RESUMO
Non-Hodgkin lymphoma (NHL) is a genetically heterogeneous disease with several oncogenic events implicated in the transformation of normal developing B lymphocytes. The objective of this study was to elucidate the signal transduction-based antitumor mechanism(s) of action for the anti-CD40 monoclonal antibody dacetuzumab (SGN-40) in NHL. We report that dacetuzumab activates two distinct proapoptotic signaling pathways, overcoming transformation events key to the pathogenesis of NHL. Dacetuzumab-mediated CD40 signaling constitutively activated the nuclear factor-κB and mitogen-activated protein kinase signaling pathways producing the sustained downregulation of B-cell lymphoma 6 (BCL-6), an oncoprotein implicated in lymphomagenesis. Loss of BCL-6 resulted in c-Myc downregulation and activation of a transcriptional program characteristic of early B-cell maturation, concomitant with reduced proliferation and cell death. In a second mechanism, dacetuzumab signaling induced the expression of the proapoptotic p53 family member TAp63α and downstream proteins associated with the intrinsic and extrinsic apoptotic machinery. Dacetuzumab was synergistic in combination with DNA-damaging chemotherapeutic drugs, correlating with TAp63α upregulation. Furthermore, dacetuzumab augmented the activity of rituximab in combination with multiple chemotherapies in the xenograft models of NHL. The ability of dacetuzumab signaling to circumvent oncogenic events and potentiate the activity of chemotherapy regimens provides a unique therapeutic approach to NHL.
Assuntos
Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linfoma não Hodgkin/tratamento farmacológico , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma não Hodgkin/patologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
BACKGROUND: CD70 is an ideal target for antibody-based therapies because of its aberrant high expression in renal carcinomas and non-Hodgkin lymphomas and its highly restricted expression in normal tissues. The expression profiling of CD70 in carcinomas has been limited because of the lack of a CD70-specific reagent that works in formalin-fixed paraffin-embedded (FFPE) tissues. METHODS: We generated murine monoclonal antibodies (mAbs) specific for CD70 and validated their specificity by western blot analysis and developed a protocol for immunohistochemistry on FFPE tissues. CD70+ tumour cell lines were used for testing the anti-tumour activity of the anti-CD70 antibody-drug conjugate, SGN-75. RESULTS: We report novel detection of CD70 expression in multiple cancers including pancreatic (25%), larynx/pharynx (22%), melanoma (16%), ovarian (15%), lung (10%), and colon (9%). Our results show that pancreatic and ovarian tumour cell lines, which express high levels of endogenous or transfected CD70, are sensitive to the anti-tumour activity of SGN-75 in vitro and in vivo. CONCLUSION: Development of murine mAbs for robust and extensive screening of FFPE samples coupled with the detection of anti-tumour activity in novel indications provide rationale for expanding the application of SGN-75 for the treatment of multiple CD70 expressing cancers.
