Neutralizing aptamers from whole-cell SELEX inhibit the RET receptor tyrosine kinase

Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacological challenge. Specific oligonucleotide ligands (aptamers) can be generated for a variety of targets through the iterative evolution of a random pool of sequences (SELEX). Nuclease-resistant aptamers that recognize the human receptor tyrosine kinase RET were obtained using RET-expressing cells as targets in a modified SELEX procedure. Remarkably, one of these aptamers blocked RET-dependent intracellular signaling pathways by interfering with receptor dimerization when the latter was induced by the physiological ligand or by an activating mutation. This strategy is generally applicable to transmembrane receptors and opens the way to targeting other members of this class of proteins that are of major biomedical importance.     

RTKdb: database of Receptor Tyrosine Kinase

Receptor Tyrosine Kinases (RTK) are transmembrane receptors specifically found in metazoans. They represent an excellent model for studying evolution of cellular processes in metazoans because they encompass large families of modular proteins and belong to a major family of contingency generating molecules in eukaryotic cells: the protein kinases. Because tyrosine kinases have been under close scrutiny for many years in various species, they are associated with a wealth of information, mainly in mammals. Presently, most categories of RTK were identified in mammals, but in a near future other model species will be sequenced, and will bring us RTKs from other metazoan clades. Thus, collecting RTK sequences would provide a good starting point as a new model for comparative and evolutionary studies applying to multigene families. In this context, we are developing the Receptor Tyrosine Kinase database (RTKdb), which is the only database on tyrosine kinase receptors presently available. In this database, protein sequences from eight model metazoan species are organized under the format previously used for the HOVERGEN, HOBACGEN and NUREBASE systems. RTKdb can be accessed through the PBIL (P?le Bioinformatique Lyonnais) World Wide Web server at http://pbil.univ-lyon1.fr/RTKdb/, or through the FamFetch graphical user interface available at the same address.     

Regulation of Receptor Tyrosine Kinase Ligand Processing

A primary mode of regulating receptor tyrosine kinase (RTK) signaling is to control access of ligand to its receptor. Many RTK ligands are synthesized as transmembrane proteins. Frequently, the active ligand must be released from the membrane by proteolysis before signaling can occur. Here, we discuss RTK ligand shedding and describe the proteases that catalyze it in flies and mammals. We focus principally on the control of EGF receptor ligand shedding, but also refer to ligands of other RTKs. Two prominent themes emerge. First, control by regulated trafficking and cellular compartmentalization of the proteases and their ligand substrates plays a key role in shedding. Second, many external signals converge on the shedding proteases and their control machinery. Proteases therefore act as regulatory hubs that integrate information that the cell receives and translate it into precise outgoing signals. The activation of signaling by proteases is therefore an essential element of the cellular communication machinery.Cells must talk to one another. This principle applies throughout the tree of life: from unicellular bacteria, to the trillions of cells that coordinate to make a mammal. Communication between cells requires dedicated machinery, capable of relaying information across membranes. Transmembrane proteins are therefore essential for signaling. Understanding how this is regulated is paramount. In mammals, receptor tyrosine kinases (RTKs) and their ligands are important examples of such machinery (Schlessinger 2000), controlling many biological processes including development, immunity, tissue repair, and metabolic homeostasis (Ullrich and Schlessinger 1990). They are transmembrane proteins with an extracellular ligand-binding motif and an intracellular kinase domain. As discussed in other chapters, a common mode of RTK activation involves receptor dimerization induced by ligand binding (Lemmon and Schlessinger 2010).Regulated access of ligand to receptor, over distance and time, is key to controlling signaling. Ligands are frequently synthesized as transmembrane forms; when they remain membrane-tethered and cannot diffuse, the range over which they can operate is limited to adjacent cells (Massague and Pandiella 1993; Singh and Harris 2005). Other ligands are soluble secretory proteins. This enables paracrine and endocrine signaling—communication between nonadjacent cells. A more complex mode of signaling exploits the characteristics of both of the above. Ligand is synthesized as a transmembrane precursor, which is then shed from the cell surface by proteolysis. This adds an additional and stringent regulatory step to a signaling network (Massague and Pandiella 1993).This chapter will focus on RTK ligand cleavage and its regulation. We shall highlight how shedding is often critical for signaling, and describe the protease families that catalyze ligand release in flies and mammals. An emergent theme is that regulated trafficking and compartmentalization of ligand and protease modulate signaling. Another theme will be the range of stimuli that impinge on shedding.The epidermal growth factor receptor (EGFR) is an excellent model RTK to illustrate the regulation of ligand proteolysis because the requirement for ligand cleavage in signaling is well established, and the major physiological sheddases have been identified (Blobel 2005). Where warranted, physiological evidence for the role of ligand shedding in the regulation of other RTKs will also be discussed. Whereas we shall deal mostly with ADAM proteases (“a disintegrin and metalloprotease”), which represent the canonical mammalian RTK ligand shedding machinery, the rhomboid family of intramembrane proteases will also be discussed.     

