RESUMO
Btk is a member of the Tec family of protein tyrosine kinases expressed in B cells. It is stimulated following cross-linking of the B cell receptor which leads to the autophosphorylation of a specific residue in the SH3 domain, Y223. Previous work using Btk-derived fusion proteins has shown that the Btk SH3 domain binds to c-Cbl and Wiskott-Aldrich syndrome protein (WASP) in vitro. We show here that when the Btk SH3 domain fusion protein is autophosphorylated, its ability to take part in protein interactions is altered as compared to the non-phosphorylated fusion protein. Although the phosphorylated Btk SH3 domain still binds c-Cbl, it no longer binds WASP and instead acquires a high affinity for kinase-active Syk. The region of Syk responsible for this interaction is contained within its C terminus, suggesting a novel mechanism by which these proteins may interact.
Assuntos
Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Ubiquitina-Proteína Ligases , Tirosina Quinase da Agamaglobulinemia , Sequência de Aminoácidos , Animais , Linfócitos B/enzimologia , Linfócitos B/imunologia , Baculoviridae/genética , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Precursores Enzimáticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Fosforilação , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera , Quinase Syk , Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich , Domínios de Homologia de srcRESUMO
Studies of X chromosome inactivation patterns are central to many aspects of our understanding of the pathogenesis of haematological malignancies. In patients with myeloproliferative disorders and myelodysplastic syndromes the demonstration of skewed X inactivation patterns in multiple haemopoietic lineages has been taken to indicate a stem cell origin for these groups of diseases. However, stem cell depletion or selection pressures can also produce skewed X inactivation patterns and might increase with age. We have therefore used the HUMARA assay to study X inactivation patterns of elderly patients with myeloproliferative disorders together with an age-matched control group of normal elderly women. A clonal pattern (clonal granulocytes and polyclonal T cells) was observed in 23.1% of normal women and 63.4% of patients with myeloproliferative disorders. This is the first report of X inactivation patterns in purified subpopulations of blood cells in normal elderly women. These results have three significant implications. Firstly, the finding of clonal granulocytes and polyclonal T cells in normal elderly women is likely to reflect age-related stem cell depletion or selection pressures. Secondly, the demonstration of clonal granulocytes and polyclonal T cells is not a useful diagnostic marker for myeloproliferative disorders or myelodysplastic syndromes in elderly women. Thirdly, our data raise the possibility that clonal blood cell patterns may precede rather than follow mutations which subsequently give rise to myelodysplastic or myeloproliferative phenotypes.
Assuntos
Hematopoese/genética , Síndromes Mielodisplásicas/genética , Transtornos Mieloproliferativos/genética , Cromossomo X/genética , Idoso , Idoso de 80 Anos ou mais , Células Clonais , Feminino , Granulócitos/patologia , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Linfócitos T/patologiaAssuntos
Medicina de Família e Comunidade , Metadona/uso terapêutico , Entorpecentes/uso terapêutico , Transtornos Relacionados ao Uso de Opioides/reabilitação , Aconselhamento , Custos de Medicamentos , Educação Médica Continuada , Feminino , Humanos , Capacitação em Serviço , Masculino , Sistemas Computadorizados de Registros Médicos , Metadona/economia , Entorpecentes/economia , Farmácias , Relações Médico-Paciente , Encaminhamento e Consulta , Escócia , Apoio SocialRESUMO
