RESUMO
A complementation strategy was developed to define the signaling pathways activated by the Bcr-Abl tyrosine kinase. Transformation inactive point mutants of Bcr-Abl were tested for complementation with c-Myc. Single point mutations in the Src-homology 2 (SH2) domain, the major tyrosine autophosphorylation site of the kinase domain, and the Grb-2 binding site in the Bcr region impaired the transformation of fibroblasts by Bcr-Abl. Hyperexpression of c-Myc efficiently restored transformation activity only to the Bcr-Abl SH2 mutant. These data support a model in which Bcr-Abl activates at least two independent pathways for transformation. This strategy may be useful for discerning signaling pathways activated by other oncogenes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Transformação Celular Neoplásica , Proteínas de Fusão bcr-abl/genética , Genes abl , Genes myc , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Proteínas de Fusão bcr-abl/fisiologia , Proteína Adaptadora GRB2 , Expressão Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Fosforilação , Mutação Puntual , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/fisiologia , Ratos , Retroviridae/fisiologia , Transdução de Sinais , Transfecção , Tirosina/metabolismoRESUMO
The human bcr gene encodes a protein with serine/threonine kinase activity, CDC24/dbl homology, a GAP domain, and an SH2-binding region. However, the precise physiological functions of BCR are unknown. Coexpression of BCR with the cytoplasmic protein-tyrosine kinase encoded by the c-fes proto-oncogene in Sf-9 cells resulted in stable BCR-FES protein complex formation and tyrosine phosphorylation of BCR. Association involves the SH2 domain of FES and a novel binding domain localized to the first 347 amino acids of the FES N-terminal region. Deletion of the homologous N-terminal BCR-binding domain from v-fps, a fes-related transforming oncogene, abolished transforming activity and tyrosine phosphorylation of BCR in vivo. Tyrosine phosphorylation of BCR in v-fps-transformed cells induced its association with GRB-2/SOS, the RAS guanine nucleotide exchange factor complex. These data provide evidence that BCR couples the cytoplasmic protein-tyrosine kinase and RAS signaling pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Receptores ErbB/metabolismo , Proteínas de Fusão gag-onc/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas , Animais , Linhagem Celular , Transformação Celular Neoplásica , Proteína Adaptadora GRB2 , Fatores de Troca do Nucleotídeo Guanina , Humanos , Proteínas Oncogênicas/biossíntese , Proteínas Oncogênicas/isolamento & purificação , Oncogenes , Fosforilação , Fosfotirosina , Ligação Proteica , Proteínas Tirosina Quinases/biossíntese , Proteínas/isolamento & purificação , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcr , Proteínas Recombinantes de Fusão , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Deleção de Sequência , Spodoptera , Transfecção , Tirosina/análogos & derivados , Tirosina/metabolismo , Fatores ras de Troca de Nucleotídeo GuaninaRESUMO
Loss of function of Bruton's tyrosine kinase (Btk) results in X-linked immunodeficiencies characterized by a broad spectrum of signaling defects, including those dependent on Src family kinase-linked cell surface receptors. A gain-of-function mutant, Btk*, induces the growth of fibroblasts in soft agar and relieves the interleukin-5 dependence of a pre-B-cell line. To genetically define Btk signaling pathways, we used a strategy to either activate or inactivate Src family kinases in fibroblasts that express Btk*. The transformation potential of Btk* was dramatically increased by coexpression with a partly activated c-Src mutant (E-378 --> G). This synergy was further potentiated by deletion of the Btk Src homology 3 domain. Downregulation of Src family kinases by the C-terminal Src kinase (Csk) suppressed Btk* activation and biological potency. In contrast, kinase-inactive Csk (K-222 --> R), which functioned as a dominant negative molecule, synergized with Btk* in biological transformation. Activation of Btk* correlated with increased phosphotyrosine on transphosphorylation and autophosphorylation sites. These findings suggest that the Src and Btk kinase families form specific signaling units in tissues in which both are expressed.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Western Blotting , Proteína Tirosina Quinase CSK , Linhagem Celular , Humanos , Mapeamento de Peptídeos , Fosfopeptídeos/química , Fosfopeptídeos/isolamento & purificação , Fosforilação , Mutação Puntual , Proteínas Tirosina Quinases/isolamento & purificação , Ratos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transfecção , Quinases da Família srcRESUMO
We have cloned a novel kinase (STY) from an embryonal carcinoma cell line. Sequence analysis of the STY cDNA reveals that it shares sequence homology with serine/threonine-type kinases and yet the bacterial expression product of the STY cDNA appears to have serine-, threonine-, and tyrosine-phosphorylating activities. The predicted STY protein is highly basic and contains a putative nuclear localization signal. During differentiation, two new mRNAs were detected in addition to the embryonic transcript.