The Insulin Receptor: Both a Prototypical and Atypical Receptor Tyrosine Kinase

Unlike prototypical receptor tyrosine kinases (RTKs), which are single-chain polypeptides, the insulin receptor (InsR) is a preformed, covalently linked tetramer with two extracellular α subunits and two membrane-spanning, tyrosine kinase-containing β subunits. A single molecule of insulin binds asymmetrically to the ectodomain, triggering a conformational change that is transmitted to the cytoplasmic kinase domains, which facilitates their trans-phosphorylation. As in prototypical RTKs, tyrosine phosphorylation in the juxtamembrane region of InsR creates recruitment sites for downstream signaling proteins (IRS [InsR substrate] proteins, Shc) containing a phosphotyrosine-binding (PTB) domain, and tyrosine phosphorylation in the kinase activation loop stimulates InsR’s catalytic activity. For InsR, phosphorylation of the activation loop, which contains three tyrosine residues, also creates docking sites for adaptor proteins (Grb10/14, SH2B2) that possess specialized Src homology-2 (SH2) domains, which are dimeric and engage two phosphotyrosines in the activation loop.Insulin is a highly potent anabolic hormone that is critical for tissue development and for glucose homeostasis (Taniguchi et al. 2006). Released from the β cells of the pancreas, insulin regulates glucose output from the liver and glucose uptake into (primarily) skeletal muscle and adipose tissue. In addition, insulin promotes the synthesis and storage of carbohydrates, lipids, and protein. Insulin’s actions are mediated by the insulin receptor (InsR), a plasma membrane-resident glycoprotein and member of the receptor tyrosine kinase (RTK) family. Other members of the InsR subfamily of RTKs include the insulinlike growth factor-1 receptor (IGF1R) and insulin receptor-related receptor, the latter of which has no known ligand. As an RTK, InsR is ligand-activated through mechanisms that are both prototypical and atypical of RTKs. These mechanisms will be the focus of this article.     

ErbB2受体酪氨酸激酶激酶区的表达与纯化

以GFP融合表达的形式在毕赤酵母中表达具有生物活性的受体酪氨酸激酶ErbB2的激酶区.构建受体酪氨酸激酶激酶区与GFP的融合表达载体pPIC3.5K,转化毕赤酵母GS115,通过组氨酸营养缺陷型筛选,G418高拷贝菌株筛选,以及摇瓶诱导表达筛选,选取较高水平表达菌株进行5升罐培养,以镍亲和层析手段纯化得到蛋白表达产物,进行SDS-PAGE分析和酶联免疫反应检测酶活.结果表明在毕赤酵母中成功诱导表达了约100kD的激酶融合蛋白并具有激酶活性.该研究为筛选ErbB2的抑制剂奠定了基础.     

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     

Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr⁶⁸⁷ in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr⁶⁸⁷ and association with components of the Tyr¹⁰⁶² signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser⁶⁹⁶, a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr⁶⁸⁷ as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.     

受体酪氨酸激酶对自噬的调控及其研究进展*

自噬是真核生物进化上保守的溶酶体降解的生物学过程,在维护细胞内的稳态、消除有害组分等方面起到了重要作用。受体酪氨酸激酶家族(receptor tyrosine kinase,RTKs)是一类激酶蛋白,在正常细胞和癌症细胞的运动和侵袭中起着重要作用。RTKs蛋白既能促进自噬,也能抑制自噬。研究显示,RTKs能够在肿瘤和相关疾病中发挥自噬作用,比如表皮生长因子受体(epidermal growth factor receptor,EGFR)可以抑制自噬,从而促进肿瘤生长、增殖;还能通过RTK/Ras/ERK信号通路诱导自噬,进而参与诸如细胞免疫反应之类的相关疾病。主要综述了RTKs对自噬的调控作用和相关研究成果,为靶点靶向疗法的理论依据提供了基础。     

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.     