Polycythemia vera (PV) is an acquired clonal disorder characterized by increased production of mature red cells and growth of erythroid colonies in the absence of erythropoietin. Mutation of the erythropoietin receptor has been demonstrated to cause familial polycythemia, but no mutations have been found in PV. Moreover, both erythroid and myeloid progenitors from patients with PV have been reported to be hypersensitive to a number of different growth factors. Attention has therefore focused on post-receptor signal transduction pathways. The SHP-1 gene is an especially attractive candidate gene. Firstly, SHP-1 binds to and negatively regulates signalling from the erythropoietin receptor and is likely to regulate other cytokine receptors in a similar manner. Secondly, absence of SHP-1 protein in the motheaten mouse is accompanied by increased sensitivity of hematopoietic progenitors to a number of cytokines including erythropoietin. Thirdly, familial or sporadic polycythemia in man may result from mutations of the SHP-1 binding domain of the erythropoietin receptor. We have therefore searched for mutations of the SHP-1 gene in genomic DNA from patients with PV. In this disease the majority of peripheral blood lymphocytes are not part of the malignant clone and a variable proportion of myeloid cells may arise from normal progenitors. We have therefore chosen to study DNA from purified peripheral blood granulocytes obtained from nine women in whom the granulocytes were clonally derived. Southern analysis was used to show that the gene was not rearranged and densitometry confirmed the presence of two copies of the gene in each DNA sample. Sequencing of the entire coding region and all splice junctions revealed no mutations. Hematopoietic transcription factor binding sites in the SHP-1 promoter region were intact and the methylation status of the two SHP-1 promoters in PV patients was identical to that in three normal controls. Finally, we showed that levels of SHP-1 protein in granulocytes from patients was similar to those from normal controls. These results demonstrate that the SHP-1 gene is structurally and transcriptionally intact in patients with PV.
Assuntos
Policitemia Vera/enzimologia , Policitemia Vera/genética , Proteínas Tirosina Fosfatases/sangue , Proteínas Tirosina Fosfatases/genética , Idoso , Idoso de 80 Anos ou mais , Southern Blotting , DNA/sangue , DNA/genética , DNA/metabolismo , Metilação de DNA , Éxons , Feminino , Rearranjo Gênico , Granulócitos/enzimologia , Células-Tronco Hematopoéticas/enzimologia , Heterozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Pessoa de Meia-Idade , Policitemia Vera/sangue , Regiões Promotoras Genéticas , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Splicing de RNA , Linfócitos T/enzimologia , Transcrição GênicaRESUMO
Polycythaemia vera (PV) is thought to result from clonal expansion of a transformed multipotent stem cell. Progenitors from patients with PV display abnormal responses to several growth factors, suggesting the presence of a defect in a signalling pathway common to different growth factors. A number of approaches are now focused on defining the molecular lesion or lesions. Identification of causal genes will be of considerable interest both to clinicians, who currently lack a specific and sensitive diagnostic test, and to scientists interested in fundamental issues of stem cell behaviour.
Assuntos
Policitemia Vera/etiologia , Humanos , Policitemia Vera/genéticaRESUMO
Mutations in the gene encoding CD40 ligand have been shown to be the cause of X-linked hypogammaglobulinemia with hyper IgM (HIGM1). We have used the technique of single strand conformational polymorphism (SSCP) analysis to screen for mutations in this gene in affected boys from nineteen unrelated families. Sixteen novel mutations were identified in patients, comprising six patients with single base substitutions, two patients with single base insertions, six patients with deletions ranging from one to seven bases and two patients with large deletions at the 5' end of the gene. These mutations were distributed throughout the gene SSCP band shifts and/or alterations in restriction enzyme digestion sites could be used for unambiguous determination of carrier status in at-risk female relatives of most of the affected boys and, in some cases, prenatal diagnosis also can be offered.
