RESUMO
The tyrosine phosphatase Shp2 is recruited into tyrosine-kinase signalling pathways through binding of its two amino-terminal SH2 domains to specific phosphotyrosine motifs, concurrent with its re-localization and stimulation of phosphatase activity. Shp2 can potentiate signalling through the MAP-kinase pathway and is required during early mouse development for gastrulation. Chimaeric analysis can identify, by study of phenotypically normal embryos, tissues that tolerate mutant cells (and therefore do not require the mutated gene) or lack mutant cells (and presumably require the mutated gene during their developmental history). We therefore generated chimaeric mouse embryos to explore the cellular requirements for Shp2. This analysis revealed an obligatory role for Shp2 during outgrowth of the limb. Shp2 is specifically required in mesenchyme cells of the progress zone (PZ), directly beneath the distal ectoderm of the limb bud. Comparison of Ptpn11 (encoding Shp2)-mutant and Fgfr1 (encoding fibroblast growth factor receptor-1)-mutant chimaeric limbs indicated that in both cases mutant cells fail to contribute to the PZ of phenotypically normal chimaeras, leading to the hypothesis that a signal transduction pathway, initiated by Fgfr1 and acting through Shp2, is essential within PZ cells. Rather than integrating proliferative signals, Shp2 probably exerts its effects on limb development by influencing cell shape, movement or adhesion. Furthermore, the branchial arches, which also use Fgfs during bud outgrowth, similarly require Shp2. Thus, Shp2 regulates phosphotyrosine-signalling events during the complex ectodermal-mesenchymal interactions that regulate mammalian budding morphogenesis.
Assuntos
Membro Anterior/embriologia , Membro Posterior/embriologia , Botões de Extremidades/enzimologia , Proteínas Tirosina Fosfatases/genética , Domínios de Homologia de src/genética , Animais , Região Branquial/citologia , Região Branquial/enzimologia , Adesão Celular/genética , Divisão Celular/genética , Movimento Celular/genética , Tamanho Celular/genética , Quimera/genética , Ectoderma/citologia , Ectoderma/enzimologia , Membro Anterior/enzimologia , Genes Reporter , Membro Posterior/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular , Botões de Extremidades/citologia , Botões de Extremidades/embriologia , Mesoderma/citologia , Mesoderma/enzimologia , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , RNA Mensageiro/biossíntese , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Transdução de Sinais/genética , Células-Tronco/citologia , Transgenes , beta-Galactosidase/genéticaRESUMO
Cellular asymmetry is critical for the development of multicellular organisms. Here we show that homologues of proteins necessary for asymmetric cell division in Caenorhabditis elegans associate with each other in mammalian cells and tissues. mPAR-3 and mPAR-6 exhibit similar expression patterns and subcellular distributions in the CNS and associate through their PDZ (PSD-95/Dlg/ZO-1) domains. mPAR-6 binds to Cdc42/Rac1 GTPases, and mPAR-3 and mPAR-6 bind independently to atypical protein kinase C (aPKC) isoforms. In vitro, mPAR-3 acts as a substrate and an inhibitor of aPKC. We conclude that mPAR-3 and mPAR-6 have a scaffolding function, coordinating the activities of several signalling proteins that are implicated in mammalian cell polarity.
Assuntos
Proteínas de Caenorhabditis elegans , Polaridade Celular , Proteínas de Helminto/metabolismo , Proteína Quinase C/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Animais , Encéfalo/metabolismo , Fracionamento Celular , Linhagem Celular , Clonagem Molecular , Ativação Enzimática , Proteínas de Helminto/química , Proteínas de Helminto/genética , Imuno-Histoquímica , Substâncias Macromoleculares , Camundongos , Mutação , Fosforilação , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Proteínas Serina-Treonina Quinases , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/químicaRESUMO
Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that causes infantile diarrhea worldwide. EPEC injects a bacterial protein, translocated intimin receptor (Tir), into the host-cell plasma membrane where it acts as a receptor for the bacterial outer membrane protein, intimin. The interaction of Tir and intimin triggers a marked rearrangement of the host actin cytoskeleton into pedestals beneath adherent bacteria. On delivery into host cells, EPEC Tir is phosphorylated on tyrosine 474 of the intracellular carboxy-terminal domain, an event that is required for pedestal formation. Despite its essential role, the function of Tir tyrosine phosphorylation has not yet been elucidated. Here we show that tyrosine 474 of Tir directly binds the host-cell adaptor protein Nck, and that Nck is required for the recruitment of both neural Wiskott-Aldrich-syndrome protein (N-WASP) and the actin-related protein (Arp)2/3 complex to the EPEC pedestal, directly linking Tir to the cytoskeleton. Cells with null alleles of both mammalian Nck genes are resistant to the effects of EPEC on the actin cytoskeleton. These results implicate Nck adaptors as host-cell determinants of EPEC virulence.
