Elena Pasquale - US grants

Protein tyrosine kinases are thought to be involved in the processes of cell division, migration and differentiation occurring during embryonic development, but their physiological functions, regulatory properties and mechanism of action are largely unknown. Proteins phosphorylated on tyrosine have been detected in embryonic tissues using antibodies specific for phosphotyrosine residues. However, a large fraction of the protein tyrosine kinases involved in developmental processes may not be known at this time. Using anti-phosphotyrosine antibodies to screen chicken embryo cDNA libraries will allow the isolation of the tyrosine kinase genes that are expressed during development. The cloned protein tyrosine kinases will then be identified by sequencing the appropriate cDNAs and characterized using specific polyclonal antibodies to detect them in embryonic tissues or cultured cells. Antibodies will be prepared by injecting the cloned kinases expressed in bacteria as fusion proteins and used in immunoprecipitation, immunoblotting and immunofluorescence microscopy experiments. Finally, the effects that one of the newly identified tyrosine kinases has on cellular growth and differentiation will be investigated by producing eukaryotic cell lines that express the chosen tyrosine kinase cDNA, in its wild type and mutated forms, and by characterizing their phenotypes. The DNA and antibody probes generated in the course of the proposed project will be useful for further studies leading to a detailed characterization of protein tyrosine kinases and their role in embryonic development. The cloning and characterization of novel protein tyrosine kinases may also contribute to the recognition of new oncogenes encoding tyrosine kinases and of their roles in the events leading to oncogenic transformation.

Protein tyrosine kinases are thought to be involved in the processes of cell division, migration and differentiation occurring during embryonic development, but their physiological functions, regulatory properties and mechanism of action are largely unknown. Proteins phosphorylated on tyrosine have been detected in embryonic tissues using antibodies specific for phosphotyrosine residues. However, a large fraction of the protein tyrosine kinases involved in developmental processes may not be known at this time. Using anti-phosphotyrosine antibodies to screen chicken embryo cDNA libraries will allow the isolation of the tyrosine kinase genes that are expressed during development. The cloned protein tyrosine kinases will then be identified by sequencing the appropriate cDNAs and characterized using specific polyclonal antibodies to detect them in embryonic tissues or cultured cells. Antibodies will be prepared by injecting the cloned kinases expressed in bacteria as fusion proteins and used in immunoprecipitation, immunoblotting and immunofluorescence microscopy experiments. Finally, the effects that one of the newly identified tyrosine kinases has on cellular growth and differentiation will be investigated by producing eukaryotic cell lines that express the chosen tyrosine kinase cDNA, in its wild type and mutated forms, and by characterizing their phenotypes. The DNA and antibody probes generated in the course of the proposed project will be useful for further studies leading to a detailed characterization of protein tyrosine kinases and their role in embryonic development. The cloning and characterization of novel protein tyrosine kinases may also contribute to the recognition of new oncogenes encoding tyrosine kinases and of their roles in the events leading to oncogenic transformation.

Neoplastic transformation is usually caused by multiple independent lesions that interfere with normal cell proliferation and differentiation. Abnormalities in tyrosine kinase genes are often implicated in the generation of malignant phenotypes. The broad long term objective of this proposal is to characterize a novel receptor-type tyrosine kinase (Cek5) that we have recently identified as part of a systematic survey of developmentally expressed protein tyrosine kinases. Its amino acid sequence indicates that Cek5 is a member of the Eph family, which at present represents the least characterized of the major families of integral membrane tyrosine kinases. Cek5 is expressed primarily in the adult central nervous system and during embryonic development. The cellular and subcellular distribution of Cek5 in the central nervous and in other tissues will be investigated using immunoelectron microscopy and in situ hybridization techniques. The normal and pathological functions of Cek5 as an intermediate signal transduction pathways will be investigated by characterizing its ligand and substrates. The ligand will be partially purified using conventional chromatography and its activity assayed using cells transfected with Cek5 cDNA. Substrates will be studied by taking advantage of their expected association with the activated Cek5 kinase and phosphorylation on tyrosine. To gain insight into the regulation of Cek5 gene expression, the genomic organization of the Cek5 locus will be determined. The oncogenic potential of Cek5 will be examined by over expressing normal and modified Cek5 cDNAs in eukaryotic cells and by investigating the expression of Cek5 in various tumor cell lines. Cek5 produced in insect cells and monoclonal antibodies specific for Cek5 will represent useful reagents to accomplish the studies proposed. Since tyrosine kinase genes are highly conserved during evolution, the characterization of Cek5 and the preparation of Cek5-specific reagents will also be useful for future studies focused on the possible role of Cek5 in: (i) human cancers and other pathological conditions, (ii) embryonic development, and (iii) nerve regeneration.

