RESUMO
The lymphocyte-specific phosphoprotein LSP1 associates with the cytoplasmic face of the plasma membrane and with the cytoskeleton. Mouse LSP1 protein contains 330 amino acids and contains an NH2-terminal acidic domain of approximately 177 amino acids. The COOH-terminal half of the LSP1 protein is rich in basic residues. In this paper we show that LSP1 protein which is immunoprecipitated with anti-LSP1 antibodies from NP-40-soluble lysates of the mouse B-lymphoma cell line BAL17 is associated with actin. In vitro binding experiments using recombinant LSP1 (rLSP1) protein and rabbit skeletal muscle actin show that LSP1 binds along the sides of F-actin but does not bind to G-actin. rLSP1 does not alter the initial polymerization kinetics of actin. The highly conserved COOH-terminal basic domains of mouse and human LSP1 share a significant homology with the 20-kD COOH-terminal F-actin binding fragment of caldesmon. A truncated rLSP1 protein containing the entire COOH-terminal basic domain from residue 179 to 330, but not the NH2-terminal acidic domain binds to F-actin at least as well as rLSP1. When LSP1/CAT fusion proteins are expressed in a LSP1-negative T-lymphoma cell line, only fusion proteins containing the basic COOH-terminal domain associate with the NP-40-insoluble cytoskeleton. These data show that LSP1 binds F-actin through its COOH-terminal basic domain and strongly suggest that LSP1 interacts with the cytoskeleton by direct binding to F-actin. We propose that LSP1 plays a role in mediating cytoskeleton driven responses in lymphocytes such as receptor capping, cell motility, or cell-cell interactions.
Assuntos
Actinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Actinas/ultraestrutura , Sequência de Aminoácidos , Animais , Western Blotting , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/ultraestrutura , Citoesqueleto/ultraestrutura , Imunofluorescência , Cinética , Camundongos , Proteínas dos Microfilamentos , Microscopia Eletrônica , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/ultraestrutura , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: Signals from the B-cell antigen receptor (BCR) help to determine B-cell fate, directing either proliferation, differentiation, or growth arrest/apoptosis. The protein tyrosine phosphatase SHP-1 is known to regulate the strength of BCR signaling. Although the B-cell co-receptor CD22 binds SHP-1, B cells in CD22-deficient mice are much less severely affected than those in SHP-1-deficient mice, suggesting that SHP-1 may also regulate B-cell signaling by affecting other signaling molecules. Moreover, direct substrates of SHP-1 have not been identified in any B-cell signaling pathway. RESULTS: We identified the B-cell transmembrane protein CD72 as a new SHP-1 binding protein and as an in vivo substrate of SHP-1 in B cells. We also defined the binding sites for SHP-1 and the adaptor protein Grb2 on CD72. Tyrosine phosphorylation of CD72 correlated strongly with BCR-induced growth arrest/apoptosis in B-cell lines and in primary B cells. Preligation of CD72 attenuated BCR-induced growth arrest/death signals in immature and mature B cells or B-cell lines, whereas preligation of CD22 enhanced BCR-induced growth arrest/apoptosis. CONCLUSIONS: We have identified CD72 as the first clear in vivo substrate of SHP-1 in B cells. Our results suggest that tyrosine-phosphorylated CD72 may transmit signals for BCR-induced apoptosis. By dephosphorylation CD72. SHP-1 may have a positive role in B-cell signaling. These results have potentially important implications for the involvement of CD72 and SHP-1 in B-cell development and autoimmunity.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Animais , Linfócitos B/imunologia , Divisão Celular , Linhagem Celular , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Fosfotirosina , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Transdução de Sinais , Baço/imunologia , Especificidade por Substrato , Transfecção , Domínios de Homologia de srcRESUMO
The gene for LSP1 is a lymphocyte-specific gene previously isolated by us using a subtractive hybridization technique. LSP1 mRNA is found in normal and transformed B lymphocytes and in normal T lymphocytes but not in transformed T lymphocytes. To study the expression of the mouse LSP1 protein, we prepared a polyclonal antiserum specific for the LSP1 protein. Here we report that the gene for LSP1 was expressed in transformed B-lymphoma cell lines and in normal mouse thymocytes as a protein doublet with apparent molecular masses of 52 and 50.5 kilodaltons when analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel. BW5147 cells transfected with an LSP1 cDNA clone expressed only the 52-kilodalton protein. No LSP1 protein was expressed in nine T-lymphoma cell lines tested. Immunofluorescence studies of intact and permeabilized cells and subcellular fractionation experiments showed that the LSP1 protein was associated with the cytoplasmic side of the plasma membrane in transformed B-lymphoma cell lines and in normal thymocytes. Using a simple filter-binding assay, we showed that recombinant LSP1 protein was Ca2+ binding, as predicted on the basis of its deduced amino acid sequence. On the basis of the particular expression pattern, the subcellular localization, and the Ca2+-binding property of the LSP1 protein, we hypothesize that the LSP1 protein is a lymphocyte-specific component of a signal transduction pathway involved in the regulation of lymphocyte growth.