Assuntos
Actinas/metabolismo , Adesinas Bacterianas , Proteínas de Transporte , Membrana Celular/microbiologia , Escherichia coli O157/fisiologia , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Linhagem Celular , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Escherichia coli O157/genética , Fibroblastos/citologia , Fibroblastos/microbiologia , Fibroblastos/fisiologia , Fibroblastos/ultraestrutura , Genes Reporter , Proteínas de Fluorescência Verde , Humanos , Cinética , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Mamíferos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosforilação , Fosfotirosina/metabolismo , Proteínas Recombinantes de Fusão/análise , Transfecção , Domínios de Homologia de srcRESUMO
Mutations in the gene encoding the phosphotyrosine phosphatase PTP1C, a cytoplasmic protein containing a COOH-terminal catalytic and two NH2-terminal Src homology 2 (SH2) domains, have been identified in motheaten (me) and viable motheaten (mev) mice and are associated with severe hemopoietic dysregulation. The me mutation is predicted to result in termination of the PTP1C polypeptide within the first SH2 domain, whereas the mev mutation creates an insertion or deletion in the phosphatase domain. No PTP1C RNA or protein could be detected in the hemopoietic tissues of me mice, nor could PTP1C phosphotyrosine phosphatase activity be isolated from cells homozygous for the me mutation. In contrast, mice homozygous for the less severe mev mutation expressed levels of full-length PTP1C protein comparable to those detected in wild type mice and the SH2 domains of mev PTP1C bound normally to phosphotyrosine-containing ligands in vitro. Nevertheless, the mev mutation induced a marked reduction in PTP1C activity. These observations provide strong evidence that the motheaten phenotypic results from loss-of-function mutations in the PTP1C gene and imply a critical role for PTP1C in the regulation of hemopoietic differentiation and immune function.
Assuntos
Hematopoese , Camundongos Mutantes/genética , Proteínas Tirosina Fosfatases/metabolismo , Animais , Sequência de Bases , Primers do DNA/química , Receptores ErbB/metabolismo , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Linhagem , Fosfoproteínas/metabolismo , Mutação Puntual , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de SinaisRESUMO
Stimulation of the T cell antigen receptor (TCR) activates a protein tyrosine kinase and leads to the tyrosine phosphorylation of phosphoinositide-specific phospholipase C-gamma 1 (PLC gamma 1). The molecular interactions involved in this phosphorylation are not known. After stimulation of the TCR on Jurkat T cells, tyrosine-phosphorylated proteins of 36, 38, 58, and 63 kD coprecipitate with PLC gamma 1. An identical pattern of proteins precipitate with TrpE fusion proteins that contain the Src homology (SH) 2 domains of PLC gamma 1, indicating that these regions of PLC gamma 1 are responsible for binding. TCR stimulation leads to an association between the SH2 domains of PLC gamma 1 and a protein tyrosine kinase, which, by peptide mapping, is identical to p56lck. These studies establish that p56lck associates with PLC gamma 1 as a result of TCR stimulation of Jurkat cells, suggesting that p56lck plays a central role in coupling the TCR to the activation of PLC gamma 1.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Fosfolipases Tipo C/metabolismo , Linhagem Celular , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Mapeamento de Peptídeos , Fosforilação , Linfócitos T/enzimologia , Tirosina/metabolismoRESUMO
SH2 domains serve as the prototype for a growing family of protein-interaction modules, characteristic of polypeptides involved in transmitting signals from external and internal cues. The specific interactions of proteins with one another, and with other cellular components such as phospholipids and nucleic acids, provide a very general device to organize cellular behavior. We discuss the idea that rewiring of the cell's interaction network by pathogenic microorganisms and mutant cellular proteins contributes to dysregulation of cell signaling and thus to disease.