Cek8 (Chicken embryo kinase 8) is a recently identified receptor-type tyrosine kinase of the Eph subclass which is preferentially expressed in neural tissues. The extracellular domain of Cek8 contains motifs characteristic of cell adhesion molecules, while the cytoplasmic domain comprises a typical tyrosine kinase catalytic domain. Preliminary data indicate that Cek8 and its mammalian homologs may function to regulate cellular interactions and axonal growth in the developing nervous system. Furthermore, Cek8 may be implicated in the formation of tumors of the nervous system. The objectives of the proposed research are to provide insight into the neural functions of Cek8 by using a variety of different molecular approaches. The cellular and subcellular localization of Cek8 in the nervous system of the developing embryo and in dissociated neural cultures will be studied in detail, together with the developmental regulation and level of catalytic activation of Cek8 in vivo. For example, the possibility that Cek8 may be concentrated in growth cones and implicated in axonal guidance will be explored. These studies will provide the initial clues about the functions of Cek8. The molecular components of the signaling pathways in which Cek8 is implicated will be dissected by investigating the interactions of Cek8 with molecules that have a similar localization in the nervous system. Neuronal adhesion molecules, proteoglycans and cytoplasmic tyrosine kinases are among the known molecules that may participate in functional interactions with Cek8. Other molecules interacting with Cek8, such s ligands or substrates, will also be searched for based on their ability to bind to Cek8. Finally, the expression and activity of Cek8 will be perturbed with antibodies, a dominant negative form of Cek8, Cek8 antisense sequences and, if identified, the Cek8 activating lignad. These studies will be performed in vivo and in vitro to address directly the functions of Cek8, particularly during axonal growth. The characterization of Cek8 and of the molecules that interact with it, which will be addressed in the proposed studies, will provide the information necessary to manipulate the neural signal transduction pathways in which Cek8 is implicated. Thus, the studies proposed are likely to form the basis for the development of treatments to ameliorte specific developmental neural pathologies and degenerative diseases of the nervous system.

The Eph subfamily represents the largest known group of receptor-type tyrosine kinases. The extracellular domains of the members of the Eph subfamily contain motifs characteristic of cell adhesion molecules, while the cytoplasmic domains comprise typical tyrosine kinase catalytic domains. The Eph-related kinases are preferentially expressed during embryonic development rather than in adult tissues. A common characteristic of many Eph-related kinases is the predominant expression in the nervous system. Presumably the Eph-related kinases are all derived from the same ancestral gene, and as members of a gene family they share common general characteristics. Different members of the Eph subfamily presumably carry out related, but specialized functions. The widespread expression, distinctive tissue distribution and developmental regulation of the kinases of the Eph subfamily suggest multiple key roles in development. The broad long term objectives of the proposed research are to elucidate the developmental roles of the Eph gene family as a whole and identify the specialized roles of individual Eph-related kinases by using a variety of different molecular approaches. The specific aims of this proposal will focus on Cek9 as the prototype of the Eph subfamily. Cek9 is developmentally regulated, expressed in embryonic neural as well as non- neural tissues, but undetectable in most adult tissues, with the exception of the thymus, retina and brain. Its expression in developing neuronal processes suggests that Cek9 may be important in axon growth or pathfinding. A distinctive characteristic of Cek9 compared to other Eph- related kinases is its ubiquitous phosphorylation on tyrosine in chicken embryonic tissues during specific developmental stages. Since enhanced phosphorylation on tyrosine of Eph-related kinases is indicative of catalytic activation, its in vivo phosphorylation suggests that Cek9 is highly active and thus plays an important role during development. The expression pattern of Cek9, and its alternatively spliced and phosphorylated forms, will be further characterized and compared to that of potential ligand(s) and substrates. The molecular components of the signaling pathways in which Cek9 is implicated will be dissected by searching for the molecules that functionally interact with Cek9. The Cek9 ligand(s) and cytoplasmic targets which are identified will also be examined for their ability to interact with other Eph-related kinases. Finally, the developmental role of Cek9 will be investigated by gene knock out through homologous recombination. These studies will elucidate whether abnormalities in the Eph subfamily of tyrosine kinase genes are likely to be the cause of any human congenital disease or developmental pathology. The identification of the cellular processes that are regulated by the Eph-related kinases, which may include cell fate determination, cell proliferation and differentiation, nerve cell survival and regeneration, and axon growth and pathfinding, will allow the manipulation of such processes with possible therapeutic outcomes.

The Eph subfamily represents the largest branch of membrane-spanning tyrosine kinases known to date. Because it has been identified only recently, it has not yet been extensively characterized. The broad long term objectives of this proposal are to elucidate the functions of the Eph subfamily of tyrosine kinases during retinal morphogenesis and in the mature retina. These studies will suggest possible implications of the Eph-related kinases in the etiology of pathological conditions affecting the retina. The retina also represents a convenient model system to elucidate the roles of the Eph-related kinases in the central nervous system. Different Eph-related kinases have distinct tissue distributions in the adult and, thus, presumably have distinct functions. At least six of the Eph-related kinases that we have identified, namely Cek4, Cek5, Cek6, Cek8, Cek9 and Cek10, are expressed in the developing and adult retina suggesting an important role for the Eph subfamily in the retina. The catalytic activity of the Eph-related kinases is likely to be responsible, at least in part, for the elevated protein tyrosine phosphorylation that is observed both in the developing and mature retina. Cek5, for example, is highly expressed and catalytically active during the differentiation of the retina. This suggests that Cek5 plays an active role during retinal morphogenesis and that mutations of the Cek5 gene are likely to disrupt the normal development of the retina. In addition, the gradient-like distribution of Cek5 in the retina and optic nerve indicates that Cek5 may be involved in the determination of the positional identity and synaptic specificity of retinal ganglion cells. Similarly, the elevated expression of Cek8 throughout the developing optic nerve suggests a role in axonal growth and guidance. To gather information about their possible functions, the spectrum of Eph-related kinases that are expressed in the retina will be identified and their expression levels, spatial distribution and extent of activation in the developing and mature retina will be characterized. The expression and activity of specific Cek Eph- related kinases will be perturbed with antibodies, dominant negative forms, anti sense sequences and ligands. These studies will be performed in vivo and in vitro to address directly the functions of the Eph subfamily in the retina. The identification of the cellular processes that are regulated by the Eph-related kinases, which may include cell fate determination, cell proliferation and differentiation, nerve cell survival and regeneration, cell adhesion and axonal guidance, will allow the manipulation of such processes with possible therapeutic outcomes.