Assuntos
Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Diferenciação Celular , Clonagem Molecular , DNA/genética , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
A novel protein kinase, the Esk kinase, has been isolated from an embryonal carcinoma (EC) cell line by using an expression cloning strategy. Sequence analysis of two independent cDNA clones (2.97 and 2.85 kb) suggested the presence of two Esk isoforms in EC cells. The esk-1 cDNA sequence predicted an 857-amino-acid protein kinase with a putative membrane-spanning domain, while the esk-2 cDNA predicted an 831-amino-acid kinase which lacked this domain. In adult mouse cells, esk mRNA levels were highest in tissues possessing a high proliferation rate or a sizeable stem cell compartment, suggesting that the Esk kinase may play some role in the control of cell proliferation or differentiation. As anticipated from the screening procedure, bacterial expression of the Esk kinase reacted with antiphosphotyrosine antibodies on immunoblots. Furthermore, in in vitro kinase assays, the Esk kinase was shown to phosphorylate both itself and the exogenous substrate myelin basic protein on serine, threonine, and tyrosine residues, confirming that the Esk kinase is a novel member of the serine/threonine/tyrosine family of protein kinases.
Assuntos
Proteínas Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Compartimento Celular , Membrana Celular/enzimologia , Clonagem Molecular , Citoplasma/enzimologia , DNA/genética , Expressão Gênica , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/genética , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Fosfotirosina , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , Alinhamento de Sequência , Especificidade por Substrato , Células Tumorais Cultivadas , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
TEL is a member of the Ets family of transcription factors which are frequently rearranged in human leukemia. The mechanism of TEL-mediated transformation, however, is unknown. We report the cloning and characterization of a chromosomal translocation associated with acute myeloid leukemia which fuses TEL to the ABL tyrosine kinase. The TEL-ABL fusion confers growth factor-independent growth to the marine hematopoietic cell line Ba/F3 and transforms Rat-1 fibroblasts and primary murine bone marrow cells. TEL-ABL is constitutively tyrosine phosphorylated and localizes to the cytoskeleton. A TEL-ABL mutant containing an ABL kinase-inactivating mutation is not constitutively phosphorylated and is nontransforming but retains cytoskeletal localization. However, constitutive phosphorylation, cytoskeletal localization, and transformation are all dependent upon a highly conserved region of TEL termed the helix-loop-helix (HLH) domain. TEL-ABL formed HLH-dependent homo-oligomers in vitro, a process critical for tyrosine kinase activation. These experiments suggest that oligomerization of TEL-ABL mediated by the TEL HLH domain is required for tyrosine kinase activation, cytoskeletal localization, and transformation. These data also suggest that oligomerization of Ets proteins through the highly conserved HLH domain may represent a previously unrecognized phenomenon.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Leucemia Mieloide/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 12 , Proteínas do Citoesqueleto/metabolismo , Primers do DNA/química , Regulação Neoplásica da Expressão Gênica , Sequências Hélice-Alça-Hélice , Humanos , Masculino , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-ets , RNA Mensageiro/genética , RNA Neoplásico/genética , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Translocação Genética , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Functional human carcinoembryonic antigen (CEA)-like genes have been shown to be present in the mouse. Southern analyses of murine DNA using both human and murine CEA complementary DNA probes have revealed the presence of multiple CEA-like genes, while analyses of RNA from different mouse tissues showed CEA-like transcripts in adult colon and liver. Furthermore, a CEA-like protein, immunoprecipitable with a rabbit polyclonal serum raised against human CEA, has been detected in adult murine colon tissue. Several murine CEA complementary DNA clones have been isolated from a murine colon complementary DNA library, and characterization of one such clone demonstrates that both the N-terminal and the internal domains have been conserved between the two species. The existence of a murine counterpart of CEA strengthens the case for an essential function for this human tumor marker and provides an experimentally amenable system for elucidation of its biological properties.