Assuntos
Proteínas de Ligação ao Cálcio/análise , Linfócitos/análise , Animais , Western Blotting , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Membrana Celular/metabolismo , Transformação Celular Neoplásica , DNA/biossíntese , Imunofluorescência , Linfócitos/ultraestrutura , Proteínas dos Microfilamentos , RNA Mensageiro/biossíntese , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
We have isolated and sequenced a novel lambda 1 constant region related cDNA clone which might represent an allelic variant of the recently described lambda 5 gene. This lambda 5 transcript is present in pre-B cell lines and bone marrow cells, but not in B cell lines, plasma cell lines or in spleen cells. In vitro translation studies show that the translation product contains a signal peptide of approx. 30 amino acids at its N-terminus.
Assuntos
Linfócitos B/metabolismo , Genes de Imunoglobulinas , Cadeias Leves de Imunoglobulina/isolamento & purificação , Sinais Direcionadores de Proteínas/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Recombinante , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , RNA , Translocação GenéticaRESUMO
Expression of LSP1, a 330 amino acid intracellular phosphoprotein, is restricted to lymphocytes, macrophages and neutrophils. In B-lymphoma cell lines LSP1 co-caps with membrane IgM after stimulation with anti-IgM. We used the LSP1+ B-lymphoma cell line WEHI-231/89 and normal lipopolysaccharide treated immature B-cells from Lsp1-/- and wild type mice to determine a role for LSP1 in signaling through membrane IgM. WEHI-231/89 cells were transfected with a truncated LSP1 protein containing the COOH-terminal residues 179-330. The three transfectants expressing the LSP1 truncate were significantly more susceptible to anti-IgM induced apoptosis than the parental cells or G418r control cell lines, while anti-IgM induced growth arrest was not affected. Expression of the LSP1 truncate increased the extent of anti-IgM induced loss of mitochondrial membrane potential, delta(psi)m indicating that LSP1 acts at a early stage in BCR mediated apoptosis. Expression of the LSP1 truncate in WEHI-231/89 cells increased susceptibility to ionomycin induced apoptosis but had no effect on apoptosis induced by nocodazole, sorbitol, C2-ceramide or H2O2. A role for LSP1 in anti-IgM induced apoptosis was confirmed using normal immature B-cells from 129/SvJ-Lsp1-/- mice which were less susceptible to anti-IgM induced apoptosis than those isolated from wild-type 129/SvJ mice. These results suggest that LSP1 regulates a Ca2+-dependent step in the induction phase of anti-IgM mediated apoptosis.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Apoptose , Linfócitos B/patologia , Proteínas de Ligação ao Cálcio/imunologia , Animais , Anticorpos Anti-Idiotípicos/biossíntese , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Feminino , Citometria de Fluxo , Camundongos , Proteínas dos Microfilamentos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Proteínas Recombinantes/biossínteseRESUMO
The in vitro translation of the capped nerve growth factor (NGF) mRNA in wheat germ extracts produced a major 34 kDa NGF precursor with translation starting at the first methionine residue and a minor 29 kDa precursor with translation starting probably at the second methionine. Digestion with the gamma-subunit generated the 22 and 18 kDa intermediates as well as a very small amount of NGF indicating that the gamma-subunit is able to carry out all the processing steps in NGF biosynthesis. The digestion also produced a large amount of a peptide from the N-terminus of the NGF precursors. The discrepancy between the yield of this peptide and of mature NGF emphasizes that inappropriate formation or the lack of formation of disulfide bridges in the NGF precursors, resulting in rapid proteolysis, probably accounts for the low yield of the NGF peptide chains.