Assuntos
Aminobenzoatos/administração & dosagem , Ligante CD27/imunologia , Imunoconjugados/uso terapêutico , Oligopeptídeos/administração & dosagem , Neoplasias Ovarianas/terapia , Neoplasias Pancreáticas/terapia , Animais , Anticorpos Monoclonais/uso terapêutico , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos NusRESUMO
SGN-40 is a therapeutic antibody targeting CD40, which induces potent anti-lymphoma activities via direct apoptotic signalling cells and by cell-mediated cytotoxicity. Here we show antibody-dependent cellular phagocytosis (ADCP) by macrophages to contribute significantly to the therapeutic activities and that the antitumour effects of SGN-40 depend on Fc interactions.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Antígenos CD40/imunologia , Linfoma/tratamento farmacológico , Macrófagos/imunologia , Receptores Fc/fisiologia , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos SCID , FagocitoseRESUMO
SGN-30, a monoclonal antibody with activity against CD30+ malignancies, is currently in phase II clinical evaluation for treatment of Hodgkin's disease (HD) and anaplastic large cell lymphoma. The mechanisms underlying SGN-30's antitumor activity were investigated using cDNA array of L540 cells. SGN-30 treatment activated NF-kappaB and modulation of several messages including the growth regulator p21WAF1/CIP1 (p21) and cellular adhesion marker ICAM-1. p21 protein levels increased coincident with growth arrest and Annexin V/PI staining in treated HD cells. To determine if SGN-30-induced growth arrest would sensitize tumor cells to chemotherapeutics used against HD, L540cy and L428 cells were exposed to SGN-30 in combination with a panel of cytotoxic agents and resultant interactions quantified by the Combination Effects Method. Interactions between SGN-30 and all cytotoxic agents examined were additive or better. These in vitro data translated to increased efficacy of SGN-30 and bleomycin against L540cy tumor xenografts. In addition to direct cell killing, SGN-30 affects growth arrest and drug sensitization through growth regulating and proapoptotic machinery. Importantly, these data suggest that SGN-30 can enhance the efficacy of standard chemotherapies used to treat patients with CD30+ malignancies.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Doença de Hodgkin/tratamento farmacológico , Doença de Hodgkin/imunologia , Antígeno Ki-1/imunologia , Transdução de Sinais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Apoptose/efeitos dos fármacos , Bleomicina/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/imunologia , Linhagem Celular Tumoral , Quimioterapia Combinada , Doença de Hodgkin/genética , Humanos , Antígeno Ki-1/efeitos dos fármacos , Camundongos , Camundongos SCID , NF-kappa B/efeitos dos fármacos , NF-kappa B/imunologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sensibilidade e Especificidade , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Using glutathione S-transferase Syk fusion proteins, we evaluated the mode of platelet FcgammaRII tyrosine phosphorylation induced by FcgammaRII cross-linking or anti-CD9 monoclonal antibodies (mAb). The N-terminal SH2 domain of Syk (Syk-N-SH2), the C-terminal SH2 domain of Syk (Syk-C-SH2), and the domain having both the N- and C-terminal SH2 of Syk (Syk-NC-SH2) all bound to tyrosine-phosphorylated FcgammaRII with FcgammaRII cross-linking. In the case of anti-CD9 mAb-induced platelet activation, only Syk-C-SH2 and Syk-NC-SH2 bound to tyrosine-phosphorylated FcgammaRII. Since the SH2 domain is specific for a particular structure containing phosphotyrosine, these findings suggest that only one tyrosine residue in the immunoreceptor tyrosine-based activation motif (ITAM) is phosphorylated with anti-CD9 mAb, and that both are phosphorylated with FcgammaRII cross-linking. Synthetic peptides corresponding to the ITAM of human platelet FcgammaRII with the N-terminal tyrosine residue phosphorylated (N-P) or the C-terminal tyrosine residue phosphorylated (C-P), were used. N-P more potently dissociated Syk-C-SH2 from tyrosine-phosphorylated FcgammaRII than C-P, suggesting that the N-terminal tyrosine residue is phosphorylated upon anti-CD9 mAb-induced activation. Furthermore, these findings imply that Syk-N-SH2 binds to the phosphorylated C-terminal tyrosine residue of ITAM, and Syk-C-SH2 to the N-terminal tyrosine. Taken together, our findings suggest that FcgammaRII-dependent platelet activation without FcgammaRII dimerization, such as with anti-CD9 mAb, is distinct from that induced by FcgammaRII cross-linking.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Plaquetas/imunologia , Receptores de IgG/imunologia , Sequência de Aminoácidos , Antígenos CD/química , Ligação Competitiva , Precursores Enzimáticos/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Fosforilação , Ativação Plaquetária , Proteínas Tirosina Quinases/química , Receptores de IgG/química , Proteínas Recombinantes de Fusão/química , Quinase Syk , Tirosina/química , Domínios de Homologia de srcRESUMO
CDw150, a receptor up-regulated on activated T or B lymphocytes, has a key role in regulating B cell proliferation. Patients with X-linked lymphoproliferative disease have mutations in a gene encoding a protein, DSHP/SAP, which interacts with CDw150 and is expressed in B cells. Here we show that CDw150 on B cells associates with two tyrosine-phosphorylated proteins, 59 kDa and 145 kDa in size. The 59-kDa protein was identified as the Src-family kinase Fgr. The 145-kDa protein is the inositol polyphosphate 5'-phosphatase, SH2-containing inositol phosphatase (SHIP). Both Fgr and SHIP interact with phosphorylated tyrosines in CDw150's cytoplasmic tail. Ligation of CDw150 induces the rapid dephosphorylation of both SHIP and CDw150 as well as the association of Lyn and Fgr with SHIP. CD95/Fas-mediated apoptosis is enhanced by signaling via CDw150, and CDw150 ligation can override CD40-induced rescue of CD95-mediated cell death. The ability of CDw150 to regulate cell death does not correlate with serine phosphorylation of the Akt kinase, but does correlate with SHIP tyrosine dephosphorylation. Thus, the CDw150 receptor may function to regulate the fate of activated B cells via SHIP as well as via the DSHP/SAP protein defective in X-linked lymphoproliferative disease patients.