Assuntos
Antígeno Carcinoembrionário/genética , Animais , Sequência de Bases , Antígeno Carcinoembrionário/análise , DNA/análise , Glicosilação , Humanos , Camundongos , Dados de Sequência Molecular , Transcrição GênicaRESUMO
We identified TMPRSS2 as a gene that is down-regulated in androgen-independent prostate cancer xenograft tissue derived from a bone metastasis. Using specific monoclonal antibodies, we show that the TMPRSS2-encoded serine protease is expressed as a Mr 70,000 full-length form and a cleaved Mr 32,000 protease domain. Mutation of Ser-441 in the catalytic triad shows that the proteolytic cleavage is dependent on catalytic activity, suggesting that it occurs as a result of autocleavage. Mutational analysis reveals the cleavage site to be at Arg-255. A consequence of autocatalytic cleavage is the secretion of the protease domain into the media by TMPRSS2-expressing prostate cancer cells and into the sera of prostate tumor-bearing mice. Immunohistochemical analysis of clinical specimens demonstrates the highest expression of TMPRSS2 at the apical side of prostate and prostate cancer secretory epithelia and within the lumen of the glands. Similar luminal staining was detected in colon cancer samples. Expression was also seen in colon and pancreas, with little to no expression detected in seven additional normal tissues. These data demonstrate that TMPRSS2 is a secreted protease that is highly expressed in prostate and prostate cancer, making it a potential target for cancer therapy and diagnosis.
Assuntos
Androgênios/fisiologia , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Serina Endopeptidases/metabolismo , Animais , Catálise , Meios de Cultura , Regulação para Baixo , Epitélio/enzimologia , Epitélio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos SCID , Transplante de Neoplasias , Neoplasias Hormônio-Dependentes/enzimologia , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Próstata/enzimologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Receptores Androgênicos/fisiologia , Serina Endopeptidases/genética , Transdução de Sinais/fisiologia , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Bcr - Abl is the molecule responsible for both the transformation phenotype and the resistance to chemotherapeutic drugs found in chronic myelogenous leukemia (CML) cells. Wild-type HL-60, a transformed pro-myelocytic cell line, is very susceptible to apoptosis-inducing agents. We show here that expression of Bcr - Abl in HL-60 cells rendered them extremely resistant to apoptosis induced by a wide variety of agents. The anti-apoptotic effect of Bcr - Abl was found to be independent of the phase of the cell cycle. Treatment with antisense oligonucleotides directed to bcr decreased the expression of the ectopic bcr - abl and restored susceptibility to apoptosis. Double mutations affecting the autophosphorylation site and the phosphotyrosine-binding motif (FLVRES) have been previously shown to impair the transforming activity of Bcr - Abl in fibroblasts and hematopoietic cells, however HL-60 cells expressing this double mutant molecule exhibited the same level of resistance to apoptosis as those expressing the wild-type Bcr - Abl. Interestingly, wild type and mutant Bcr - Abl induced in HL-60 cells a dramatic down regulation of Bcl-2 and increased the levels of Bcl-xL. The level of Bax did not change in response to the presence of Bcr - Abl. Antisense oligonucleotides targeted to bcl-x downregulated the expression of Bcl-x, and increased the susceptibility of HL-60. Bcr - Abl cells to staurosporine. Importantly, HL-60 cells overexpressing Bcl-xL showed higher expression of Bcl-xL but lower resistance to apoptosis when compared to HL-60. Bcr - Abl cells. The results described here show that Bcr - Abl is a powerful mammalian anti-apoptotic molecule and can act independently of Bcl-2. Bcl-xL, however, seems to participate in part in Bcr - Abl-mediated resistance to apoptosis in HL-60 cells.