DESCRIPTION (Adapted from applicant's abstract): There are three specific aims in the present proposal. Aim 1 seeks to determine whether the complementary distribution of EphB2 and its ligand, ephrin B1 regulate dorso-ventral patterning in the retina. Ephrin B1 is in dorsal retina and EphB2 is in ventral retina. Studies include: (a) in vivo misexpression using RCAS virus, using ephrin-B1DC +/- EGFP and EphB2DC and examining retinas between E6 and E16. The investigator(s) will look for growth abnormalities and axon patterning using DiI labeling. (b) in vivo misexpression using replication incompetent virus (RSV based with lacZ and internal ribosome entry site (IRES) to misexpress Eph B2 in hostile ephrin B1 domain and vice versa to test the hypothesis of bidirectional signaling, and (c) in vitro assays with cultured retinal cells and transfected lines. The investigator has generated CHO cells transfected with EphB2 and will generate ephrin-B1 expressing cells. The investigator(s) will look for segregation between them and patching of receptors/ligands and will analyze both directional migration of retinal cells in transfilter assays and growth cone collapse. Aim 2: Eph A3 is expressed in posterior retina and ephrin A5 in anterior retina. The investigator will determine whether the complementary pattern regulates AP (anterior-posterior) patterning of visual structures. Studies include: (a) misexpression of EphA3 and ephrin A5 using replication incompetent viruses, (b) in vitro assays using CHO cells transfected with EphA3 or ephrin A5, and (c) examination of directional migration and axon collapse. Aim 3 will determine whether the polarized tyrosine phosphorylation of EphA4 regulates patterning. Preliminary results show that EphA4 and B2 exhibit a polarized pattern of phosphorylation in retina. A4 expression is high dorsoanteriorly and low ventroposteriorly. Studies include (a) alteration of activation using a constitutively active myc-EphA3 lacking most of extracellular domain inserted into RCAS and myc-ephrin A5 and (b) examination of axon trajectories using DiI.

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, regulate cell movement and neuronal axon guidance during development, contributing to the establishment of the embryonic body plan. Eph/ephrin signaling pathways, however, are poorly understood. A unique feature is that binding of Eph receptors to transmembrane ephrins activates signals mediated by the ephrin cytoplasmic domain. EphB2 with ephrin-B1 results in tyrosine phosphorylation of the cytoplasmic ligands. Interaction of EphB2 with ephrin-B1 results in tyrosine phosphorylation of the cytoplasmic domains of both proteins. Effector proteins that bind to distinct tyrosine phosphorylated sequences propagate EphB2/ephrin-B1 signals by activating cytoplasmic signaling cascades. Ultimately, the signaling pathways activated by the EphB2/ephrin-B1 complex cause changes in cytoskeletal organization, cell-cell adhesion, and cell-matrix adhesion. Our goal is to characterize at the molecular level the signaling pathways activated by EphB2 and ephrin-B, and understand how they control the behavior of both EphB2 expressing cells and ephrin-B1 expressing cells. This will be accomplished by (i) mapping the tyrosine- phosphorylated sequence motifs that are created upon formation of the EphB2/ephrin-B1 complex; (ii) identifying signaling effectors that interact with the EphB2/ephrin-B1 complex; and (iii) dissecting EphB2 and ephrin-B1 signaling pathways that underlie different biological motifs of EphB2 and ephrin-B1 to specific biological activities, and contribute to elucidate how these Eph family proteins govern tissue patterning. These studies will also provide the knowledge necessary for modulating EphB2/ephrin-B1 signals when their malfunction causes disease. Aberrant EphB2/ephrin-B1 signaling could play a role in congenital neurological disorders and organ malformations, as well as other diseases involving tissue disorganization, including malignant tumor progression.

