RESUMO
The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (Ig) molecule as antigen-binding subunit and the Ig-alpha/Ig-beta heterodimer as signaling subunit. BCR signal transduction involves activation of protein tyrosine kinases (PTKs) and phosphorylation of several proteins, only some of which have been identified. The phosphorylation of these proteins can be induced by exposure of B cells either to antigen or to the tyrosine phosphatase inhibitor pervanadate/H2O2. One of the earliest substrates in B cells is a 65-kD protein, which we identify here as a B cell adaptor protein. This protein, named SLP-65, is part of a signaling complex involving Grb-2 and Vav and shows homology to SLP-76, a signaling element of the T cell receptor. In pervanadate/H2O2-stimulated cells, SLP-65 becomes phosphorylated only upon expression of the BCR. These data suggest that SLP-65 is part of a BCR transducer complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Linfócitos B/metabolismo , Proteínas de Transporte , Proteínas de Ciclo Celular , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Linfócitos B/efeitos dos fármacos , Sequência de Bases , DNA Complementar , Inibidores Enzimáticos/farmacologia , Proteína Adaptadora GRB2 , Humanos , Peróxido de Hidrogênio/farmacologia , Cadeias delta de Imunoglobulina/metabolismo , Cadeias mu de Imunoglobulina/metabolismo , Dados de Sequência Molecular , Fosfoproteínas/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-vav , Coelhos , Células Tumorais Cultivadas , Vanadatos/farmacologiaAssuntos
Doenças Autoimunes/imunologia , Autoimunidade/imunologia , Autorreceptores/imunologia , Linfócitos B/imunologia , Imunidade Inata/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Doenças Autoimunes/patologia , Humanos , Camundongos , Modelos ImunológicosRESUMO
Perpetual V(D)J recombinase activity involving multiple DNA double-strand break events in B-cell lineage leukemia and lymphoma cells may introduce secondary genetic aberrations leading towards malignant progression. Here, we investigated defective negative feedback signaling through the (pre-) B-cell receptor as a possible reason for deregulated V(D)J recombinase activity in B-cell malignancy. On studying 28 cases of pre-B-lymphoblastic leukemia and 27 B-cell lymphomas, expression of the (pre-) B-cell receptor-related linker molecule SLP65 (SH2 domain-containing lymphocyte protein of 65 kDa) was found to be defective in seven and five cases, respectively. SLP65 deficiency correlates with RAG1/2 expression and unremitting V(H) gene rearrangement activity. Reconstitution of SLP65 expression in SLP65-deficient leukemia and lymphoma cells results in downregulation of RAG1/2 expression and prevents both de novo V(H)-DJ(H) rearrangements and secondary V(H) replacement. We conclude that iterative V(H) gene rearrangement represents a frequent feature in B-lymphoid malignancy, which can be attributed to SLP65 deficiency in many cases.
Assuntos
Linfoma de Burkitt/genética , Proteínas de Transporte/genética , Linfoma de Células B/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , VDJ Recombinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Linfoma de Burkitt/enzimologia , Linhagem Celular Tumoral , Dano ao DNA , Rearranjo Gênico , Genes de Imunoglobulinas , Humanos , Dados de Sequência Molecular , Deleção de Sequência , VDJ Recombinases/genéticaRESUMO
SLP65 represents a critical component in (pre-) B cell receptor signal transduction but is compromised in a subset of pre-B cell-derived acute lymphoblastic leukemia. Based on these findings, we investigated (i.) whether SLP65-deficiency also occurs in mature B cell-derived lymphoma and (ii.) whether SLP65-deficient B cell lymphoma cells use an alternative B cell receptor signaling pathway in the absence of SLP65. Indeed, expression of SLP65 protein was also missing in a fraction of B cell lymphoma cases. While SLP65 is essential for B cell receptor-induced Ca2+ mobilization in normal B cells, B cell receptor engagement in SLP65-deficient as compared to SLP65-reconstituted B cell lymphoma cells resulted in an accelerated yet shortlived Ca2+-signal. B cell receptor engagement of SLP65-deficient lymphoma cells involves SRC kinase activation, which is critical for B cell receptor-dependent Ca2+-mobilisation in the absence but not in the presence of SLP65. As shown by RNA interference, the SRC