Assuntos
Apoptose , Proteínas de Fusão bcr-abl/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima , Resistência a Múltiplos Medicamentos/genética , Proteínas de Fusão bcr-abl/genética , Células HL-60 , Humanos , Oligonucleotídeos Antissenso/farmacologia , Proteína bcl-XRESUMO
Biliary-glycoprotein (BGP), a cell adhesion molecule related to carcinoembryonic antigen (CEA), has been shown to exist as several alternatively spliced isoforms. Here we show that BGPa and BGPb are phosphorylated in the chronic myelogenous leukaemia cell line KG-1, which constitutively expresses several BGP isoforms, and Chinese hamster LR-73 cells transfected with the cDNAs encoding BGPa and BGPb. The phosphorylation can be augmented with the protein tyrosine phosphatase inhibitor ammonium vanadate and with TPA (an activator of protein kinase C). Phospho-amino acid analysis of phosphorylated BGPs demonstrated that phosphorylation occurs on serine, threonine and tyrosine residues. Phosphorylation reactions carried out in in vitro membrane preparations from KG-1 cells revealed a close association of BGP proteins with membrane associated protein tyrosine kinases. These observations suggest an association of BGP proteins with signal transduction molecules which is regulated by alternative splicing of the cytoplasmic domain.
Assuntos
Moléculas de Adesão Celular/metabolismo , Glicoproteínas/metabolismo , Tirosina/análogos & derivados , Sequência de Aminoácidos , Antígenos CD , Antígeno Carcinoembrionário/metabolismo , Moléculas de Adesão Celular/química , Glicoproteínas/química , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosfoproteínas/metabolismo , Fosfotirosina , Alinhamento de Sequência , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
We used genetic strategies which have been proven valuable to decipher signaling pathways in comparatively simple organisms such as Drosophila and Caenorhabditis elegans, to dissect signaling network activated by tyrosine kinases in mammals. The strategy was developed further towards a generally applicable expression cloning system to identify signal transducers in tyrosine kinase pathways. This system is based on the ability of downstream acting genes to rescue the transformation phenotype of partial loss-of-function mutants of BCR-ABL which still retain tyrosine kinase activity. Using this strategy we have previously shown that overexpression of c-Myc and Cyclin D1 can rescue a signaling defective SH2 mutant of BCR-ABL for transformation. In an unbiased approach to identify new compensating genes, a cDNA library was introduced by retroviral infection into fibroblasts which express the BCR-ABL SH2 mutant. CDNA clones, capable of rescuing the SH2 mutant for transformation should result in colony formation in soft agar. A PCR approach was used to recover these compensating genes from the genomic DNA of the transformed fibroblasts. Sequencing analysis of the initial cDNAs identified three known genes, the adapter molecule Shc, the kinases SPRK and p38 MAPK. These genes have been found to interact functionally with BCR-ABL for fibroblast and hematopoietic cell transformation. Currently, we are constructing and screening new libraries to identify novel genes which complement the BCR-ABL SH2 mutant. Our results demonstrate that this cloning approach is an effective means of identifying and characterizing signaling molecules that function in specific signaling pathways. This in turn may identify specific targets for mechanism-based therapeutic intervention to block altered signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Proteínas de Fusão bcr-abl/genética , Proteínas Tirosina Quinases/fisiologia , Transdução de Sinais/genética , Animais , Bioensaio , Caenorhabditis elegans , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Transformação Celular Viral/genética , Drosophila melanogaster , Proteínas de Fusão bcr-abl/fisiologia , Camundongos , Oncogenes/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas/genética , Proteínas/fisiologia , Retroviridae/genética , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