Receptor Tyrosine Kinase Signaling and Primordial Germ Cell Development

Primordial germ cells (PGCs) give rise to sperms and eggs. Their development is crucial to species propagation and has to be precisely controlled. Studies in several model organisms have identified many genes involved in the specification and guided migration of PGCs. However, the mechanisms governing the behaviors of this unique type of cells remain to be investigated. Interestingly, PGCs share certain cellular properties with metastasizing cancer cells including proliferation, invasion of other tissues, survival, and migration. Recently we have shown that in Drosophila the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development. In later stages, activation of both STAT and Ras, likely by other molecules, is required continuously for PGC migration. The requirement for RTK suggests molecular conservation between flies and mice in PGC development and also suggests that germ cells and cancer cells share certain intracellular signaling strategies.     

Polymorphism in the Immunoglobulin-like Domains of the Receptor Tyrosine Kinase from the Sponge Geodia Cydonium

Sponges [Porifera] are the phylogenetically oldest phylum of the Metazoa. They are provided with both cellular and humoral allorecognition systems. The underlying molecules are not yet known. To study allorecognition in sponges we first determined the frequency of graft rejection in a natural population of the marine sponge Geodia cvdonium. We then determined, for the first time at the molecular level, the degree of sequence polymorphism in segments of one molecule which may be related to sponge allorecognition and host defense: the Ig-like domains from the receptor tyrosine kinase [RTK]. Thirty six pairs of auto- and allografts were assayed, either by parabiotic attachment or insertion of grafts. All of the autografts fused, while only two allografts fused and 34 pairs were incompatibile. Rejection among the parabiotic allografts was characterized by the formation of a collagenous barrier, while the allografts that were inserted into the host underwent destruction. At the molecular level we first cloned to completion the 5'-end of sponge RTK, which displays a Pro-Ser-Thr-rich sequence; this is thought to act as a module of cell adhesion proteins. Then we analyzed RT-PCR products of amplification across the two Ig-like domains of RTK (about 500 bp), from two pairs of fusing sponges and one pair of rejecting sponges. High levels of polymorphism were recorded, including 18 nucleotide-substitution positions and a tri-nucleotide deletion, which translate into 13 polymorphic amino acid positions. Two of the six sponges were scored as heterozygotes. Among 9 informative polymorphic sites that were tested for linkage disequilibrium, 11 pairwise comparisons were found to be significant, implying the possibility of distinguishable alleles in this locus. To the best of our knowledge this is the first report of polymorphism in Ig-like domains of a receptor from invertebrates that may be associated with allorecognition. This data attests also that fusion in sponges is not confined to genetically identical individuals.     

Polymorphism in the Immunoglobulin-like Domains of the Receptor Tyrosine Kinase from the Sponge Geodia Cydonium

Sponges [Porifera] are the phylogenetically oldest phylum of the Metazoa. They are provided with both cellular and humoral allorecognition systems. The underlying molecules are not yet known. To study allorecognition in sponges we first determined the frequency of graft rejection in a natural population of the marine sponge Geodia cvdonium. We then determined, for the first time at the molecular level, the degree of sequence polymorphism in segments of one molecule which may be related to sponge allorecognition and host defense: the Ig-like domains from the receptor tyrosine kinase [RTK]. Thirty six pairs of auto- and allografts were assayed, either by parabiotic attachment or insertion of grafts. All of the autografts fused, while only two allografts fused and 34 pairs were incompatibile. Rejection among the parabiotic allografts was characterized by the formation of a collagenous barrier, while the allografts that were inserted into the host underwent destruction. At the molecular level we first cloned to completion the 5′-end of sponge RTK, which displays a Pro-Ser-Thr-rich sequence; this is thought to act as a module of cell adhesion proteins. Then we analyzed RT-PCR products of amplification across the two Ig-like domains of RTK (about 500 bp), from two pairs of fusing sponges and one pair of rejecting sponges. High levels of polymorphism were recorded, including 18 nucleotide-substitution positions and a tri-nucleotide deletion, which translate into 13 polymorphic amino acid positions. Two of the six sponges were scored as heterozygotes. Among 9 informative polymorphic sites that were tested for linkage disequilibrium, 11 pairwise comparisons were found to be significant, implying the possibility of distinguishable alleles in this locus. To the best of our knowledge this is the first report of polymorphism in Ig-like domains of a receptor from invertebrates that may be associated with allorecognition. This data attests also that fusion in sponges is not confined to genetically identical individuals.     