Assuntos
Fatores de Crescimento Neural/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Camundongos , Peso Molecular , RNA Mensageiro/metabolismoRESUMO
Cross-linking-induced association of membrane IgM (mIgM) with the cytoskeleton is well documented. However, its functional significance during B cell activation is not yet understood. One possible need for mIgM/cytoskeleton interactions may be to recruit the B cell receptor (BCR)-associated signaling molecules to the cytoskeletal matrix for the propagation of downstream signaling. We first verified whether BCR-associated Ig alpha translocates to the cytoskeleton together with mIgM in polyclonal anti-IgM-treated murine B lymphoma cell line, BAL17.7.1. Co-capping experiments and the purification of the membrane skeleton under conditions that preserve IgM-Ig alphabeta) interactions confirmed that Ig alpha translocates to the cytoskeleton as part of the BCR complex. Furthermore, two BCR-associated kinases that are known to play critical roles in anti-IgM-induced B cell signaling, the src family kinase Lyn and the non-src family kinase Syk, accumulate in the membrane skeleton shortly after BCR cross-linking, when most of IgM and Ig alpha accumulate in this fraction. The kinetics of recruitment of the bulk of Ig alpha, Lyn, and Syk into the membrane skeleton appeared to precede the accumulation of their hypertyrosine-phosphorylated forms, suggesting that activation of the BCR-associated signaling molecules occurs in this fraction. These data suggest that cross-linked mIgM translocating to the membrane skeleton serves as a vehicle for active signaling molecules to be recruited to this vicinity. This may promote B cell activation events by providing high affinity interactions between signaling molecules and their substrates supported by the cytoskeletal matrix.
Assuntos
Antígenos CD/metabolismo , Citoesqueleto/enzimologia , Precursores Enzimáticos/metabolismo , Imunoglobulina M/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores Fc/metabolismo , Quinases da Família src/metabolismo , Animais , Transporte Biológico/imunologia , Antígenos CD79 , Membrana Celular/enzimologia , Membrana Celular/imunologia , Citoesqueleto/imunologia , Precursores Enzimáticos/imunologia , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Ativação Linfocitária , Camundongos , Fosforilação , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Quinase Syk , Quinases da Família src/imunologiaRESUMO
Cross-linking induced interactions between the membrane form of immunoglobulin (mIg) and the cytoskeletal matrix have been described by several groups. To date, the function of mIgM association with the cytoskeleton is not yet understood. Delineation of the molecular basis of these interactions will be instrumental in elucidating their function. We have previously shown that the Ig alpha/beta heterodimer is not required for ligand-induced mIgM binding to the cytoskeleton. In this study, we have investigated the role of other B cell-specific proteins in mediating these interactions. For this, we expressed mIgM in the non-hematopoietic human cervical carcinoma cell line HeLa S3 and verified the capacity of the surface-expressed IgM to interact with the cytoskeletal matrix upon cross-linking with anti-mu chain antibodies. We show here that only the mIgM molecule itself and no other B cell-specific protein(s) is required in mediating mIgM interactions with actin filaments. In an attempt to determine the cytoskeleton-binding site of mIgM we investigated the role of the cytoplasmic tail of mIgM (KVK) in binding the receptor to actin-based microfilaments. Using mutated forms of mIgM expressed in J558L cells, we show here that KVK plays a role in mediating these interactions. The absence of KVK did not, however, completely abrogate mIgM-cytoskeletal interactions, suggesting that there are additional molecular requirements for the ligand-induced mIgM binding to the cytoskeletal matrix.