We examined the potential of generating an immune response against Philadelphia chromosome-positive acute lymphoblastic leukemia. The immunostimulatory molecules chosen for this study were the cytokines IL-2 and GM-CSF and the costimulatory ligand CD80 (B7.1). We used a murine model based on a BALB/c pre-B cell line, BM185wt, in which leukemia is induced by the p185 BCR-ABL oncogenic product, which reproduces Philadelphia chromosome-positive ALL. BM185wt cells were transduced with retroviral vectors and the transduced clones expressing mIL-2, mGM-CSF, or mCD80 were used for challenge. Expression of the immunomodulators by BM185 cells was correlated with delay in leukemia development in immunocompetent mice, but not in immunodeficient mice, indicating an immune response against the modified leukemia cells. Expression of CD80 caused leukemia rejection in 50% of the cohort, which was associated with the CD4+ and CD8+ T cell-dependent development of anti-leukemia cytotoxic T lymphocytes. Furthermore, mice surviving the BM185/CD80 challenge or preimmunized with irradiated BM185/CD80 cells developed an immune response against subsequent challenge with the parental leukemia. These studies provide evidence that immunotherapeutic approaches can be developed for the treatment of ALL.
Assuntos
Antígeno B7-1/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Interleucina-2/genética , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Vacinas/imunologia , Adjuvantes Imunológicos/genética , Animais , Antígenos CD4/imunologia , Antígenos CD8/imunologia , Divisão Celular/imunologia , Testes Imunológicos de Citotoxicidade , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID/imunologia , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Transdução Genética/genética , Células Tumorais CultivadasRESUMO
BCR is an interesting signaling protein, whose cellular function is currently unknown. Its biochemical properties include serine kinase activity, SH2-binding activity, and a GTPase-activating activity. The SH2-binding activity is particularly interesting because it may link BCR to signaling pathways involving SH2-containing molecules. Since tyrosine phosphorylation of BCR has been detected in CML-derived cell lines and since tyrosine-phosphorylated BCR shows increased affinity toward certain SH2 domains, it seems particularly important to further characterize this activity. This chapter described a simple purification scheme for partial purification of BCR, which can be used to assess in vitro kinase and SH2-binding activities.
Assuntos
GTP Fosfo-Hidrolases/análise , GTP Fosfo-Hidrolases/metabolismo , Proteínas Oncogênicas/análise , Proteínas Oncogênicas/metabolismo , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Cromatografia de Afinidade/métodos , Cromossomos Humanos Par 22 , Cromossomos Humanos Par 9 , Eletroforese em Gel de Poliacrilamida/métodos , Éxons , GTP Fosfo-Hidrolases/isolamento & purificação , Humanos , Cinética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Metionina/metabolismo , Proteínas Oncogênicas/genética , Radioisótopos de Fósforo , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcr , Técnica de Diluição de Radioisótopos , Proteínas Recombinantes/análise , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Spodoptera , Radioisótopos de Enxofre , Transfecção/métodos , Translocação GenéticaRESUMO
Treatment of primary cultures of oligodendrocyte precursors with calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, caused the phosphorylation of two intermediate filament components, nestin and vimentin. Phosphoamino acid analysis demonstrated that phosphorylation took place mainly on serine and to a lesser extent on threonine residues. In addition, calyculin A treatment caused a shift in the distribution of the two proteins from the Triton-X-100 insoluble fraction to the detergent soluble fraction as demonstrated by immunoblotting. This redistribution, which was evident within 15 min after treatment and was nearly completed by 90 min, was accompanied by a disruption of the intermediate filament network. Thus, both nestin and vimentin retracted from the cytoplasmic processes to form a large perikaryal ring as shown by immunocytochemical analysis. Both morphological and biochemical changes were reversed 2-5 hr after removal of calyculin A from the culture medium.