Assuntos
Citoesqueleto/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/fisiologia , Domínios de Homologia de src/fisiologia , Animais , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Evolução Molecular , Humanos , Ligação Proteica/fisiologiaRESUMO
We have used cell lines deficient in p120 Ras GTPase activating protein (Ras-GAP) to investigate the roles of Ras-GAP and the associated p190 Rho-GAP (p190) in cell polarity and cell migration. Cell wounding assays showed that Ras-GAP-deficient cells were incapable of establishing complete cell polarity and migration into the wound. Stimulation of mutant cells with growth factor rescued defects in cell spreading, Golgi apparatus fragmentation, and polarized vesicular transport and partially rescued migration in a Ras-dependent manner. However, for directional movement, the turnover of stress fibers and focal adhesions to produce an elongate morphology was dependent on the constitutive association between Ras-GAP and p190, independent of Ras regulation. Disruption of the phosphotyrosine-mediated Ras-GAP/p190 complex by microinjecting synthetic peptides derived from p190 sequences in wild-type cells caused a suppression of actin filament reorientation and migration. From these observations we suggest that although Ras-GAP is not directly required for motility per se, it is important for cell polarization by regulating actin stress fiber and focal adhesion reorientation when complexed with 190. This observation suggests a specific function for Ras-GAP separate from Ras regulation in cell motility.
Assuntos
Movimento Celular/fisiologia , Complexo de Golgi/fisiologia , Fatores de Troca do Nucleotídeo Guanina , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteína p120 Ativadora de GTPase/metabolismo , Animais , Polaridade Celular/fisiologia , Células Cultivadas , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Proteínas de Ligação a DNA , Embrião de Mamíferos/citologia , Proteínas Ativadoras de GTPase , Complexo de Golgi/ultraestrutura , Camundongos , Camundongos Knockout , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Proteínas Nucleares/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Fosfoproteínas/química , Proteínas Recombinantes/metabolismo , Proteínas Repressoras , Transfecção , Saco Vitelino/citologia , Proteína p120 Ativadora de GTPase/deficiência , Proteína p120 Ativadora de GTPase/genética , ras-GRF1RESUMO
Signaling proteins can be regulated by their interactions with other proteins and phospholipids. As Fawcett and Pawson discuss in their Perspective, activation of the N-WASP protein (which coordinates formation of actin filaments) is far more complex, depending on the interaction of N-WASP with both a protein and a phospholipid. The authors explain new results (Prehoda et al.) demonstrating that cooperative binding of the phospholipid PIP2 and the small GTPase Cdc42 to N-WASP results in its activation. The Arp2/3 complex is then able to bind to N-WASP and to proceed with its job of initiating the assembly of actin monomers into actin filaments.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas do Citoesqueleto , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Motivos de Aminoácidos , Animais , Sítios de Ligação , Biopolímeros , GTP Fosfo-Hidrolases/metabolismo , Ligantes , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Xenopus , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
The process by which extracellular signals are relayed from the plasma membrane to specific intracellular sites is an essential facet of cellular regulation. Many signaling pathways do so by altering the phosphorylation state of tyrosine, serine, or threonine residues of target proteins. Recently, it has become apparent that regulatory mechanisms exist to influence where and when protein kinases and phosphatases are activated in the cell. The role of scaffold, anchoring, and adaptor proteins that contribute to the specificity of signal transduction events by recruiting active enzymes into signaling networks or by placing enzymes close to their substrates is discussed.