Assuntos
Agamaglobulinemia/genética , Imunoglobulina M , Síndromes de Imunodeficiência/genética , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Mutação , Cromossomo X , Agamaglobulinemia/imunologia , Antígenos CD40/metabolismo , Ligante de CD40 , Criança , Análise Mutacional de DNA , Éxons , Feminino , Mutação da Fase de Leitura , Triagem de Portadores Genéticos , Humanos , Síndromes de Imunodeficiência/imunologia , Masculino , Núcleo Familiar , Linhagem , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Deleção de SequênciaRESUMO
X-linked agammaglobulinemia, a B cell immunodeficiency, is caused by mutations in the Bruton's tyrosine kinase (Btk) gene. The absence of a functional Btk protein leads to a failure of B cell differentiation and antibody production. B cell receptor stimulation leads to the phosphorylation of the Btk protein and it is, therefore, likely that Btk is involved in B cell receptor signaling. As a nonreceptor tyrosine kinase, Btk is likely to interact with several proteins within the context of a signal transduction pathway. To understand such interactions, we have generated glutathione S-transferase fusion proteins corresponding to different domains of the human Btk protein. We have identified a 120-kD protein present in human B cells as being bound by the SH3 domain of Btk and which, after B cell receptor stimulation, is one of the major substrates of tyrosine phosphorylation. We have shown that this 120-kD protein is the protein product of c-cbl, a protooncogene, which is known to be phosphorylated in response to T cell receptor stimulation and to interact with several other tyrosine kinases. Association of the SH3 domain of Btk with p120cbl provides evidence for an analogous role for p120cbl in B cell signaling pathways. The p120cbl protein is the first identified ligand of the Btk SH3 domain.
Assuntos
Linfócitos B/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Ubiquitina-Proteína Ligases , Tirosina Quinase da Agamaglobulinemia , Sequência de Bases , Linhagem Celular , Primers do DNA/química , Humanos , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-cbl , Transdução de SinaisRESUMO
X-linked agammaglobulinemia is a primary inherited immunodeficiency resulting in a lack of or dramatic reduction in the number of mature B lymphocytes and, thus, greatly reduced levels of serum immunoglobulin. The defect results from mutations in the gene for Bruton's tyrosine kinase (Btk). Using rabbit antisera generated against Btk, we have demonstrated an increase in the level of in vitro kinase activity present in anti-Btk immunoprecipitates from B cells following stimulation with anti-immunoglobulin antibody. This increase in immune complex kinase activity is detectable 1 to 2 min following stimulation and remains elevated for over 30 min. A similar increase was not seen with two late pre-B cell lines investigated in the same way. This stimulation of activity may suggest a role for Btk in signalling through the B cell receptor or associated proteins, in mature B cells.
Assuntos
Agamaglobulinemia/imunologia , Linfócitos B/imunologia , Proteínas Tirosina Quinases/metabolismo , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/enzimologia , Anticorpos Anti-Idiotípicos/farmacologia , Linfócitos B/efeitos dos fármacos , Ativação Enzimática , Humanos , Ativação Linfocitária , Fosforilação , Proteínas Tirosina Quinases/imunologiaRESUMO
Defects in the gene encoding Bruton's tyrosine kinase (Btk), normally expressed in B cells, cause X-linked agammaglobulinemia (XLA). The phenotype of XLA is characterized by a lack of circulating B cells and immunoglobulin. It has been suggested that B cell maturation from the pre-B cell stage to more mature stages is dependent on the appropriate expression of this gene. The Btk mRNA is expressed in B cells and myeloid cells, but protein expression in relation to B cell maturation has not been determined. Moreover, expression of the Btk protein has so far only been investigated in human Epstein-Barr virus-transformed B cell lines, and in murine splenocytes and B cell lines. We have developed an antiserum which recognizes the human Btk protein and shown that normal human tonsillar B cells, peripheral blood monocytes and myeloid cells express the protein, whereas tonsil-derived T cells do not. We also show that the protein is present in early and mature human B cell lines, but is absent in terminally differentiated plasma cell lines. Furthermore, expression is reduced or absent in three B lineage cell lines derived from two patients with defined genetic mutations in Btk and suffering from XLA.