kinase LYN is required for B cell receptor-induced Ca2+ release in SLP65-deficient B cell lymphoma cells but dispensable after SLP65-reconstitution. B cell receptor engagement in SLP65-deficient B cell lymphoma cells also resulted in tyrosine-phosphorylation of the proliferation- and survival-related MAPK1 and STAT5 molecules, which was sensitive to silencing of the SRC kinase LYN. Inhibition of SRC kinase activity resulted in growth arrest and cell death specifically in SLP65-deficient lymphoma cells. These findings indicate that LYN can short-circuit conventional B cell receptor signaling in SLP65-deficient B cell lymphoma cells and thereby promote activation of survival and proliferation-related molecules.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Linfoma de Células B/enzimologia , Transdução de Sinais , Quinases da Família src/metabolismo , Humanos , Linfoma de Células B/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Fator de Transcrição STAT5/metabolismoRESUMO
Pre-B lymphoblastic leukemia cells carrying a BCR-ABL1 gene rearrangement exhibit an undifferentiated phenotype. Comparing the genome-wide gene expression profiles of normal B-cell subsets and BCR-ABL1+ pre-B lymphoblastic leukemia cells by SAGE, the leukemia cells show loss of B lymphoid identity and aberrant expression of myeloid lineage-specific molecules. Consistent with this, BCR-ABL1+ pre-B lymphoblastic leukemia cells exhibit defective expression of IKAROS, a transcription factor needed for early lymphoid lineage commitment. As shown by inducible expression of BCR-ABL1 in human and murine B-cell precursor cell lines, BCR-ABL1 induces the expression of a dominant-negative IKAROS splice variant, termed IK6. Comparing matched leukemia sample pairs from patients before and during therapy with the BCR-ABL1 kinase inhibitor STI571 (Imatinib), inhibition of BCR-ABL1 partially corrected aberrant expression of IK6 and lineage infidelity of the leukemia cells. To elucidate the contribution of IK6 to lineage infidelity in BCR-ABL1+ cell lines, IK6 expression was silenced by RNA interference. Upon inhibition of IK6, BCR-ABL1+ leukemia cells partially restored B lymphoid lineage commitment. Therefore, we propose that BCR-ABL1 induces aberrant splicing of IKAROS, which interferes with lineage identity and differentiation of pre-B lymphoblastic leukemia cells.
Assuntos
Processamento Alternativo , Fator de Transcrição Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Tirosina Quinases/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas , Linhagem Celular Tumoral , Linhagem da Célula/genética , Núcleo Celular/química , Proteínas de Fusão bcr-abl , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Fator de Transcrição Ikaros/análise , Fator de Transcrição Ikaros/metabolismo , Mesilato de Imatinib , Camundongos , Piperazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/dietoterapia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/análise , Pirimidinas/farmacologiaRESUMO
SRp20 is a splicing factor belonging to the highly conserved family of SR proteins [1] [2] [3] [4], which have multiple roles in the regulation of constitutive and alternative splicing in vivo. It has been suggested that SR proteins are involved in bringing together the splice sites during spliceosome assembly [5]. SR proteins show partial redundancy, as each single SR protein can restore splicing activity to a splicing-deficient cytoplasmic extract (termed S-100 extract). Nevertheless, several studies demonstrate that individual SR proteins have different effects on the selection of specific alternative splice sites, and they recognize distinct RNA sequences [6] [7] [8] [9] [10] [11] [12]. Also, inactivation of two SR proteins, B52/SRp55 in Drosophila [13] or ASF/SF2 in the chicken cell line DT40 [14], is lethal, indicating the existence of nonredundant functions. Here, using Cre-loxP-mediated recombination in mice to inactivate the SRp20 gene, we found that it is essential for mouse development. Mutant preimplantation embryos failed to form blastocysts and died at the morula stage. Immunofluorescent staining showed that SRp20 is present in oocytes and early stages of embryonic development. This is the first report of mice deficient for a member of the SR protein family. Our experiments confirm that, although similar in structure, the SR proteins are not functionally redundant.