Assuntos
Fosfoproteínas/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Ligantes , Fosfolipídeos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Fosfotirosina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/química , Domínios de Homologia de srcRESUMO
A mouse phosphotyrosine phosphatase containing two Src homology 2 (SH2) domains, Syp, was identified. Syp bound to autophosphorylated epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors through its SH2 domains and was rapidly phosphorylated on tyrosine in PDGF- and EGF-stimulated cells. Furthermore, Syp was constitutively phosphorylated on tyrosine in cells transformed by v-src. This mammalian phosphatase is most closely related, especially in its SH2 domains, to the corkscrew (csw) gene product of Drosophila, which is required for signal transduction downstream of the Torso receptor tyrosine kinase. The Syp gene is widely expressed throughout embryonic mouse development and in adult tissues. Thus, Syp may function in mammalian embryonic development and as a common target of both receptor and nonreceptor tyrosine kinases.
Assuntos
Genes src , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Transformada , Transformação Celular Neoplásica , Embrião de Mamíferos , Desenvolvimento Embrionário e Fetal , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Poli A/isolamento & purificação , Poli A/metabolismo , Reação em Cadeia da Polimerase , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Ratos , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
The ras proto-oncogene products appear to relay intracellular signals via the Ras guanosine triphosphatase (GTPase) activator protein, GAP. In dog epithelial cells expressing human platelet-derived growth factor (PDGF) receptors, binding of PDGF caused approximately one-tenth of the total GAP molecules to complex with the receptor. Studies with mutant PDGF receptors showed that maximum association required both receptor kinase activity and phosphorylatable tyrosine residues at both the identified sites of receptor autophosphorylation.
Assuntos
Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Células Cultivadas , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , GTP Fosfo-Hidrolases/metabolismo , Proteínas Ativadoras de GTPase , Humanos , Immunoblotting , Proteína Oncogênica p21(ras)/metabolismo , Mapeamento de Peptídeos , Fosfopeptídeos/análise , Fosforilação , Testes de Precipitina , Proteínas Quinases/análise , Proto-Oncogene Mas , Receptores do Fator de Crescimento Derivado de Plaquetas , Tirosina/metabolismo , Proteínas Ativadoras de ras GTPaseRESUMO
Phospholipase C gamma 1 (PLC gamma 1) and p21ras guanosine triphosphatase (GTPase) activating protein (GAP) bind to and are phosphorylated by activated growth factor receptors. Both PLC gamma 1 and GAP contain two adjacent copies of the noncatalytic Src homology 2 (SH2) domain. The SH2 domains of PLC gamma 1 synthesized individually in bacteria formed high affinity complexes with the epidermal growth factor (EGF)- or platelet derived growth factor (PDGF)-receptors in cell lysates, and bound synergistically to activated receptors when expressed together as one bacterial protein. In vitro complex formation was dependent on prior growth factor stimulation and was competed by intracellular PLC gamma 1. Similar results were obtained for binding of GAP SH2 domains to the PDGF-receptor. The isolated SH2 domains of other signaling proteins, such as p60src and Crk, also bound activated PDGF-receptors in vitro. SH2 domains, therefore, provide a common mechanism by which enzymatically diverse regulatory proteins can physically associate with the same activated receptors and thereby couple growth factor stimulation to intracellular signal transduction pathways.
Assuntos
Receptores ErbB/metabolismo , Genes src/genética , Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Fosfolipases Tipo C/metabolismo , Receptores ErbB/genética , Proteínas Ativadoras de GTPase , Fosforilação , Proteínas/genética , Receptores de Superfície Celular/genética , Receptores do Fator de Crescimento Derivado de Plaquetas , Homologia de Sequência do Ácido Nucleico , Fosfolipases Tipo C/genéticaRESUMO
Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein-tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation.