Assuntos
Apoptose , Linfócitos B/metabolismo , Glicoproteínas/metabolismo , Imunoglobulinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases , Receptor fas/metabolismo , Antígenos CD , Proteínas de Transporte/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Transtornos Linfoproliferativos/imunologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Agregação de Receptores , Receptores de Superfície Celular , Transdução de Sinais , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Domínios de Homologia de src , Quinases da Família srcRESUMO
Propagation of signals from the T cell antigen receptor (TCR) involves a number of adaptor molecules. SH2 domain-containing protein 76 (SLP-76) interacts with the guanine nucleotide exchange factor Vav to activate the nuclear factor of activated cells (NF-AT), and its expression is required for normal T cell development. We report the cloning and characterization of a novel Grb2-like adaptor molecule designated as Grb2-related protein of the lymphoid system (GrpL). Expression of GrpL is restricted to hematopoietic tissues, and it is distinguished from Grb2 by having a proline-rich region. GrpL can be coimmunoprecipitated with SLP-76 but not with Sos1 or Sos2 from Jurkat cell lysates. In contrast, Grb2 can be coimmunoprecipitated with Sos1 and Sos2 but not with SLP-76. Moreover, tyrosine-phosphorylated LAT/pp36/38 in detergent lysates prepared from anti-CD3 stimulated T cells associated with Grb2 but not GrpL. These data reveal the presence of distinct complexes involving GrpL and Grb2 in T cells. A functional role of the GrpL-SLP-76 complex is suggested by the ability of GrpL to act alone or in concert with SLP-76 to augment NF-AT activation in Jurkat T cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares , Fosfoproteínas/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Complexo CD3/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica/genética , Humanos , Células Jurkat , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFATC , Prolina/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Ativação Transcricional/genética , Domínios de Homologia de src/genéticaRESUMO
Histologic evidence of intestinal spirochetosis (IS) was found in 22 of 41 (53.7%) rectal biopsy specimens from homosexual men attending a sexually transmitted diseases clinic. Serpulina pilosicoli was cultured from 11 of the IS-positive biopsy specimens (50%) and from 2 specimens (10.5%) in which spirochetes were not observed. The association between seeing spirochetes in biopsy specimens and isolating S. pilosicoli was statistically significant, clearly indicating that this spirochete is the agent of IS.