DESCRIPTION (provided by applicant): Communication between glial cells and neurons is emerging as a critical parameter of synaptic function. However, the molecular mechanisms that glial cells use to modify synaptic structure and physiology are poorly understood. We have found that the receptor tyrosine kinase EphA4 has remarkably high expression in the adult hippocampus, a major brain center for learning and memory. EphA4 is discretely localized on dendritic spines of hippocampal pyramidal neurons. Dendritic spines are small protrusions on the surface of neurons that mediate contact with presynaptic excitatory terminals and are believed to be important for memory and cognitive processes. Spines can undergo geometrical remodeling, which has been linked to physiological effectiveness. However, the factors that regulate spine motility and organization remain unclear. Interestingly ephrin-A3, a ligand that stimulates the signaling ability of EphA4, is expressed on the surface of glial processes that surround dendritic spines. Furthermore, activation of EphA4 induces spine retraction whereas inhibiting ephrin/EphA4 interaction distorts spine shape and organization in hippocampal slices. Spine irregularities in EphA4 knock-out mice and hippocampal neurons expressing a kinase-inactive form of EphA4 further indicate that EphA4 signaling is critical for spine morphology. Thus, our data support a model where ephrin-A3/EphA4 receptor signaling between glia and neurons regulates the shape and organization of dendritic spines. Our goal is to characterize this novel form of cross-talk between glial cells and neurons. We will use organotypic cultures of hippocampal slices as a model to examine the effects of EphA4 activity on dendritic spine morphology and identify the signaling pathways activated downstream of EphA4 in dendritic spines. We will also examine whether other EphA receptors as well as EphB receptors function in concert with EphA4 to impart changes in dendritic spine morphology and organization. This project will elucidate a new and intriguing mechanism that could be critical for modulating synaptic function and perhaps the processes of learning and memory formation. Furthermore, it may clarify the neurological aspects of diseases characterized by abnormal dendritic spine structure, such as mental retardation, William's syndrome, Down's syndrome, and autism.

DESCRIPTION (provided by applicant): This program project application focuses on signaling networks that regulate cell survival and motility/invasion. Eight Burnham Institute laboratories participate, and multidisciplinary approaches will be used. Cell survival and motility signals are intimately connected and can be coordinately reprogrammed, for example by certain transforming mutations in cancer. Many of these pathways culminate in activation of the small GTPase Rac and the downstream Jun N-terminal kinase (JNK) pathway. Rac is a central coordinator of the actin cytoskeleton and controls both cell migration/invasion and JNK. Depending on the circumstances, JNK can either support cell survival or promote cell death, and can also affect invasiveness through matrix metalloproteinase expression. Dr. Vuori will work on the regulation of Rac by the adaptor protein Crk and its partner DOCK180. Dr. Pasquale will study SHEP1, which binds receptor tyrosine kinases, Ras GTPases that promote integrin activity, and the docking protein p130Cas. p130Cas functions upstream of the Crk/DOCK180 complex, and mediates integrin-induced Rac and JNK activation. Thus, SHEP1 may integrate diverse cellular signals related to cell survival, adhesion, and migration. Dr. Ruoslahti will examine an adhesion-dependent survival pathway that involves the novel mitochondrial protein Bit1, which promotes cell death when is released in the cytoplasm and forms a complex with the transcriptional regulator AES. The regulation of the Bit1/AES complex and its relationship with other survival/apoptosis pathways will be investigated. Dr. Feng will study the docking protein Gab1, which he has recently implicated in JNK activation and apoptosis caused by UV irradiation. Gab1 may be a switch that turns on the pro-apoptotic mode of JNK activation. Two core components will enable specialized functional studies, one in structural biology of protein-protein interfaces and the other in genetic mouse tumor models. The diverse expertise of the participating laboratories and the resources provided by the cores will synergistically contribute to the characterization of novel signaling connections and the discovery of strategies to control cell survival and invasion pathways that are deregulated in cancer. These studies may allow future development of small molecules that can disrupt pathologic signals by targeting protein interfaces.

Rho-family of GTPases, such as Rac, are central coordinators of signaling pathways that regulate actin cytoskeleton, cell survival and cell motility. Integrins, which are receptors for extracellular matrix proteins, regulate Rac in two ways. First, integrins control the GTP loading of Rac. Second, integrins regulate membrane targeting of GTP-loaded, activated Rac. Both of these events are essential for Rac to activate downstream effectors. Our preliminary data demonstrate that the adapter protein Crk and its binding partner DOCK180 are crucial for both of the regulatory steps in the activation of Rac signaling. In the present application, we will elucidate the mechanisms of the Crk/DOCK180 complex leading to Rac activation. In Aim 1, we will investigate the mechanism by which integrins control the membrane targeting of active Rac. Our preliminary studies indicate that Crk and Rac become recruited to lipid rafts upon integrin ligation and that this recruitment is essential for activation of Rac signaling. We will investigate the mechanisms of Crk/Rac localization to lipid rafts and characterize the events through which cell adhesion regulates lipid rafts. It is anticipated that these studies will reveal novel aspects of not only integrin signaling, but also of lipid raft regulation and function. In Aim 2, we will study the molecular mechanism by which GTP loading and activation of Rac is controlled. Our preliminary data demonstrate that DOCK180 functions as a novel guanine nucleotide exchange factor (GEF) for Rac. In this aim, a multidisciplinary approach ranging from cellular to structural biology will be undertaken to elucidate the regulation of DOCK180 activity in detail. At present, nothing is known about the function of mammalian DOCK180 in vivo. Our goal in Aim 3 is to determine the physiological role of DOCK180 by generating a DOCK180-deficient mouse model. Subsequent studies in mouse cancer models will address the significance of DOCK180 in tumor initiation and progression in vivo. This work combined with studies proposed in the other two aims will enable us to fully understand the mechanisms and significance of the Crk/DOCK180 complex in regulating Rac signaling. These studies will contribute to our fundamental understanding of how integrins regulate Rho GTPases. These regulatory events are central to cell migration, survival and growth in normal human physiology and diseases, such as cancer.

