurotrophins responsible for the binding to Trk receptors and the mechanisms whereby this interaction results in receptor activation and biological activity are unknown. Here we show that in nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), discontinuous stretches of amino acid residues group together on one side of the neurotrophin dimer forming a continuous surface responsible for binding to and activation of TrkA and TrkB receptors. Two symmetrical surfaces are formed along the two-fold axis of the neurotrophin dimer providing a model for ligand-mediated receptor dimerization. Mutated neurotrophins inducing similar levels of receptor phosphorylation showed different biological activities, suggesting that structural differences in a ligand may result in dissimilar responses in a given tyrosine kinase receptor. Our results allowed us to combine structural elements from NGF, BDNF and neurotrophin-3 to engineer a pan-neurotrophin that efficiently activates all Trk receptors and displays multiple neurotrophic specificities.     

The ErbB kinase domain: structural perspectives into kinase activation and inhibition

Epidermal growth factor receptor (EGFR) and its family members, ErbB2, ErbB3 and ErbB4, are receptor tyrosine kinases which send signals into the cell to regulate many critical processes including development, tissue homeostasis, and tumorigenesis. Central to the signaling of these receptors is their intracellular kinase domain, which is activated by ligand-induced dimerization of the receptor and phosphorylates several tyrosine residues in the C-terminal tail. The phosphorylated tail then recruits other signaling molecules and relays the signal to downstream pathways. A model of the autoinhibition, activation and feedback inhibition mechanisms for the ErbB kinase domain has emerged from a number of recent structural studies. Meanwhile, recent clinical studies have revealed the relationship between specific ErbB kinase mutations and the responsiveness to kinase inhibitor drugs. We will review these regulation mechanisms of the ErbB kinase domain, and discuss the binding specificity of kinase inhibitors and the effects of kinase domain mutations found in cancer patients from a structural perspective.     

A transmembrane leucine zipper is required for activation of the dimeric receptor tyrosine kinase DDR1

Receptor tyrosine kinases of the discoidin domain family, DDR1 and DDR2, are activated by different types of collagen and play important roles in cell adhesion, migration, proliferation, and matrix remodeling. In a previous study, we found that collagen binding by the discoidin domain receptors (DDRs) requires dimerization of their extracellular domains (Leitinger, B. (2003) J. Biol. Chem. 278, 16761-16769), indicating that the paradigm of ligand-induced receptor dimerization may not apply to the DDRs. Using chemical cross-linking and co-immunoprecipitation of differently tagged DDRs, we now show that the DDRs form ligand-independent dimers in the biosynthetic pathway and on the cell surface. We further show that both the extracellular and the cytoplasmic domains are individually dispensable for DDR1 dimerization. The DDR1 transmembrane domain contains two putative dimerization motifs, a leucine zipper and a GXXXG motif. Mutations disrupting the leucine zipper strongly impaired collagen-induced transmembrane signaling, although the mutant DDR1 proteins were still able to dimerize, whereas mutation of the GXXXG motif had no effect. A bacterial reporter assay (named TOXCAT) showed that the DDR1 transmembrane domain has a strong potential for self-association in a biological membrane and that this interaction occurs via the leucine zipper and not the GXXXG motif. Our results demonstrate that the DDRs exist as stable dimers in the absence of ligand and that receptor activation requires specific interactions made by the transmembrane leucine zipper.     

Structural analysis of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the γ-phosphate of ATP to tyrosine residues in protein substrates. RTKs are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. Included in this large protein family are the insulin receptor and the receptors for growth factors such as epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor. Receptor activation occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins. Over the past several years, structural studies employing X-ray crystallography have advanced our understanding of the molecular mechanisms by which RTKs recognize their ligands and are activated by dimerization and tyrosine autophosphorylation. This review will highlight the key results that have emerged from these structural studies.     

Dimerization mediated through a leucine zipper activates the oncogenic potential of the met receptor tyrosine kinase.