Assuntos
Brachyspira/isolamento & purificação , Enteropatias/diagnóstico , Reto/microbiologia , Infecções por Spirochaetales/diagnóstico , Biópsia , Homossexualidade Masculina , Humanos , MasculinoRESUMO
CD22 is a B cell-restricted surface molecule which may play an important role in interactions between B cells and other cells and in regulating signals through the B cell receptor (BCR) complex. Here we have examined whether the mouse is a suitable in vivo model for studying CD22 functions. In primary and secondary lymphoid organs of adult mice CD22 is on all mature B cells, including resting IgM+IgD+ B cells, IgG+ HSA(lo) memory B cells, syndecan+ plasma cells and CD5+ B cells, but it is not on immature IgM+IgD- B cells. Biochemical analysis revealed that murine CD22 is associated with the IgM receptor in some, but not all, CD22+ B leukemic and lymphoma cell lines; as with human CD22, murine CD22 is rapidly phosphorylated on tyrosine after ligation of the BCR. In the CD22- murine pro-B cell line, FEMCL, CD22 expression was inducible by treatment with phorbol 12-myristate 13-acetate. A genomic fragment of the cd22b allele containing 1.3 kb 5' of exon 1 was sequenced in order to identify potential DNA regulatory elements in the CD22 promoter region. Consensus sequences for transcription factor binding sites including PU.1, AP-1, AP-2, C/EBP and SP-1 were present, but no classical TATA elements or initiator motifs were evident at relevant positions. The 1.3-kb promoter fragment 5' of exon 1 was sufficient for directing basal promoter activity in B and T cells. There was no significant sequence similarity between the murine and human cd22 gene promoters, although both contain repetitive elements and Sp-1 and AP1 binding sites. Thus, murine CD22 shares a number of features with human CD22 and the mouse provides a suitable model system for elucidating the function of CD22 in vivo.
Assuntos
Antígenos CD/química , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/química , Antígenos de Diferenciação de Linfócitos B/genética , Linfócitos B/metabolismo , Moléculas de Adesão Celular , Lectinas , Regiões Promotoras Genéticas/imunologia , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos B/biossíntese , Linfócitos B/efeitos dos fármacos , Sequência de Bases , Linhagem Celular , Éxons/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Mensageiro/análise , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The B-lymphocyte-restricted glycoprotein CD22 is expressed on mature IgM+IgD+ B cells, and is capable of binding to ligands on T and B cells. CD22 can interact with both the B-cell antigen receptor (BCR) complex and signalling molecules, including the protein tyrosine phosphatase SHP1 (PTP1C, SHP), a putative negative regulator of BCR signalling. Thus CD22 may facilitate interactions with lymphocytes and regulate the threshold of BCR signalling. To define the in vivo function of CD22, we generated CD22-deficient mice. Here we show that CD22 is required for normal antibody responses to thymus-independent antigens and regulates the lifespan of mature B cells.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Linfócitos B/fisiologia , Moléculas de Adesão Celular , Lectinas , Animais , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/genética , Apoptose , Linfócitos B/imunologia , Cálcio/metabolismo , Senescência Celular/genética , Senescência Celular/fisiologia , Deleção de Genes , Ativação Linfocitária , Camundongos , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Baço/citologia , Timo/citologiaAssuntos
Antígenos CD/metabolismo , Linfócitos B/imunologia , Comunicação Celular , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Modelos Imunológicos , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
We have identified a Ser/Thr kinase associated with the B cell receptor (BCR) complex as protein kinase C mu (PKC mu). PKC mu activity is up-regulated after cross-linking the BCR and CD19 on B cells, and PKC mu co-precipitates with Syk and phospholipase C-gamma 1/2 (PLC gamma 1/2). In vitro phosphorylation of fusion proteins showed that both Syk and PLC gamma 1 are potential substrates of PKC mu in vivo. Analysis of mutants of the chicken B cell line DT40 deficient in either Syk, Lyn, Btk, or PLC gamma 2 revealed that BCR-induced activation of PKC mu, like activation of PLC gamma 2, requires Syk and is partially regulated by Btk, but is Lyn independent. PKC mu can down-regulate the ability of Syk to phosphorylate PLC gamma 1 in vitro. Thus, PKC mu may function in a negative feedback loop regulating BCR-initiated signaling cascades.