Assuntos
Citoplasma/metabolismo , Sinais Direcionadores de Proteínas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Sítios de Ligação , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Proteínas Ativadoras de GTPase , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina , Fator de Crescimento Derivado de Plaquetas/farmacologia , Sinais Direcionadores de Proteínas/química , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteínas/química , Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas , Homologia de Sequência do Ácido Nucleico , Fosfolipases Tipo C/química , Fosfolipases Tipo C/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Proteínas Ativadoras de ras GTPaseRESUMO
The EPH-related transmembrane tyrosine kinases constitute the largest known family of receptor-like tyrosine kinases, with many members displaying specific patterns of expression in the developing and adult nervous system. A family of cell surface-bound ligands exhibiting distinct, but overlapping, specificities for these EPH-related kinases was identified. These ligands were unable to act as conventional soluble factors. However, they did function when presented in membrane-bound form, suggesting that they require direct cell-to-cell contact to activate their receptors. Membrane attachment may serve to facilitate ligand dimerization or aggregation, because antibody-mediated clustering activated previously inactive soluble forms of these ligands.
Assuntos
Membrana Celular/metabolismo , Proteínas de Ligação a DNA , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases/metabolismo , Receptor EphA5 , Proteínas Oncogênicas de Retroviridae/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Linhagem Celular , Efrina-A1 , Efrina-B1 , Humanos , Ligantes , Proteínas de Membrana/química , Dados de Sequência Molecular , Neurônios/metabolismo , Fosforilação , Proteínas/química , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Transfecção , Células Tumorais Cultivadas , Proteínas Elk-1 do Domínio etsRESUMO
Src homology 2 (SH2) domains have been identified in a large number of proteins involved in signal transduction downstream of receptor tyrosine kinases. They allow cytoplasmic signalling proteins to bind specifically to other polypeptides that are phosphorylated on tyrosine in response to growth factor stimulation. A novel phosphotyrosine-binding (PTB) domain has been identified recently in the amino terminus of Shc, an adaptor molecule that appears to be involved in Ras activation PTB domains are longer than SH2 domains, and recognize phosphotyrosine in the context of amino-terminal residues, in contrast to SH2 domains, which recognize them in the context of carboxy-terminal residues.
Assuntos
Proteínas/metabolismo , Transdução de Sinais/fisiologia , Tirosina/análogos & derivados , Sequência de Aminoácidos , Antígenos Transformantes de Poliomavirus/metabolismo , Dados de Sequência Molecular , Fosfotirosina , Proteínas Tirosina Quinases/metabolismo , Proteínas/química , Receptor de Insulina/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Tirosina/metabolismo , Proteínas ras/metabolismoRESUMO
Signaling pathways in mammalian cells are assembled and regulated by a finely controlled network of protein-protein and protein-phospholipid interactions, mediated by dedicated signaling domains and their cognate binding motifs. The domain-based modular architecture of signaling proteins may have facilitated the evolution of complex biological systems, and can be exploited experimentally to generate synthetic signaling pathways and artificial mechanisms of autoregulation. Pathogenic proteins, such as those encoded by bacteria and viruses, frequently form ectopic signaling complexes to respecify cellular behavior. In a similar fashion, proteins expressed as a consequence of oncogenic fusions, mutations or amplifications can elicit ectopic protein-protein interactions that re-wire signaling pathways, in a fashion that promotes malignancy. Compounds that directly or indirectly reverse these aberrant interactions offer new possibilities for therapy in cancer.