Animal cells depend on signals from the extracellular matrix and growth factors to survive, proliferate, and find their position in the organism. De-regulation of these signals is a hallmark of malignant cell transformation. SHEP1 is the prototype of a novel family of cytoplasmic proteins that likely coordinate survival and migratory signals from extracellular matrix receptors (integrins) and growth factor receptor tyrosine kinases. SHEP family proteins contain a bifunctional domain that binds both Ras proteins that positively regulate integrins and the docking protein p130Cas (Cas), which transmits signals downstream of integrins. In addition, SHEP proteins bind activated receptor tyrosine kinases. Our goal is to examine the molecular mechanisms underlying SHEP 1 function in intracellular signaling. SHEP 1 activates the c-Jun N-terminal kinase (JNK) and the nuclear factor (NF)-kB transcription factor. Through these pathways, which are involved in adhesion-dependent cell survival, SHEP1 may contribute to making cells more resistant to environmental insults such as detachment from the extracellular matrix and exposure to cytotoxic agents. Furthermore, SHEP 1 promotes the formation of membrane ruffles, suggesting a role in cell migration and invasion. Cell culture models will be used to examine how SHEP 1 activates JNK and NF-kB and whether the SHEP 1-Cas complex regulates cell adhesion and motility by linking integrins and receptor tyrosine kinases. X-ray crystallography and NMR spectroscopy will define the SHEP1 protein interfaces involved in Cas and Ras proteins binding, which we hypothesize to be critical for SHEP1 function. In a complementary approach, phage display will identify peptides that inhibit SHEP1 binding to Cas and Ras proteins by targeting these interfaces. Finally, conditional inactivation of the SHEP 1 gene in conjunction with mouse mammary tumor models will elucidate the role of SHEP 1 in cell survival/proliferation and migration/invasion in vivo in normal tissues and in tumors. This multidisciplinary approach is made possible by collaborations among the program laboratories and the resources provided by the cores. This project may yield new insight into the signaling networks that protect cells from apoptosis and promote motility and invasion, and elucidate the role of SHEP family proteins in these networks.

DESCRIPTION (provided by applicant): Damaged neuronal connections in the adult central nervous system (CMS) do not regenerate. This is in part due to the physical barrier formed by glial cells that respond by proliferating and forming a scar and to the presence of inhibitory molecules in the CMS environment. Several molecules have been implicated in the failure of the CMS to regenerate, but inhibiting the function of these molecules so far has produced modest improvements. Therefore, other factors must be important. Recent work has shown that the EphA4 receptor tyrosine kinase plays a critical role in the inhibition of axon regeneration that occurs after spinal cord injury. Remarkably, axons in EphA4 knockout mice can regenerate past the site of injury and re-establish severed connections resulting in functional recovery. Other evidence suggests that EphA4 plays an inhibitory role in axonal and dendritic growth in other regions of the central nervous system as well. Thus, inhibiting EphA4 function is a very promising new approach to promote regeneration in the CMS, with high potential for a number of therapeutic applications. However, EphA4 has not yet been exploited as a target for small molecules. The signaling activity of EphA4 is stimulated by binding several membrane-anchored ligands, called ephrins. As we have recently shown, peptides that antagonize ephrin binding without stimulating the signaling ability of EphA4 block the physiological activity of the receptor. In this application we propose to develop an assay system that can be used for high throughput screening of small molecule antagonists for EphA4, perform a pilot screen, and develop assays to characterize the potency and selectivity of the active compounds identified. The methodology developed will in the future allow full scale high throughput screens for small molecule EphA4 antagonists and their subsequent characterization. An advantage of reagents that target the extracellular domain of EphA4 is that they can be highly selective, unlike most kinase domain inhibitors, and that they can act without having to penetrate inside the cell. A particularly promising therapeutic application of EphA4 antagonists is in spinal cord injury, where these reagents could be applied locally to improve functional recovery.