Assuntos
Citoplasma/imunologia , Citoesqueleto/imunologia , Imunoglobulina M/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Actinas/metabolismo , Animais , Fracionamento Celular , Citoplasma/química , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Gelsolina/farmacologia , Células HeLa , Humanos , Imunoglobulina M/biossíntese , Imunoglobulina M/genética , Camundongos , Plasmocitoma , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos B/biossíntese , Receptores de Antígenos de Linfócitos B/genética , Transfecção , Células Tumorais CultivadasRESUMO
The LSP1 gene is a new lymphocyte-specific gene which is expressed in normal mouse B and T lymphocytes and in transformed B cells but not (or in much smaller amounts) in nine T lymphoma lines tested. No LSP1 mRNA is found in myeloid cells or in liver, kidney, or heart tissue. Inspection of the predicted LSP1 protein sequence reveals the presence of two putative Ca2+-binding domains in the LSP1 protein. Southern blotting analysis of genomic DNA from mouse liver suggests that the LSP1 gene is present as one copy per haploid genome. Similar analysis of genomic DNA extracted from three transformed B cell lines and five transformed T cell lines shows that the absence of LSP1 mRNA in T cell lines is not due to deletion or gross rearrangements of the LSP1 locus. With the use of the mouse LSP1 cDNA as a probe we can detect a cross-hybridizing RNA species in four normal human functional T cell lines but not in three transformed human T cell lines. This suggests that at least part of the DNA sequence and the expression pattern of the LSP1 gene is conserved between mouse and man. These conserved features, together with the particular expression pattern and the protein sequence homologies, suggest that the LSP1 protein is involved in a Ca2+-dependent aspect of normal T cell growth.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Genes , Ativação Linfocitária , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Transformada , Rearranjo Gênico do Linfócito T , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos , Dados de Sequência Molecular , RNA Mensageiro/isolamento & purificação , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
With use of the mouse LSP1 cDNA we isolated a human homologue of the mouse LSP1 gene from a human CTL cDNA library. The predicted protein sequence of human LSP1 is compared with the predicted mouse LSP1 protein sequence and regions of homology are identified in order to predict structural features of the LSP1 protein that might be important for its function. Both the human and mouse LSP1 proteins consist of two domains, an N-terminal acidic domain and a C-terminal basic domain. The C-terminal domains of the mouse and human LSP1 proteins are highly conserved and include several conserved, putative serine/threonine phosphorylation sites. Immunoprecipitation of LSP1 protein from 32P-orthophosphate-loaded cells show that both the mouse and human LSP1 proteins are phosphoproteins. The sequences of the putative Ca2(+)-binding sites present in the N-terminal domain of the mouse LSP1 protein are not conserved in the human LSP1 protein; however, a different Ca2(+)-binding site may exist in the human protein, indicating a functional conservation rather than a strict sequence conservation of the two proteins. The expression of the human LSP1 gene follows the same pattern as the expression of the mouse LSP1 gene. Southern analysis of human genomic DNA shows multiple LSP1-related fragments of varying intensity in contrast to the simple pattern found after similar analysis of mouse genomic DNA. By using different parts of the human LSP1 cDNA as a probe, we show that most of these multiple bands contain sequences homologous to the conserved C-terminal region of the LSP1 cDNA. This suggests that there are several LSP1-related genes present in the human genome.
Assuntos
Linfócitos B/fisiologia , Proteínas de Ligação ao Cálcio/genética , Fosfoproteínas/genética , Linfócitos T/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Clonagem Molecular , DNA/genética , Genes , Humanos , Camundongos , Proteínas dos Microfilamentos , Dados de Sequência Molecular , RNA Mensageiro/genética , Mapeamento por RestriçãoRESUMO
To determine whether B cells from New Zealand Black (NZB) and (New Zealand Black x New Zealand White)F1 (NZB/W) mice possess intrinsic defects that lead to altered immune responsiveness, we purified resting B cells from these mice and compared their surface phenotype and function with those of resting B cells isolated from BALB/c and DBA/2 nonautoimmune mouse strains. Flow cytometric analysis of freshly isolated resting B cells revealed that NZB and NZB/W resting B cells are conventional B2-type cells similar to their nonautoimmune counterparts. Despite this, resting B cells from young NZB and NZB/W mice express lower levels of CD23 on their surface and aberrant levels of intracellular IgM. Upon stimulation, resting B cells from young NZB and NZB/W mice demonstrate increased proliferation, IgM secretion, or enhanced expression of costimulatory molecules in response to a variety of different T cell-derived stimuli, including cytokines and signals generated through CD40. Therefore, B cell hyper-responsiveness to T cell stimuli is immunodominant or codominant in NZB/W mice. Taken together, our results suggest that intrinsic B cell hyper-responsiveness may play a role in the pathogenesis of autoimmune disease in NZB and NZB/W mice. The increased clonal expansion of these B cells together with increased Ig production and enhanced costimulatory capacity serve to amplify the immune response. In the context of normal but incomplete T cell tolerance, B cell hyperresponsiveness to the limited signals provided by partially tolerant T cells may be sufficient to yield an autoantibody response.