Assuntos
Oncogenes , Transdução de Sinais/genética , Animais , HumanosRESUMO
The Epstein-Barr virus latency-associated membrane protein LMP2A has been shown to activate the survival kinase Akt in epithelial and B cells in a phosphoinositide 3-kinase-dependent fashion. In this study, we demonstrate that the signalling scaffold Shb associates through SH2 and PTB domain interactions with phosphorylated tyrosine motifs in the LMP2A N-terminal tail. Additionally, we show that mutation of tyrosines in these motifs as well as shRNA-mediated downregulation of Shb leads to a loss of constitutive Akt-activation in LMP2A-expressing cells. Furthermore, utilization by Shb of the LMP2A ITAM motif regulates stability of the Syk tyrosine kinase in LMP2A-expressing cells. Our data set the precedent for viral utilization of the Shb signalling scaffold and implicate Shb as a regulator of LMP2A-dependent Akt activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/virologia , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas da Matriz Viral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sítios de Ligação , Linhagem Celular , Linhagem Celular Transformada , Herpesvirus Humano 4/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo , Quinase Syk , Tirosina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas da Matriz Viral/químicaRESUMO
Shc proteins possess SH2 and PTB domains and serve a scaffolding function in signaling by a variety of receptor tyrosine kinases. There are three known mammalian Shc genes, of which ShcB and ShcC are primarily expressed in the nervous system. We have generated null mutations in ShcB and ShcC and have obtained mice lacking either ShcB or ShcC or both gene products. ShcB-deficient animals exhibit a loss of peptidergic and nonpeptidergic nociceptive sensory neurons, which is not enhanced by additional loss of ShcC. Mice lacking both ShcB and ShcC exhibit a significant loss of neurons within the superior cervical ganglia, which is not observed in either mutant alone. The results indicate that these Shc family members possess both unique and overlapping functions in regulating neural development and suggest physiological roles for ShcB/ShcC in TrkA signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Proteínas do Tecido Nervoso/metabolismo , Neurônios Aferentes/metabolismo , Neuropeptídeos , Sistema Nervoso Simpático/metabolismo , Domínios de Homologia de src/genética , Animais , Células COS , Diferenciação Celular/genética , Células Cultivadas , Clonagem Molecular , Marcação de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Neurônios Aferentes/citologia , Especificidade de Órgãos , Fosfotirosina/metabolismo , Proteínas/genética , Homologia de Sequência de Aminoácidos , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Proteína 2 de Transformação que Contém Domínio 2 de Homologia de Src , Proteína 3 de Transformação que Contém Domínio 2 de Homologia de Src , Sistema Nervoso Simpático/citologiaRESUMO
Members of the Eph family of receptor tyrosine kinases control many aspects of cellular interactions during development, including axon guidance. Here, we demonstrate that EphB2 also regulates postnatal synaptic function in the mammalian CNS. Mice lacking the EphB2 intracellular kinase domain showed wild-type levels of LTP, whereas mice lacking the entire EphB2 receptor had reduced LTP at hippocampal CA1 and dentate gyrus synapses. Synaptic NMDA-mediated current was reduced in dentate granule neurons in EphB2 null mice, as was synaptically localized NR1 as revealed by immunogold localization. Finally, we show that EphB2 is upregulated in hippocampal pyramidal neurons in vitro and in vivo by stimuli known to induce changes in synaptic structure. Together, these data demonstrate that EphB2 plays an important role in regulating synaptic function.
Assuntos
Receptores Proteína Tirosina Quinases/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/fisiologia , Animais , Giro Denteado/citologia , Giro Denteado/fisiologia , Efrina-B2 , Agonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Ácido Glutâmico/metabolismo , Técnicas In Vitro , Ácido Caínico/farmacologia , Potenciação de Longa Duração/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Plasticidade Neuronal/fisiologia , Receptores Proteína Tirosina Quinases/genética , Receptor EphB2 , Sinapses/ultraestrutura , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
In response to NGF, the Trk receptor tyrosine kinase forms a complex with SHC, a protein that couples receptor tyrosine kinases to p21ras. Complex formation between Trk and SHC, SHC tyrosine phosphorylation, and association of SHC with Grb2 were mediated by autophosphorylation at Y490 in Trk [sequence: see text]. To determine the role of SHC and other Trk substrates in NGF signaling, Trk receptors with mutations in Y490 and Y785 (the PLC-gamma 1 association site) were introduced into PC12nnr5 cells. NGF treatment of PC12nnr5 cells expressing Trk with mutations in either substrate-binding site resulted in normal neurite outgrowth and Erk1 activity and tyrosine phosphorylation. However, PC12nnr5 cells expressing Trk with mutations at both sites failed to stably extend neurites and efficiently induce Erk1 activity and tyrosine phosphorylation in response to NGF. We postulate that Trk receptors can activate Erk1 by either SHC- or PLC-gamma 1-dependent signaling pathways. These results suggest a model whereby Trk receptors utilize at least partially redundant signal transduction pathways to mediate NGF responses.