Oncogenic activation of the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase involves a genomic rearrangement that generates a hybrid protein containing tpr-encoded sequences at its amino terminus fused directly to the met-encoded receptor kinase domain. Deletion of Tpr sequences abolishes the transforming ability of this protein, implicating this region in oncogenic activation. We demonstrate, by site-directed mutagenesis and coimmunoprecipitation experiments, that a leucine zipper motif within Tpr mediates dimerization of the tpr-met product and is essential for the transforming activity of the met oncogene. By analogy with ligand-stimulated activation of receptor tyrosine kinases, we propose that constitutive dimerization mediated by a leucine zipper motif within Tpr is responsible for oncogenic activation of the Met kinase. The possibility that this mechanism of activation represents a paradigm for a class of receptor tyrosine kinase oncogenes activated by DNA rearrangement is discussed.     

An oncogenic point mutation confers high affinity ligand binding to the neu receptor. Implications for the generation of site heterogeneity.

The neu protooncogene encodes a receptor tyrosine kinase homologous to the receptor for the epidermal growth factor. The oncogenic potential of neu is released upon chemical carcinogenesis, which replaces a glutamic acid for a valine residue, within the single transmembrane domain. This results in constitutive receptor dimerization and activation of the intrinsic catalytic function. To study the implications of the oncogenic mutation and the consequent receptor dimerization on the interaction with the yet incompletely characterized ligand of p185neu, we constructed chimeric proteins between the ligand binding domain of the epidermal growth factor receptor and the transmembrane and cytoplasmic domains of the normal or the transforming Neu proteins. The chimeric receptors displayed cellular and biochemical differences characteristic of the normal and the transforming Neu proteins and therefore may reliably represent the ligand binding functions of the two receptor forms. Analyses of ligand binding revealed qualitative and quantitative differences that were a result of the single mutation; whereas the normal chimera (valine version) displayed two populations of binding sites with approximately 90% of the receptors in the low affinity state, the transforming receptor (glutamic acid version) showed a single population of binding sites with relatively high affinity. Kinetics measurements indicated that the difference in affinities was because of slower rates of both ligand association and ligand dissociation from the constitutively dimerized mutant receptor. It therefore appears that the oncogenic mutation, by permanently dimerizing the receptor, establishes a high affinity ligand binding state which is functionally equivalent to the ligand-occupied normal receptor. Our conclusion is further supported by the rates of endocytosis of the wild-type and the mutant receptor. Hence, these results provide the first experimental evidence from living cells which supports a model that attributes the heterogeneity of ligand binding sites to the state of oligomerization of receptor tyrosine kinases.     

Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor beta receptor

Growth factor receptors are typically activated by the binding of soluble ligands to the extracellular domain of the receptor, but certain viral transmembrane proteins can induce growth factor receptor activation by binding to the receptor transmembrane domain. For example, homodimers of the transmembrane 44-amino acid bovine papillomavirus E5 protein bind the transmembrane region of the PDGF beta receptor tyrosine kinase, causing receptor dimerization, phosphorylation, and cell transformation. To determine whether it is possible to select novel biologically active transmembrane proteins that can activate growth factor receptors, we constructed and identified small proteins with random hydrophobic transmembrane domains that can bind and activate the PDGF beta receptor. Remarkably, cell transformation was induced by approximately 10% of the clones in a library in which 15 transmembrane amino acid residues of the E5 protein were replaced with random hydrophobic sequences. The transformation-competent transmembrane proteins formed dimers and stably bound and activated the PDGF beta receptor. Genetic studies demonstrated that the biological activity of the transformation-competent proteins depended on specific interactions with the transmembrane domain of the PDGF beta receptor. A consensus sequence distinct from the wild-type E5 sequence was identified that restored transforming activity to a non-transforming poly-leucine transmembrane sequence, indicating that divergent transmembrane sequence motifs can activate the PDGF beta receptor. Molecular modeling suggested that diverse transforming sequences shared similar protein structure, including the same homodimer interface as the wild-type E5 protein. These experiments have identified novel proteins with transmembrane sequences distinct from the E5 protein that can activate the PDGF beta receptor and transform cells. More generally, this approach may allow the creation and identification of small proteins that modulate the activity of a variety of cellular transmembrane proteins.     

Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia.

Achondroplasia, the most common genetic form of dwarfism, is an autosomal dominant disorder whose underlying mechanism is a defect in the maturation of the cartilage growth plate of long bones. Achondroplasia has recently been shown to result from a Gly to Arg substitution in the transmembrane domain of the fibroblast growth factor receptor 3 (FGFR3), although the molecular consequences of this mutation have not been investigated. By substituting the transmembrane domain of the Neu receptor tyrosine kinase with the transmembrane domains of wild-type and mutant FGFR3, the Arg380 mutation in FGFR3 is shown to activate both the kinase and transforming activities of this chimeric receptor. Residues with side chains capable of participating in hydrogen bond formation, including Glu, Asp, and to a lesser extent, Gln, His and Lys, were able to substitute for the activating Arg380 mutation. The Arg380 point mutation also causes ligand-independent stimulation of the tyrosine kinase activity of FGFR3 itself, and greatly increased constitutive levels of phosphotyrosine on the receptor. These results suggest that the molecular basis of achondroplasia is unregulated signal transduction through FGFR3, which may result in inappropriate cartilage growth plate differentiation and thus abnormal long bone development. Achondroplasia may be one of the number of cogenital disorders where constitutive activation of a member of the FGFR family leads to development abnormalities.     

Growth factor-induced binding of dynamin to signal transduction proteins involves sorting to distinct and separate proline-rich dynamin sequences.

Dynamin, a 100 kDa GTPase, is critical for endocytosis, synaptic transmission and neurogenesis. Endocytosis accompanies receptor processing and plays an essential role in attenuating receptor tyrosine kinase signal transduction. Dynamin has been demonstrated to be involved in the endocytic processing at the cell surface and may play a general role in coupling receptor activation to endocytosis. Src homology (SH) domain dependent protein-protein interactions are important to tyrosine kinase receptor signal transduction. The C-terminus of dynamin contains two clusters of SH3 domain binding proline motifs; these motifs may interact with known SH3 domain proteins during tyrosine kinase receptor activation. We demonstrate here that SH3 domain-containing signal transduction proteins, such as phospholipase C gamma-1 (PLC gamma-1), do indeed bind to dynamin in a growth factor inducible manner. The induction of PLC gamma-1 binding to dynamin occurs within minutes of the addition of platelet derived growth factor (PDGF) to cells. Binding of these signal transduction proteins to dynamin involves specific sorting to individual proline motif clusters and appears to be responsible for co-immunoprecipitation of tyrosine phosphorylated PDGF receptors with dynamin following PDGF stimulation of mammalian cells. The binding of dynamin to SH3 domain-containing proteins may therefore be important for formation of the protein complex required for the endocytic processing of activated tyrosine kinase receptors.     

Glycosylation-induced conformational modification positively regulates receptor-receptor association: a study with an aberrant epidermal growth factor receptor (EGFRvIII/DeltaEGFR) expressed in cancer cells

The epidermal growth factor receptor (EGFR) is a multisited and multifunctional transmembrane glycoprotein with intrinsic tyrosine kinase activity. Upon ligand binding, the monomeric receptor undergoes dimerization resulting in kinase activation. The consequences of kinase stimulation are the phosphorylation of its own tyrosine residues (autophosphorylation) followed by association with and activation of signal transducers. Deregulation of signaling resulting from aberrant expression of the EGFR has been implicated in a number of neoplasms including breast, brain, and skin tumors. A mutant epidermal growth factor (EGF) receptor missing 267 amino acids from the exoplasmic domain is common in human glioblastomas. The truncated receptor (EGFRvIII/DeltaEGFR) lacks EGF binding activity; however, the kinase is constitutively active, and cells expressing the receptor are tumorigenic. Our studies revealed that the high kinase activity of the DeltaEGFR is due to self-dimerization, and contrary to earlier reports, the kinase activity per molecule of the dimeric DeltaEGFR is comparable to that of the EGF-stimulated wild-type receptor. Furthermore, the phosphorylation patterns of both receptors are similar as determined by interaction with a conformation-specific antibody and by phosphopeptide analysis. This eliminates the possibility that the defective down-regulation of the DeltaEGFR is due to its altered phosphorylation pattern as has been suggested previously. Interestingly, the receptor-receptor self-association is highly dependent on a conformation induced by N-linked glycosylation. We have identified four potential sites that might participate in self-dimerization; these sites are located in a domain that plays an important role in EGFR functioning.     

Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor

The crystal structure of the kinase domain from the epidermal growth factor receptor (EGFRK) including forty amino acids from the carboxyl-terminal tail has been determined to 2.6-A resolution, both with and without an EGFRK-specific inhibitor currently in Phase III clinical trials as an anti-cancer agent, erlotinib (OSI-774, CP-358,774, Tarceva(TM)). The EGFR family members are distinguished from all other known receptor tyrosine kinases in possessing constitutive kinase activity without a phosphorylation event within their kinase domains. Despite its lack of phosphorylation, we find that the EGFRK activation loop adopts a conformation similar to that of the phosphorylated active form of the kinase domain from the insulin receptor. Surprisingly, key residues of a putative dimerization motif lying between the EGFRK domain and carboxyl-terminal substrate docking sites are found in close contact with the kinase domain. Significant intermolecular contacts involving the carboxyl-terminal tail are discussed with respect to receptor oligomerization.     

Multimerization of polyomavirus middle-T antigen.

The oncogenic protein of polyomavirus, middle-T antigen, associated with cell membranes and interacts with a variety of cellular proteins involved in mitogenic signalling. Middle-T antigen may therefore mimic the function of cellular tyrosine kinase growth factor receptors, like the platelet-derived growth factor or epidermal growth factor receptor. Growth factor receptor signalling is initiated upon the binding of a ligand to the extracellular domain of the receptor. This results in activation of the intracellular tyrosine kinase domain of the receptor, followed by receptor phosphorylation, presumably as a consequence of dimerization of two receptor molecules. Similar to middle-T antigen, phosphorylation of growth factor receptors leads to recruitment of cellular signalling molecules downstream in the signalling cascade. In this study, we investigated whether middle-T antigen, similar to tyrosine kinase growth factor receptors, is able to form dimeric signalling complexes. We found that association with cellular membranes was a prerequisite for multimerization, most likely dimer formation. A chimeric middle-T antigen carrying the membrane-targeting sequence of the vesicular stomatitis virus G protein instead of the authentic polyomavirus sequence still dimerized. However, mutants of middle-T antigen unable to associate with 14-3-3 proteins, like d18 and S257A, did not form dimers but were still oncogenic. This indicates that both membrane association and binding of 14-3-3 are necessary for dimer formation of middle-T antigen but that only the former is essential for cell transformation.     

Death receptors bind SHP-1 and block cytokine-induced anti-apoptotic signaling in neutrophils.

Death domain-containing receptors of the tumor necrosis factor (TNF)/nerve growth factor (NGF) family can induce apoptosis upon activation in many cellular systems. We show here that a conserved phosphotyrosine-containing motif within the death domain of these receptors can mediate inhibitory functions. The Src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), SHP-2 and SH2-containing inositol phosphatase (SHIP) bound to this motif in a caspase-independent but cell-dependent manner. We also found that stimulation of death receptors disrupted anti-apoptosis pathways initiated (at least under certain conditions) by survival factors in neutrophils. In these cells, activation of the tyrosine kinase Lyn, an important anti-apoptotic event, was prevented as a consequence of death-receptor stimulation, most likely through association of the receptor with activated SHP-1. Thus, we provide molecular and functional evidence for negative signaling by death receptors.