Assuntos
Subpopulações de Linfócitos B/imunologia , Interfase/imunologia , Nefrite Lúpica/imunologia , Ativação Linfocitária , Camundongos Endogâmicos NZB/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Subpopulações de Linfócitos B/metabolismo , Antígenos CD40/imunologia , Cruzamentos Genéticos , Citocinas/farmacologia , Suscetibilidade a Doenças , Feminino , Imunoglobulina M/biossíntese , Imunofenotipagem , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Nefrite Lúpica/genética , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Cooperação Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos Endogâmicos NZB/genética , Baço/citologiaRESUMO
The effect of poly(ADP-ribosylation) on calf thymus topoisomerase type II reactions has been investigated. Unknotting of phage P4 head DNA, and relaxation and catenation of supercoiled PM2 DNA are inhibited. We conclude that the inhibition results from poly(ADP-ribosylation) on the following grounds. Firstly, the enzyme poly(ADP-ribose) (PADPR) synthetase and NAD are required, secondly, the competitive synthetase inhibitor nicotinamide abolishes topoisomerase inhibition, and thirdly, the polymer alone is not inhibitory. The mechanism of inhibition appears to be disruption of the strand cleavage reaction. A topoisomerase-DNA complex can be formed that upon treatment with protein denaturant at low ionic strength results in strand cleavage. The amount of DNA present in such a cleavable-complex progressively decreased following pretreatment of topoisomerase type II with PADPR synthetase and increasing concentrations of NAD. Treatment of the pre-formed complex with NAD and PADPR synthetase had no effect on its salt-induced dissociation. This suggests that either poly(ADP-ribosylation) has no influence on dissociation of topoisomerase, in contrast to association, or topoisomerase is not accessible to the synthetase when bound to DNA. Similar data were obtained with calf thymus type I topoisomerase.
Assuntos
Açúcares de Nucleosídeo Difosfato/biossíntese , Poli Adenosina Difosfato Ribose/biossíntese , Timo/enzimologia , Inibidores da Topoisomerase II , Animais , Bovinos , DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Cinética , NAD/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Nicotinamide (10 mM) or 3-aminobenzamide (5 mM) added at the onset of phytohemagglutinin (PHA) treated human lymphocyte cultures provoke a marked inhibition of the PHA induced DNA synthesis and cell proliferation as well as of poly(ADPR) polymerase activity. When the inhibitors of poly(ADPR) polymerase are added at a later stage of culture (48 h) no inhibition of the stimulation of DNA synthesis and cell proliferation by PHA in human lymphocyte cultures is observed. The intervention of ADP ribosylation at the initiation of DNA synthesis is suggested.
Assuntos
Ativação Linfocitária/efeitos dos fármacos , NAD+ Nucleosidase/metabolismo , Fito-Hemaglutininas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Benzamidas/farmacologia , Replicação do DNA/efeitos dos fármacos , Humanos , Niacinamida/farmacologiaRESUMO
When rat pancreatic polynucleosomes were poly(ADP-ribosyl)ated with purified calf thymus poly(ADP-ribose) polymerase and examined by electron microscopy, a relaxation of their native zigzag structure was observed. At high ionic strengths control nucleosomes condensed into 250-A-thick fibers, but poly(ADP-ribosyl)ated polynucleosomes did not; they showed a close resemblance to chromatin depleted of histone H1. The relaxed state of poly(ADP-ribosyl)ated polynucleosomes was also confirmed by sedimentation velocity analysis. Histone H1 was found to be the major histone acceptor of poly(ADP-ribose). Poly(ADP-ribose) linked to histone H1 did not seem to cause its dissociation from the chromatin, but it impaired significantly its effect on chromatin condensation.