Affect; Ammon Horn; Astrocytes; Astrocytus; Astroglia; Autism; Autism, Early Infantile; Autism, Infantile; Autistic Disorder; Axon; Brain; CSPG4; CSPG4 gene; Cek-8 Kinase; Cell Communication and Signaling; Cell Function; Cell Process; Cell Signaling; Cell physiology; Cellular Function; Cellular Physiology; Cellular Process; Chromosome Pairing; Communication; Cornu Ammonis; Dendrites; Dendritic Spines; Development; Disease model; Dysfunction; EPH; EPHA1 Protein; EPHA1 Receptor Tyrosine Kinase; EPLG3; Encephalon; Encephalons; Eph Family Receptors; Eph Receptor; Eph Receptor Ligands; Eph Receptor Tyrosine Kinase; Eph Receptors; Eph-A4 Receptor Tyrosine Kinase; EphA1 Receptor; EphA4 Protein; EphA4 Receptor; Ephrin Receptor A4; Ephrin Receptors; Ephrin-A3; Ephrins; Epilepsy; Epileptic Seizures; Epileptics; Epl3 (Protein); Functional disorder; Genes; Genetic Alteration; Genetic Change; Genetic defect; Glia; Glial Cells; Goals; Head; Heparan Sulfate; Heparitin Sulfate; Hippocampus; Hippocampus (Brain); Intracellular Communication and Signaling; Kanner's Syndrome; Knockout Mice; Kolliker's reticulum; LERK-3; LERK-3 Protein; Learning; Ligands; Link; MCSP; MCSPG; MEL-CSPG; MSK16; Mediating; Memory; Mental Retardation; Mice, Knock-out; Mice, Knockout; Mutation; NG2; NG2 antigen; NG2 proteoglycan; NRVS-SYS; Neck; Nerve Cells; Nerve Unit; Nervous System; Nervous System Diseases; Nervous System, Brain; Nervous system structure; Neural Cell; Neural Transmission; Neurocyte; Neuroglia; Neuroglial Cells; Neurologic Body System; Neurologic Disorders; Neurologic Organ System; Neurological Disorders; Neurons; Non-neuronal cell; Null Mouse; Numbers; PTK Receptors; Peripheral Nervous System; Physiology; Physiopathology; Play; Property; Property, LOINC Axis 2; Pyramidal neuron; RTK; Receptor Protein-Tyrosine Kinases; Receptor, EphA1; Receptor, EphA4; Receptors, Eph Family; Regulation; Research; Role; Seizure Disorder; Sek-1 Receptor Tyrosine Kinase; Series; Shapes; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Spinal Column; Spine; Structure; Subcellular Process; Surface; Synapses; Synapsis; Synapsis, Chromosomal; Synaptic; Synaptic Transmission; Synaptic plasticity; Transmembrane Receptor Protein Tyrosine Kinase; Tyrosine Kinase Growth Factor Receptor; Tyrosine Kinase Linked Receptors; Tyrosine Kinase Receptors; Vertebral column; backbone; biological signal transduction; dendrite spine; disorder model; epilepsia; epileptiform; epileptogenic; genome mutation; hippocampal; hippocampal pyramidal neuron; long term memory; nerve cement; nervous system disorder; neurological disease; neuronal; pathophysiology; prevent; preventing; size; social role; synapse formation; synaptogenesis; tool

DESCRIPTION (provided by applicant): The Eph receptor tyrosine kinases have emerged as a new important family of cancer targets. Studies from several groups, including one from this program, have shown that disrupting the binding of Eph receptors with their ligands, the ephrins, inhibits tumor growth in preclinical mouse tumor models. Eph receptors that are upregulated in cancerous tissue can also be exploited for targeted drug delivery to tumors. Although binding interactions between Eph receptors and ephrin ligands are highly promiscuous, collaborative work between laboratories from this program has revealed that artificial ligands such as peptides and small molecules can bind selectively to the ephrin-binding pocket of different Eph receptors. Thus, specific targeting of individual Eph receptors can be achieved. However, only a few agents that inhibit ephrin binding have been identified so far. This program project aims to define the structural features conferring high affinity and controlling selectivity versus promiscuity of ligand binding to the Eph receptors as well as to optimize existing small molecule and peptide leads that inhibit Eph receptor-ephrin interaction. The anticancer effects of the optimized molecules will be evaluated using culture models and in vivo preclinical mouse cancer models. Close collaboration among the three participating laboratories, which have complementary expertise in Eph receptor biology and signal transduction, X-ray crystallography and biophysics, and NMR-based drug design and chemistry, will enable achievements that are beyond the immediate reaches of each individual component. Component 1 will evaluate strategies to modulate Eph receptor function in cancer cells and endothelial cells using chemical compounds and peptides optimized through the combined efforts of the program. Component 2 will use X-ray crystallography to characterize with high resolution the interfaces of Eph receptors in complex with high affinity peptide and small molecule ligands in comparison with the natural ephrin ligands. Component 3 will use NMR to characterize binding interfaces of Eph receptor domains in complex with chemical compounds and thus provide structural information enabling their optimization. Component 3 will also develop peptide-drug conjugates to selectively target cells expressing the EphA2 receptor in tumors, an approach complementary to using agents that interfere with Eph receptor/ephrin biological activities. The information obtained from the proposed studies is expected to enable development of new ways to effectively target the ephrin-binding pocket of Eph receptors using chemical compounds and peptides.