Assuntos
Blastocisto , Desenvolvimento Embrionário , Desenvolvimento Embrionário e Fetal/fisiologia , Proteínas de Ligação a RNA/fisiologia , Alelos , Animais , Blastocisto/química , Eletroporação , Feminino , Imunofluorescência , Deleção de Genes , Genótipo , Camundongos , Mórula/química , Reação em Cadeia da Polimerase , Gravidez , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina , Fatores de TempoRESUMO
Eukaryotic splicing factors belonging to the SR family are essential splicing factors consisting of an N-terminal RNA-binding region and a C-terminal RS domain. They are believed to be involved in alternative splicing of numerous transcripts because their expression levels can influence splice site selection. We have characterized the structure and transcriptional regulation of the gene for the smallest member of the SR family, SRp20 (previously called X16). The mouse gene encoding SRp20, termed Srp20, consists of one alternative exon and six constitutive exons and was mapped to a 2-centimorgan interval on chromosome 17. When cells are transfected with SRp20 genomic DNA, both standard and alternatively spliced transcripts and corresponding proteins are produced. Interestingly, in starved (G0) cells, the amount of SRp20 mRNA containing the alternative exon is large, whereas the amount of the standard SRp20 mRNA without the alternative exon is small. When starved cells are stimulated with serum, the alternative form is lost and the standard form is induced. These results suggest that splicing could be regulated during the cell cycle and that this could be, at least in part, due to regulated expression of SR proteins. Consistent with this, experiments with synchronized cells showed an induction of SRp20 transcripts in late G1 or early S. We have also characterized the promoter of SRp20. It lies within a GC-rich CpG island and contains two consensus binding sites for E2F, a transcription factor thought to be involved in regulating the cell cycle. These motifs may be functional since reporter constructs with the SRp20 promoter can be stimulated by cotransfection with E2F expression plasmids.
Assuntos
Proteínas de Transporte , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Transcrição Gênica , Processamento Alternativo , Animais , Sequência de Bases , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Sequência Consenso , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Genes Reporter , Luciferases/genética , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Mapeamento por Restrição , Proteína 1 de Ligação ao Retinoblastoma , Fatores de Processamento de Serina-Arginina , Fator de Transcrição DP1 , Fatores de Transcrição/metabolismoRESUMO
Precursor-B-cell receptor (pre-BCR) signaling and spleen tyrosine kinase (SYK) recently were introduced as therapeutic targets for patients with B-cell acute lymphoblastic leukemia (B-ALL), but the importance of this pathway in B-ALL subsets and mechanism of downstream signaling have not fully been elucidated. Here, we provide new detailed insight into the mechanism of pre-BCR signaling in B-ALL. We compared the effects of pharmacological and genetic disruption of pre-BCR signaling in vitro and in mouse models for B-ALL, demonstrating exquisite dependency of pre-BCR(+) B-ALL, but not other B-ALL subsets, on this signaling pathway. We demonstrate that SYK, PI3K/AKT, FOXO1 and MYC are important downstream mediators of pre-BCR signaling in B-ALL. Furthermore, we define a characteristic immune phenotype and gene expression signature of pre-BCR(+) ALL to distinguish them from other B-ALL subsets. These data provide comprehensive new insight into pre-BCR signaling in B-ALL and corroborate pre-BCR signaling and SYK as promising new therapeutic targets in pre-BCR(+) B-ALL.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Células Precursoras de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Proteína Forkhead Box O1/metabolismo , Xenoenxertos , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Quinase Syk/metabolismoRESUMO
SR proteins are essential splicing factors involved in the use of both constitutive and alternative exons. We previously showed that the SR proteins SRp20 and ASF/SF2 have antagonistic activities on SRp20 pre-mRNA splicing. SRp20 activates exon 4 recognition in its pre-mRNA, whereas ASF/SF2 inhibits this recognition. In experiments aimed at testing the specificity of SRp20 and ASF/SF2 for exon 4 splicing regulation, we show here that this specificity lies in the RNA binding domains of SRp20 and ASF/SF2 and not in the RS domains. Surprisingly, a deletion of 14 amino acids at the end of ASF/SF2-RBD2 converts ASF/SF2 from an inhibitor to an activator of exon 4 splicing. We found that ASF3 also inhibits exon 4 recognition, thus acting similarly to ASF/SF2, while SC35 activates a cryptic 5' splice site downstream of exon 3 and, in doing so, represses exon 4 use. In contrast, Tra2 and the SR proteins 9G8 and SRp40 do not appear to affect exon 4 splicing.
Assuntos
Processamento Alternativo/genética , Proteínas de Drosophila , Éxons/genética , Proteínas Nucleares/metabolismo , Precursores de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Western Blotting , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleoproteínas/metabolismo , Deleção de Sequência/genética , Fatores de Processamento de Serina-Arginina , Especificidade por Substrato , Transfecção , Células Tumorais CultivadasRESUMO
The non-receptor protein tyrosine kinase Syk (spleen tyrosine kinase) is an important mediator of signal transduction in B cells. By acting downstream of the B-cell antigen receptor, Syk promotes signaling pathways involved in proliferation, differentiation and survival of B cells. To study the oncogenic potential of Syk, we generated a mouse model for the inducible expression of the leukemia-derived TEL-Syk fusion protein exhibiting constitutive kinase activity. To achieve B-cell-specific expression of TEL-Syk in adult mice, we used a tamoxifen-inducible Cre mouse line. This study shows that inducible expression of TEL-Syk in B cells leads to transient proliferation and subsequent plasma cell differentiation. However, it does not lead to B-cell transformation. Instead, Syk activation induces the tumor suppressor B-lymphocyte-induced maturation protein-1 (Blimp-1), which interferes with the expression of the antiapoptotic protein Bcl-2. Combined induction of TEL-Syk with transgenic expression of Bcl-2 results in a severe phenotype and plasma cell expansion. Our results suggest that deregulated Syk activity by itself is not sufficient for the transformation of B cells, as downstream effectors, such as Blimp-1, limit the survival and expansion of the activated B cell.