Assuntos
Linfócitos B/enzimologia , Proteínas Tirosina Quinases/metabolismo , Tirosina Quinase da Agamaglobulinemia , Linfócitos B/citologia , Sequência de Bases , Western Blotting , Diferenciação Celular , Primers do DNA/química , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Plasmócitos/enzimologia , Proteínas Tirosina Quinases/genética , RNA Mensageiro/genéticaRESUMO
X-linked agammaglobulinemia (XLA) is an inherited human immunodeficiency disease, characterized by an arrest in B-cell development, which results in a dramatic decrease in immunoglobulin production. The gene product defective in XLA has been identified as a cytoplasmic protein tyrosine kinase, named Bruton's tyrosine kinase (Btk). The dramatic XLA phenotype indicates a critical role for Btk in the regulation of B-cell development. However, neither external stimuli leading to Btk activation nor any of its in vivo substrates have thus far been identified, and the mechanism of disease induction remains unexplained. We report here that stimulation of the B-cell antigen receptor (membrane immunoglobulin) on mature B-cells induces tyrosine phosphorylation of Btk in vivo, accompanied by an increase in its kinase activity in vitro. These results place Btk in the B-cell receptor signal transduction pathway, which is known to be essential in driving B-cell differentiation.
Assuntos
Agamaglobulinemia/enzimologia , Ligação Genética , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Cromossomo X , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/genética , Citosol/enzimologia , Ativação Enzimática , Humanos , Fosforilação , Proteínas Tirosina Quinases/deficiência , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
The btk gene has recently been identified as the causative gene in X-linked agammaglobulinemia (XLA). This has opened up many new possibilities for the treatment of this B-cell immunodeficiency. Christine Kinnon and colleagues review the high degree of sequence of homology of btk to the non-receptor tyrosine kinases and speculate on putative roles for this gene in B-cell development.
Assuntos
Agamaglobulinemia/genética , Clonagem Molecular , Ligação Genética , Cromossomo X , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/imunologia , Modelos Animais de Doenças , Previsões , Humanos , Camundongos , Camundongos Endogâmicos CBA , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/fisiologiaRESUMO
Histidine-requiring auxotrophs of Mycobacterium smegmatis were isolated following N-methyl-N'-nitro-N-nitrosoguanidine treatment. One of these mutants, his5, was transformed with an M. smegmatis shuttle cosmid library, and complementing clones were isolated at a frequency of approximately 1%. A 2.3 kb fragment was subcloned and sequenced, and found to contain the start of an operon including the hisD gene and part of the hisC gene. No hisG gene was detected upstream of hisD, suggesting that the regulation of histidine biosynthesis in mycobacteria may differ from that of Escherichia coli. The strategy used here will allow the molecular genetics of complex mycobacterial-specific biosynthetic pathways involved in the virulence of pathogenic species to be studied.
Assuntos
Oxirredutases do Álcool , Proteínas de Bactérias/genética , Clonagem Molecular , Genes Bacterianos , Histidina/genética , Mycobacterium/genética , Transaminases/genética , ATP Fosforribosiltransferase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Teste de Complementação Genética , Histidina/biossíntese , Dados de Sequência Molecular , Mutação , Mycobacterium/metabolismo , Óperon , Transaminases/químicaRESUMO
A shuttle cosmid vector, pMSC1, has been constructed which replicates in Escherichia coli and Mycobacterium smegmatis. The vector was mainly derived from the lambda ori cosmid, Lawrist4, and the Mycobacterium fortuitum cryptic plasmid, pAL5000, which replicates in M. smegmatis and Mycobacterium bovis BCG. The vector contains two cos sites which facilitates library construction, unique BamHI and HindIII sites for cloning, and a kanamycin-resistance-encoding gene for selection in mycobacteria. After packaging, the vector sequences comprise 10.3 kb, so that the theoretical size limits for inserts are 30-42 kb. A genomic library from M. smegmatis was constructed in E. coli; clones from this library were transferred into M. smegmatis by electroporation, and back again to E. coli, without any apparent rearrangements. This vector will be useful in cloning genes encoding complex pathways in mycobacteria.