Assuntos
Cromatina/ultraestrutura , Histonas/metabolismo , Açúcares de Nucleosídeo Difosfato/metabolismo , Nucleossomos/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Animais , Microscopia Eletrônica , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , UltracentrifugaçãoRESUMO
The interaction between purified calf thymus poly(ADP-ribose) polymerase and its activating co-purified DNA (sDNA) was investigated by electron microscopy. We have shown that the enzyme-DNA complex possesses a nucleosome-like structure. The enzyme-bound DNA (sDNA) was found to be enriched in single-stranded regions and branched structures, presumed to be replication forks. The auto-ribosylated polymerase as well as the branched poly(ADP-ribose) formed were visualized by dark field electron microscopy during the auto-ADP-ribosylation reaction and the possible mechanism of this phenomenon is discussed.
Assuntos
DNA/metabolismo , NAD+ Nucleosidase/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Bovinos , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Microscopia Eletrônica , Processamento de Proteína Pós-TraducionalRESUMO
A slight DNA topoisomerase I activity was detected in highly purified poly(ADP-Rib)polymerase prepared from calf thymus. This copurified activity was found to be suppressed under conditions where the poly(ADP-ribosylation) reaction occurs in the presence of NAD. Purified topoisomerase I from calf thymus was shown to be ADP-ribosylated by poly(ADP-Rib) polymerase purified from the same tissue. Poly(ADP-ribosylation) of topoisomerase I produces an inhibition of the enzymatic activity in parallel to the extent of ADP-ribosylation. The fact that a slight poly(ADP-Rib) polymerase activity was also found to copurify with a topoisomerase I preparation and that topoisomerase I activity can be modified by ADP-ribosylation, may suggest a spatial and functional correlation of these two enzymes in chromatin.
Assuntos
DNA Topoisomerases Tipo I/isolamento & purificação , NAD+ Nucleosidase/isolamento & purificação , Poli(ADP-Ribose) Polimerases/isolamento & purificação , Timo/enzimologia , Animais , Bovinos , Fenômenos Químicos , Química , Técnicas In Vitro , Cinética , Poli Adenosina Difosfato Ribose/farmacologia , Ligação Proteica , Inibidores da Topoisomerase IRESUMO
Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.
Assuntos
Linfócitos B/análise , Células-Tronco Hematopoéticas/análise , Cadeias lambda de Imunoglobulina/análise , Cadeias mu de Imunoglobulina/análise , Animais , Linhagem Celular , Rearranjo Gênico , Genes de Imunoglobulinas , Soros Imunes/imunologia , Cadeias lambda de Imunoglobulina/imunologia , CamundongosRESUMO
Expression of the COOH-terminal residues 179-330 of the LSP1 protein in the LSP1(+) B-cell line W10 increases anti-IgM- or ionomycin-induced apoptosis, suggesting that expression of this LSP1 truncate (B-LSP1) interferes with a Ca(2+)-dependent step in anti-IgM signaling. Here we show that inhibition of Ca(2+)-dependent conventional protein kinase C (cPKC) isoforms with Gö6976 increases anti-IgM-induced apoptosis of W10 cells and that expression of B-LSP1 inhibits translocation of PKCbetaI but not of PKCbetaII or PKCalpha to the plasma membrane. The increased anti-IgM-induced apoptosis is partially reversed by overexpression of PKCbetaI. This shows that the B-LSP1-mediated inhibition of PKCbetaI leads to increased anti-IgM-induced apoptosis. Expression of constitutively active PKCbetaI protein in W10 cells activates the mitogen-activated protein kinase ERK2, whereas expression of B-LSP1 inhibits anti-IgM-induced activation of ERK2, suggesting that anti-IgM-activated PKCbetaI is involved in the activation of ERK2 and that inhibition of ERK2 activation contributes to the increased anti-IgM-induced apoptosis. Pull-down assays show that LSP1 interacts with PKCbetaI but not with PKCbetaII or PKCalpha in W10 cell lysates, while in vitro LSP1 and B-LSP1 bind directly to PKCbetaI. Thus, B-LSP1 is a unique reagent that binds PKCbetaI and inhibits anti-IgM-induced PKCbetaI translocation, leading to inhibition of ERK2 activation and increased apoptosis.