Assuntos
Linfócitos B/citologia , Diferenciação Celular , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Tirosina Quinases/genética , Fatores de Transcrição/metabolismo , Animais , Apoptose , Linfócitos B/metabolismo , Biomarcadores/metabolismo , Sobrevivência Celular , Expressão Gênica/efeitos dos fármacos , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Fenótipo , Plasmócitos/citologia , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Recombinantes de Fusão/genética , Quinase Syk , Tamoxifeno/farmacologiaRESUMO
The spleen tyrosine kinase (SYK) was identified as an oncogenic driver in a broad spectrum of hematologic malignancies. The in vivo comparison of three SYK containing oncogenes, SYK(wt), TEL-SYK and IL-2-inducible T-cell kinase (ITK)-SYK revealed a general myeloexpansion and the establishment of three different hematologic (pre)diseases. SYK(wt) enhanced the myeloid and T-cell compartment, without leukemia/lymphoma development. ITK-SYK caused lethal T-cell lymphomas and the cytoplasmic TEL-SYK fusion induced an acute panmyelosis with myelofibrosis-type acute myeloid leukemia (AML) with up to 50% immature megakaryoblasts infiltrating bone marrow, spleen and liver, additional MPN features (myelofibrosis and granulocyte expansion) and MDS stigmata with megakaryocytic and erythroid dysplasia. LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates. SYK inhibitor treatment (R788) of diseased TEL-SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis. Stat5 was identified as a major downstream mediator of TEL-SYK in vitro as well as in vivo. Consequently, targeted deletion of Stat5 in vivo completely abrogated TEL-SYK-induced AML and myelofibrosis development, proving Stat5 as a major driver of SYK-induced transformation. Our experiments highlight the important role of SYK in AML and myelofibrosis and prove SYK and STAT5 inhibitors as potent treatment options for those diseases.
Assuntos
Deleção de Genes , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Proteínas de Fusão Oncogênica , Mielofibrose Primária , Fator de Transcrição STAT5 , Animais , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Síndromes Mielodisplásicas/prevenção & controle , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Mielofibrose Primária/patologia , Mielofibrose Primária/prevenção & controle , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Quinase Syk , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
The purpose of this study was the appraisal of the clinical and functional consequences of germline mutations within the gene for the IL-2 inducible T-cell kinase, ITK. Among patients with Epstein-Barr virus-driven lymphoproliferative disorders (EBV-LPD), negative for mutations in SH2D1A and XIAP (n=46), we identified two patients with R29H or D500T,F501L,M503X mutations, respectively. Human wild-type (wt) ITK, but none of the mutants, was able to rescue defective calcium flux in murine Itk(-/-) T cells. Pulse-chase experiments showed that ITK mutations lead to varying reductions of protein half-life from 25 to 69% as compared with wt ITK (107 min). The pleckstrin homology domain of wt ITK binds most prominently to phosphatidylinositol monophosphates (PI(3)P, PI(4)P, PI(5)P) and to lesser extend to its double or triple phosphorylated derivates (PIP2, PIP3), interactions which were dramatically reduced in the patient with the ITK(R29H) mutant. ITK mutations are distributed over the entire protein and include missense, nonsense and indel mutations, reminiscent of the situation in its sister kinase in B cells, Bruton's tyrosine kinase.