Assuntos
Proteínas de Filamentos Intermediários/metabolismo , Proteínas do Tecido Nervoso , Oligodendroglia/metabolismo , Oxazóis/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Células-Tronco/metabolismo , Aminoácidos/metabolismo , Animais , Animais Recém-Nascidos/fisiologia , Western Blotting , Células Cultivadas , Citoesqueleto/metabolismo , Densitometria , Imunofluorescência , Toxinas Marinhas , Nestina , Oligodendroglia/efeitos dos fármacos , Fosforilação , Ratos , Células-Tronco/efeitos dos fármacos , Frações Subcelulares/metabolismo , Vimentina/metabolismoRESUMO
A sural nerve biopsy from a patient with benign monoclonal IgM kappa gammopathy and sensory-motor demyelinative neuropathy, revealed marked loss of myelinated fibers and focal axonal degeneration as well as widespread demyelination and remyelination with onion-skin formation. Almost all myelinated fibers displayed characteristic widening of the myelin lamellae as well as excessive thickness and/or exuberant outfoldings of myelin, reminiscent of that seen in tomaculous neuropathy. Many endoneurial capillaries were lined by fenestrated endothelium, indicating breakdown of a normal blood-nerve barrier. The endoneurium contained large amounts of extracellular proteinaceous material. Immunofluorescence and immunoelectron microscopy performed on the nerve of the patient, demonstrated selective deposition of IgM kappa gammaglobulin, exclusively in the areas of splittings of the myelin lamellae. Schwann cells contained cytoplasmic myelin debris labelled with IgM kappa only. In the indirect immunofluorescence and immunoelectron microscopy, serum of the patient reacted with the whole thickness of compact peripheral myelin of a normal human nerve. There was no immunoreactivity with the central myelin, Schwannoma cells, glial cells, axons or neurons. Demonstration of the selective presence of monoclonal IgM in widened lamellae of myelinated fibers, as well as bound to the internalized myelin debris in Schwann cells and macrophages, indicates a pathogenetic role of monoclonal paraprotein in myelin injury. Demyelination is promoted by development of endothelial fenestrations in the endoneurial capillaries and breakdown of the blood-nerve barrier.
Assuntos
Anticorpos Monoclonais/análise , Doenças Desmielinizantes/complicações , Imunoglobulina M/análise , Paraproteinemias/imunologia , Doenças do Sistema Nervoso Periférico/complicações , Idoso , Doenças Desmielinizantes/patologia , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Microscopia Eletrônica , Microscopia Imunoeletrônica , Paraproteinemias/complicações , Paraproteinemias/patologia , Doenças do Sistema Nervoso Periférico/patologia , Nervo Sural/ultraestruturaRESUMO
Myelin-associated glycoprotein (MAG) is a myelin-specific cell adhesion molecule of the immunoglobulin supergene family and is tyrosine-phosphorylated in the developing brain. To define the role of MAG in signal transduction, the tyrosine phosphorylation sites were analyzed. The major tyrosine phosphorylation residue was identified as Tyr-620, which was found to interact specifically with the SH2 domains of phospholipase C (PLC gamma). This domain may represent a novel protein binding motif that can be regulated by tyrosine phosphorylation. MAG also specifically bound the Fyn tyrosine kinase, suggesting that MAG serves as a docking protein that allows the interaction between different signaling molecules.