Allosteric Communication across the Native and Mutated KIT Receptor Tyrosine Kinase

A fundamental goal in cellular signaling is to understand allosteric communication, the process by which signals originated at one site in a protein propagate dependably to affect remote functional sites. Here, we describe the allosteric regulation of the receptor tyrosine kinase KIT. Our analysis evidenced that communication routes established between the activation loop (A-loop) and the distant juxtamembrane region (JMR) in the native protein were disrupted by the oncogenic mutation D816V positioned in the A-loop. In silico mutagenesis provided a plausible way of restoring the protein communication detected in the native KIT by introducing a counter-balancing second mutation D792E. The communication patterns observed in the native and mutated KIT correlate perfectly with the structural and dynamical features of these proteins. Particularly, a long-distance effect of the D816V mutation manifested as an important structural re-organization of the JMR in the oncogenic mutant was completely vanished in the double mutant D816V/D792E. This detailed characterization of the allosteric communication in the different forms of KIT, native and mutants, was performed by using a modular network representation composed of communication pathways and independent dynamic segments. Such representation permits to enrich a purely mechanistic interaction-based model of protein communication by the introduction of concerted local atomic fluctuations. This method, validated on KIT receptor, may guide a rational modulation of the physiopathological activities of other receptor tyrosine kinases.     

原癌蛋白c-Cbl促进受体酪氨酸激酶EphA2的降解

c Cbl最近被证明是泛素 蛋白酶体 (ubiquitin proteasome)通路中的一个新的RINGFinger型泛素连接酶 (ubiquitinligase ,E3) .c Cbl可以介导受体酪氨酸激酶和非受体酪氨酸受体激酶的降解 .利用内源性表达较高EphA2的大肠癌细胞株HCT1 1 6 ,通过转染野生型c Cbl和显性负变异体(dominantnegativemutant)c Cbl 70Z ,探讨c Cbl在EphA2降解中的作用 .结果显示 ,c Cbl可促进磷酸化EphA2的降解 ,EphA2的降解必须依赖其配体ephrin A1的刺激 ;利用蛋白酶体 (proteasome)抑制剂MG1 32可抑制磷酸化的EphA2降解 ,提示EphA2的最终降解部位是在蛋白酶体 .研究的结果提示 ,c Cbl作为泛素连接酶诱导磷酸化后的EphA2在蛋白酶体中降解     

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.     

生长因子受体酪氨酸激酶的结构与功能

受体酪氨酸蛋白激酶是细胞信号转导进行的关键信号酶,在生长因子调控细胞生长、发育与功能的过程中起着重要的生理作用.本文主要介绍生长因子受体酪氨酸蛋白激酶的分类、结构与功能及其部分相关信号转导机制的研究进展.     

Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling

The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition.     

Casein Kinase 2 Dependent Phosphorylation of Neprilysin Regulates Receptor Tyrosine Kinase Signaling to Akt

Neprilysin (NEP) is a type II membrane metalloproteinase that cleaves physiologically active peptides at the cell surface thus regulating the local concentration of these peptides available for receptor binding and signal transduction. In addition, the cytoplasmic N-terminal domain of NEP interacts with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) thereby regulating intracellular signaling via Akt. Thus, NEP serves dual functions in extracellular and intracellular signal transduction. Here, we show that NEP undergoes phosphorylation at serine residue 6 within the N-terminal cytoplasmic domain. In vitro and cell culture experiments demonstrate that Ser 6 is efficiently phosphorylated by protein kinase CK2. The phosphorylation of the cytoplasmic domain of NEP inhibits its interaction with PTEN. Interestingly, expression of a pseudophosphorylated NEP variant (Ser6Asp) abrogates the inhibitory effect of NEP on insulin/insulin-like growth factor-1 (IGF-1) stimulated activation of Akt. Thus, our data demonstrate a regulatory role of CK2 in the interaction of NEP with PTEN and insulin/IGF-1 signaling.