DESCRIPTION (provided by applicant): Breast cancer is the second leading cause of cancer deaths in women, despite a number of treatment options. Most breast cancers are estrogen receptor-positive and commonly treated with anti-estrogens, while antibodies and kinase inhibitors are used to treat cancers that overexpress the HER2 receptor tyrosine kinase. Unfortunately, tumors are often intrinsically resistant or develop resistance to these drugs due to overexpression of specific genes. One of these genes is the scaffolding protein BCAR1 (breast cancer anti- estrogen resistance 1 or p130Cas), which is a well-known key component of the signaling pathways that regulate cell proliferation, survival and migration/invasion downstream of integrin adhesion receptors. BCAR1 is also a critical node in HER2 oncogenic signaling pathways and mediator of resistance to adriamycin, a drug frequently used to treat breast cancers that do not respond to anti-estrogens or HER2-targeted therapies. Thus, BCAR1 is a central player in the signaling networks that control breast cancer malignancy. Another important but poorly characterized factor in anti-estrogen resistance is the SH2 domain-containing protein BCAR3, a member of a family of three proteins that also includes SHEP1, which we identified in a screen for Eph receptor tyrosine kinase-binding molecules. Members of the BCAR1 and BCAR3 families function in a concerted manner through direct association of their C-terminal regions, but the structure of their complexes has eluded investigators. To gain insight into these crucial yet enigmatic assemblies, we have solved the first crystal structure of a BCAR1-BCAR3 family complex, that of BCAR1 and SHEP1, revealing a novel type of protein interaction. The SHEP1 C-terminal region overall resembles a Cdc25-type guanine nucleotide exchange factor domain. However, crucial regions are altered by BCAR1 binding, resulting in a closed conformation of SHEP1 that cannot bind Ras GTPases. We now seek to unravel the mechanistic basis of breast cancer malignancy and resistance to chemotherapy mediated by BCAR1 association with BCAR3, a protein related to SHEP1 but with distinct differences in key features. Thus, we propose a multidisciplinary approach that integrates a spectrum of effective tools ranging from high resolution structural and biochemical analysis to functional studies in engineered cancer cells in culture and mouse breast cancer xenograft models. Our goals will be accomplished by (1) unraveling the molecular details of the BCAR1-BCAR3 signaling association to precisely define the regulatory modifications occurring upon complex formation; and (2) elucidating the role of the BCAR-BCAR3 association in breast cancer growth, invasiveness and resistance to chemotherapy. Our studies will lead to a new understanding of the BCAR1-BCAR3 signaling node as well as complexes of related proteins, and shed light on how breast cancer cells acquire the malignant characteristics that enable them to metastasize and cope with chemotherapeutic insults. Thus, this work will ultimately provide a new basis for developing treatment options to overcome cancer drug resistance.

DESCRIPTION (provided by applicant): Among all cancers, metastatic melanoma is one of the most deadly. In most cases melanoma develops due to multiple gene mutations induced in skin melanocytes by exposure to UV light, but few of these mutations have been studied in detail. Most melanomas harbor mutations that constitutively activate the RAS-RAF-MEK-ERK pathway, such as the frequent NRAS and BRAF mutations, which are well known to play a critical role in melanoma malignancy. However, mutations in other pathways such as the PTEN, TP53 or CDKN2A tumor suppressors are also needed for melanoma development. Furthermore, recent studies have uncovered new mutations in other gene families that contribute to melanoma malignancy. The objective of the proposed research is to explore the novel hypothesis that Eph receptor mutations play a functional role in melanoma development and/or progression. Recent sequencing studies of ~300 melanoma specimens and cell lines have revealed that approximately half of the tumors carry one or more nonsynonymous mutations in genes of the Eph receptor tyrosine kinase family. The Eph receptors regulate many fundamental biological and pathological processes, such as the growth and invasiveness of cancer cells, and several of them have been implicated in melanoma pathogenesis. Various in silico analyses predict that many of the Eph receptor mutations contribute to malignancy, consistent with our preliminary studies showing that several EphA2 and EphA3 melanoma mutations analyzed so far drastically affect receptor functional properties. We therefore propose two aims to explore the functional significance of Eph mutations in melanoma. In Aim 1, we will characterize a subset of the mutations identified to determine if they affect the abilit of Eph receptors to perform their normal functions, including trafficking to the cell surface, binding ephrin ligands, assembling into receptor signaling clusters, and fulfilling their role as tyrosine kinases. We will especially focus on Eph receptors that are either known or very likely to play a role in melanoma pathogenesis and on the mutations most likely to be driver mutations. In Aim 2, we will investigate how representative Eph mutations affect melanoma cell development and/or malignancy by introducing mutant and wild-type receptors in melanocytes and melanoma cells, and measuring cell proliferation/survival and migration/invasiveness. Surveying the functional effects of Eph receptor mutations occurring in melanoma will provide valuable information on how melanoma cells acquire their malignant properties and on the involvement of the Eph receptor family in cancer development and progression, which is currently incompletely understood. This information, and the collection of research tools to be generated, will also enable future more extensive studies to characterize the importance of normal as well as mutated Eph receptors in additional cell culture and in vivo melanoma models. Cataloguing the effects of Eph gene mutations may also prove useful for identifying novel biomarkers as well as genomic alterations to help prognosis and the design of individualized therapies for melanoma patients.