Assuntos
Proteínas da Mielina/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Oligodendroglia/metabolismo , Tirosina/análogos & derivados , Animais , Sequência de Bases , Primers do DNA/química , Camundongos , Dados de Sequência Molecular , Glicoproteína Associada a Mielina , Mapeamento de Peptídeos , Fosforilação , Fosfotirosina , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn , Ratos , Proteínas Recombinantes , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Tirosina/metabolismoRESUMO
The alternative splicing of myelin-associated glycoprotein (MAG) mRNA generates two isoforms that harbor distinct potential phosphorylation sites in their cytoplasmic tails. Here we characterize the in vivo phosphorylation of MAG isoforms in NIH 3T3 cells transfected with the cDNAs encoding the two isoforms of MAG. Our results demonstrate that the longer isoform, L-MAG, is phosphorylated constitutively mainly on serine, but also on threonine and tyrosine residues. This phosphorylation is subject to change by 12-O-tetradecanoylphorbol 13-acetate (TPA) and ammonium vanadate, but not by dibutyryl-cyclic AMP. The shorter isoform, S-MAG, is constitutively phosphorylated only on serine residues. While TPA and dibutyryl-cyclic AMP have no detectable effect, ammonium vanadate induces tyrosine and threonine phosphorylation in S-MAG. 32P labeling of v-src-transformed NIH 3T3 cells that express L-MAG also show that L-MAG is likely to be an in vivo substrate for pp60v-src tyrosine kinase activity. These results demonstrate that both MAG isoforms are phosphorylated in a heterologous cell system and that this phosphorylation is subject to pharmacological manipulation.
Assuntos
Proteínas da Mielina/genética , Animais , Bucladesina/farmacologia , Linhagem Celular , Transformação Celular Neoplásica , AMP Cíclico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Proteínas da Mielina/isolamento & purificação , Proteínas da Mielina/metabolismo , Glicoproteína Associada a Mielina , Fosforilação , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
Oncogenic signals induce cellular proliferation by deregulating the cell division cycle. Cyclin D1, a regulator of G1-phase progression, acts synergistically with ABL oncogenes in transforming fibroblasts and hematopoietic cells in culture. Synergy with v-Abl depended on a motif in cyclin D1 that mediates its binding to the retinoblastoma protein, suggesting that ABL oncogenes in part mediate their mitogenic effects via a retinoblastoma protein-dependent pathway. Overexpression of cyclin D1, but not cyclin E, rescued a signaling-defective src-homology 2 (SH2) domain mutant of BCR-ABL for transformation of cells in culture and malignant tumor formation in vivo. These results demonstrate that cyclin D1 can provide essential signals for malignant transformation in concert with an activated tyrosine kinase.
Assuntos
Transformação Celular Neoplásica/genética , Ciclinas/metabolismo , Genes abl , Proteínas Oncogênicas/metabolismo , Transdução de Sinais/genética , Animais , Células da Medula Óssea , Células Cultivadas , Ciclina D1 , Ciclinas/genética , Proteínas de Fusão bcr-abl/genética , Leucemia Experimental/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Oncogênicas/genética , Ligação Proteica , Proteínas Recombinantes , Proteína do Retinoblastoma/metabolismo , Domínios de Homologia de srcRESUMO
Bcr-Abl oncoproteins are responsible for the pathogenesis of human leukemias with a reciprocal chromosome translocation t(9;22). The amino-terminal Bcr sequence has a potential to form a homotetramer (tetramer domain), and destructions of the tetramer domain cause a complete loss of biological activities in Bcr-Abl. Here we show that Bcr-Abl in which the tetramer domain is replaced with glutathione S-transferase (GST) with a dimerizing ability (GST/Bcr-Abl-(Delta1-160)) can no longer induce an interleukin-3 (IL-3) independence in Ba/F3 cells or transform mouse bone marrow cells but still retains by 30-40% the ability to transform Rat1 cells. Compared with the wild type Bcr-Abl, autophosphorylation of GST/Bcr-Abl-(Delta1-160) in vivo was reduced by more than 50%. The Grb-2 binding to GST/Bcr-Abl-(Delta1-160) was 50% reduced in Rat1 cells and undetectable in Ba/F3 cells. In Rat1 cells expressing GST/Bcr-Abl-(Delta1-160), phosphotyrosine contents of p62 and Shc were 70% decreased.