Assuntos
Mutação em Linhagem Germinativa , Herpesvirus Humano 4/fisiologia , Transtornos Linfoproliferativos/virologia , Proteínas Tirosina Quinases/genética , Sítios de Ligação , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Fosforilação , Proteínas Tirosina Quinases/metabolismoRESUMO
The mb1 gene encodes the Ig-alpha signaling subunit of the B cell antigen receptor and is expressed exclusively in B cells beginning at the very early pro-B cell stage in the bone marrow. We examine here the efficacy of the mb1 gene as a host locus for cre recombinase expression in B cells. We show that by integrating a humanized cre recombinase into the mb1 locus we obtain extraordinarily efficient recombination of loxP sites in the B cell lineage. The results from a variety of reporter genes including the splicing factor SRp20 and the DNA methylase Dnmt1 suggest that mb1-cre is probably the best model so far described for pan-B cell-specific cre expression. The availability of a mouse line with efficient cre-mediated recombination at an early developmental stage in the B lineage provides an opportunity to study the role of various genes specifically in B cell development and function.
Assuntos
Linfócitos B/fisiologia , Antígenos CD79/genética , Marcação de Genes , Integrases/metabolismo , Camundongos Mutantes/genética , Animais , Antígenos CD19/genética , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linhagem da Célula , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Deleção de Genes , Expressão Gênica , Genes Reporter , Integrases/genética , Interleucina-7/farmacologia , Camundongos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Recombinação Genética , Fatores de Processamento de Serina-ArgininaRESUMO
SRp20 is a member of the highly conserved SR family of splicing regulators. Using a variety of reporter gene constructs, we show that SRp20 regulates alternative splicing of its own mRNA. Overexpression of SRp20 results in a reduction in the level of exon 4-skipped SRp20 transcripts and activates the production of transcripts containing exon 4. These exon 4-included transcripts encode a truncated protein lacking the C-terminal RS domain. We provide evidence that SRp20 probably enhances the recognition of the otherwise unused, weak splice acceptor of exon 4. The recognition of exons with weak splice acceptor sites may be a general activity of SRp20. Unexpectedly, ASF/SF2, another member of the SR family, antagonizes the effect of SRp20 on SRp20 pre-mRNA splicing and suppresses the production of the exon 4-included form. Our results indicate that ASF/SF2 suppresses the use of the alternative exon 4, most likely by inhibiting the recognition of the splice donor of exon 4. These results demonstrate, for the first time, an auto-regulatory activity of an SR protein which is antagonized by a second SR protein.
Assuntos
Processamento Alternativo/genética , Proteínas Nucleares/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Clonagem Molecular , Éxons/genética , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Vetores Genéticos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Regiões Promotoras Genéticas/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Análise de Sequência de DNA , Fatores de Processamento de Serina-Arginina , Spliceossomos/química , Spliceossomos/metabolismo , Transfecção/genética , Células Tumorais CultivadasRESUMO
Mice deficient for the adapter protein SLP65 (BLNK) show a partial block in early B cell development, reduced numbers of mature B cells in the periphery, an absence of B1 cells and a reduction of IgM and IgG3 serum immunoglobulin levels. A strikingly similar phenotype is observed in Btk-deficient mice. To investigate the consequences of mutations in both SLP65 and Btk, we generated SLP65/ Btk double-mutant mice by crossing the single-mutant mice. Analysis of the double-mutant mice reveals a much more severe defect in B cell development. B cells in the SLP65/Btk double-mutant mice are arrested at the preB cell stage and, surprisingly, express the preB cell receptor. Normally, preB cell receptor expression in wild-type mice is restricted to a very small fraction of B cells making it difficult to identify these cells in the bone marrow. Together, the data demonstrate the synergistic role of SLP65 and Btk in B cell development and describe a situation where large numbers of preB cell receptor-positive cells accumulate in the bone marrow and spleen.
Assuntos
Antígenos CD , Linfócitos B/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Receptores de Antígenos de Linfócitos B/análise , Proteínas Adaptadoras de Transdução de Sinal , Tirosina Quinase da Agamaglobulinemia , Animais , Medula Óssea/imunologia , Diferenciação Celular , Deleção de Genes , Leucossialina , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Sialoglicoproteínas/análise , Baço/imunologia , Células-Tronco/imunologiaRESUMO
During signal transduction through the B cell antigen receptor (BCR), several signaling elements are brought together by the adaptor protein SLP-65. We have investigated the role of SLP-65 in B cell maturation and function in mice deficient for SLP-65. While the mice are viable, B cell development is affected at several stages. SLP-65-deficient mice show increased proportions of pre-B cells in the bone marrow and immature B cells in peripheral lymphoid organs. B1 B cells are lacking. The mice show lower IgM and IgG3 serum titers and poor IgM but normal IgG immune responses. Mutant B cells show reduced Ca2+ mobilization and reduced proliferative responses to B cell mitogens. We conclude that while playing an important role, SLP-65 is not always required for signaling from the BCR.