? DESCRIPTION (provided by applicant): The receptor tyrosine kinase EphA4 has been implicated in several neurodegenerative processes. Strikingly, a recent study has shown that low EphA4 expression and loss-of-function mutations are linked to late onset and prolonged survival in amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative illness for which there is no cure and the only FDA-approved therapy can increase survival by only several months. Even partial EphA4 gene inactivation has shown beneficial effects in animal models of ALS, making inhibition of EphA4 function an attractive strategy to counteract neurodegeneration. Accordingly, a peptide that we previously discovered as a selective EphA4 antagonist has shown promise in the classic rat SOD1 G93A ALS model, where it significantly dampened ALS pathogenesis demonstrating the therapeutic potential of EphA4 antagonistic agents. Despite these exciting results, our first generation EphA4 antagonists lack the required potency and stability in biological systems to make them suitable as leads for drug development. Thus, in this application we propose to develop and evaluate new potent EphA4 antagonists with pharmacological properties suitable to make them bona fide therapeutic leads for future treatment of ALS. In our preliminary work, we have developed a derivative of a cyclic dodecapeptide that specifically inhibits EphA4-ephrin binding with an IC50 value of ~25 nM. This prototype is a striking 80 fold more potent than any previously known EphA4 antagonist and has the potential for optimization to become a desired therapeutic lead. To develop related cyclic EphA4 antagonists towards this goal, we propose a highly integrated iterative strategy using peptide medicinal chemistry, structural biology and our long-standing expertise in EphA4 neurobiology. The activities of rationally designed new peptide antagonists will be evaluated using biochemical assays and neuronal cell culture models. Complementing these studies, our most promising new EphA4 antagonists will be profiled for their properties in plasma and cerebrospinal fluid and their propensity to cross the blood-brain barrier, as well as their half-lie in the blood circulation. Finally, in vivo studies in collaboration with renowned ALS expert Dr. Wim Robberecht will characterize the best antagonists in delaying disease onset and promoting survival in an ALS mouse model. We anticipate that the proposed studies will result in a potent EphA4 inhibitor with a pharmacologic profile suited for immediate development as a therapeutic agent alone or in combination with other treatments. They will also provide valuable insight into the mechanism underlying the benefits of EphA4 inhibition against neurodegeneration.

SUMMARY The mechanism behind the phenomenon of ligand functional selectivity?, defined as the ability of different ligands to differentially activate distinct signaling pathways through a common receptor, is not understood for receptor tyrosine kinases. Here we seek to obtain such mechanistic information for EphA2, a transmembrane receptor tyrosine kinase that is critically important for human health. We will investigate EphA2 signaling responses to three ligands: dimeric ephrinA1-Fc, monomeric m-ephrinA1, and the engineered monomeric YSA peptide ligand using biochemical and biophysical approaches in the context of live cells. We have already discovered differences in the extracellular configuration of the EphA2/YSA, EphA2/m- ephrinA1 and EphA2/ephrinA1-Fc oligomers in live cells, and in Aim 1 we will investigate whether these differences are transmitted across the plasma membrane, leading to differences in the configuration of the EphA2 intracellular regions. In Aim 2, we will compare EphA2 signaling responses to the three ligands to test the hypothesis that differences in downstream signaling correlate with differential phosphorylation of some of the EphA2 tyrosines and/or different kinetics of EphA2 activation/inactivation. This work will advance the general understanding of receptor tyrosine kinase signal transduction, and inform the design of ?pathway-biased? targeted drugs with improved specificity and safety profiles.

Alzheimer?s is a devastating disease involving chronic, progressive neurodegenerative processes and decline in brain cognitive function that ultimately lead to death. Accumulation of aggregates of amyloid-beta and hyperphosphorylated tau protein in the brain causes synaptic dysfunction and loss of neurons, although the precise mechanisms underlying the disease remain unknown. There are few drugs available to treat Alzheimer?s disease, and they provide only limited benefits. In fact, the economic damages from Alzheimer?s disease reach hundreds of billion dollars every year in the US alone. Many candidate drugs have been evaluated in clinical trials in the last decade, but none has been approved. Thus, it is important to identify new drugs based on novel targets. The EphA4 receptor tyrosine kinase has recently emerged as a novel promising target for counteracting neurodegeneration and cognitive deficits in Alzheimer?s disease and other neurodegenerative diseases. EphA4 receptor signaling can promote neurotoxicity when aberrantly induced by amyloid-beta oligomers and ephrin ligands. The kinase domain of EphA4, which is responsible for the neurotoxic effects, is a druggable target. However, small molecule kinase inhibitors targeting EphA4 with selectivity and high affinity remain to be identified. Here we propose to perform two high-throughput screening campaigns of EphA4. One screen will deploy Protein Thermal Shift (PTS) as the assay format and the other screen will deploy a kinase activity assay format. The goal of these screens is to identify as broad a panel of modulators of the kinase-dependent functions of EphA4 as possible. Use of two formats should maximize our ability to identify a large repertoire of modulators with diverse modes of action and ultimately develop an inhibitory compound with high selectivity for EphA4. We anticipate that the PTS screen will identify compounds that stabilize the inactive conformation of the EphA4 intracellular region by binding not only to the kinase domain but also to regulatory regions outside this domain. The second screen will deploy a novel in vitro kinase assay configuration we have developed, where the inactive EphA4 intracellular region serves as the physiological substrate for the catalytically competent EphA4 intracellular region. We anticipate that this assay will identify not only compounds that target the EphA4 ATP binding pocket, but also allosteric inhibitors with novel mechanisms of action. The top hits identified in the two screens and their derivatives will be characterized and improved through rounds of secondary and tertiary biochemical and cell-based assays already established in the laboratories of the PIs and their collaborators, complemented by structure-guided approaches such as in silico docking and X-ray crystallography. These assays will provide formal hit validation, selectivity profiling and initial characterization of mechanisms of action and activities in neurons. We anticipate that this project will result in the identification of several novel EphA4 selective inhibitors that are suitable for advancement to future studies in animal models of Alzheimer?s and other neurodegenerative diseases.