Assuntos
Anticorpos Anti-Idiotípicos/metabolismo , Apoptose , Proteínas de Ligação ao Cálcio/metabolismo , Inibidores Enzimáticos/metabolismo , Isoenzimas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Ativação Enzimática , Humanos , Proteínas dos Microfilamentos , Proteína Quinase C beta , Células Tumorais CultivadasRESUMO
The B cell Ag receptor complex consists of membrane-associated Ig (mIg), Ig alpha, and Ig beta, associated molecules that have been implicated in transducing the activation signal that occurs upon receptor cross-linking. The role of the Ig alpha beta heterodimer in mediating binding to the cytoskeleton is unknown. We studied the ligand-induced association of mIgM with the cytoskeleton following receptor cross-linking in mIgM-expressing B lymphoma lines by biochemical assays, FACS analysis, and electron microscopy. Cytoskeletal association is not detected in unstimulated cells, but occurs rapidly upon anti-IgM-mediated cross-linking. Ig alpha is absent from the cytoskeleton-mIgM complex. To further analyze the possible role of Ig alpha beta in cytoskeletal binding, a surface Ig alpha beta-negative plasmacytoma line was transfected with a mutant form of mIgM (IgM-MutA). IgM-MutA is expressed on the surface despite the lack of Ig alpha beta, and the cytoskeletal binding occurred to a similar extent as in Ig-alpha-positive cell lines. In another transfectant expressing a mutated form of human mIgM (YS:VV), which does not have the capacity to bind to Ig alpha beta, the association of the receptor with the cytoskeleton appeared to be more extensive (100%) and faster than with mouse mIgM. These data indicate that Ig-associated Ig alpha beta proteins are not required for mIgM association with the cytoskeleton.
Assuntos
Linfócitos B/metabolismo , Proteínas do Citoesqueleto/imunologia , Imunoglobulina M/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Animais , Linfócitos B/imunologia , Sítios de Ligação de Anticorpos , Western Blotting , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina M/imunologia , Cinética , Linfoma de Células B/imunologia , Substâncias Macromoleculares , Camundongos , Receptores de Antígenos de Linfócitos B/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
Interaction of calf thymus poly(ADP-ribose(ADPR] polymerase with a copurified DNA fraction (sDNA) was investigated. Electron microscopic studies of sDNA which appeared to be a powerful poly(ADPR) polymerase activator have shown that the purified poly(ADPR) polymerase-DNA complexes possess a "nucleosome like structure", with DNA wrapping around the enzyme molecule. Examination of the DNA linked poly(ADPR) polymerase preparations revealed the presence of Y-structures in sDNA. The enrichment in the sDNA fraction of the Y shape DNA suggests the existence of replication fork structures in the poly(ADPR) polymerase linked DNA and or in the vicinity of the enzyme. With increasing auto-poly(ADP-ribosyl)ation the enzyme molecule becomes much denser, increases in size and detaches from the DNA. When poly(ADPR) formed was purified and examined by electron microscopy, branched polymers of different sizes were observed. The formation of these polymers may explain the size gained by poly ADP-ribosylated enzyme molecules. When the interaction of poly(ADPR) polymerase with the plasmid pBR 322 was tested, a slight contamination of our enzyme preparation with topoisomerase I was detected. The contaminant topoisomerase I activity, however, was completely abolished by ADP-ribosylation. Further experiments with purified calf thymus topoisomerase I confirmed that this enzyme loses its activity following ADP-ribosylation with poly(ADPR) polymerase. These results may suggest that ADP-ribosylation of topoisomerase I can be one of the regulatory mechanisms of its activity. Furthermore, these results confirm that a topoisomerase I contaminant does not interfere with the ADP-ribosylation experiments of purified poly(ADPR) polymerase preparation.