Assuntos
Linfócitos B/fisiologia , Proteína Quinase C/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Precursores Enzimáticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/metabolismo , Fosfolipase C gama , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Quinase Syk , Células Tumorais Cultivadas , Fosfolipases Tipo C/metabolismoRESUMO
Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinase-dependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2 are early events in collagen-induced activation. We recently proposed that collagen-signaling in platelets involves a receptor or a receptor-associated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk. In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor gamma-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein. In addition we demonstrate an association between the Fc receptor gamma-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor gamma-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin alpha2beta1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, FcgammaRIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor gamma-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin alpha2beta1 and involves phosphorylation of the Fc receptor gamma-chain, its association with Syk and subsequent phosphorylation of phospholipase Cgamma2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.
Assuntos
Plaquetas/efeitos dos fármacos , Colágeno/farmacologia , Precursores Enzimáticos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de IgG/metabolismo , Precursores Enzimáticos/genética , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Testes de Precipitina , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão , Transdução de Sinais , Quinase Syk , Tirosina/metabolismo , Domínios de Homologia de srcRESUMO
Antigen receptor ligation on lymphocytes activates protein tyrosine kinases and phospholipase C-gamma (PLC-gamma) isoforms. Glutathione S-transferase fusion proteins containing the C-terminal Src-homology 2 [SH2(C)] domain of PLC-gamma1 bound to tyrosyl phosphorylated Syk. Syk isolated from antigen receptor-activated B cells phosphorylated PLC-gamma1 on Tyr-771 and the key regulatory residue Tyr-783 in vitro, whereas Lyn from the same B cells phosphorylated PLC-gamma1 only on Tyr-771. The ability of Syk to phosphorylate PLC-gamma1 required antigen receptor ligation, while Lyn was constitutively active. An mCD8-Syk cDNA construct could be expressed as a tyrosyl-phosphorylated chimeric protein tyrosine kinase in COS cells, was recognized by PLC-gamma1 SH2(C) in vitro, and induced tyrosyl phosphorylation of endogenous PLC-gamma1 in vivo. Substitution of Tyr-525 and Tyr-526 at the autophosphorylation site of Syk in mCD8-Syk substantially reduced the kinase activity and the binding of this variant chimera to PLC-gamma1 SH2(C) in vitro; it also failed to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. In contrast, substitution of Tyr-348 and Tyr-352 in the linker region of Syk in mCD8-Syk did not affect the kinase activity of this variant chimera but almost completely eliminated its binding to PLC-gamma1 SH(C) and completely eliminated its ability to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. Thus, an optimal kinase activity of Syk and an interaction between the linker region of Syk with PLC-gamma1 are required for the tyrosyl phosphorylation of PLC-gamma1.
Assuntos
Linfócitos B/metabolismo , Precursores Enzimáticos/metabolismo , Isoenzimas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Fosfolipases Tipo C/metabolismo , Domínios de Homologia de src/genética , Sequência de Aminoácidos , Animais , Linfócitos B/enzimologia , Sequência de Bases , Linhagem Celular , Sequência Conservada , Ativação Enzimática , Precursores Enzimáticos/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/genética , Dados de Sequência Molecular , Mutagênese Insercional , Fosfolipase C gama , Fosforilação , Plasmídeos , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos B/genética , Especificidade por Substrato , Quinase Syk , Fosfolipases Tipo C/genética , Tirosina/genética , Proteína-Tirosina Quinase ZAP-70RESUMO
Cross-linking B cell antigen receptor (BCR) elicits early signal transduction events, including activation of protein tyrosine kinases, phosphorylation of receptor components, activation of phospholipase C-gamma (PLC-gamma), and increases in intracellular free Ca2+. In this article, we report that cross-linking the BCR led to a rapid translocation of cytosolic protein tyrosine phosphatase (PTP) 1C to the particulate fraction, where it became associated with a 140-150-kD tyrosyl-phosphorylated protein. Western blotting analysis identified this 140-150-kD protein to be CD22. The association of PTP-1C with CD22 was mediated by the NH2-terminal Src homology 2 (SH2) domain of PTP-1C. Complexes of either CD22/PTP-1C/Syk/PLC-gamma(1) could be isolated from B cells stimulated by BCR engagement or a mixture of hydrogen peroxidase and sodium orthovanadate, respectively. The binding of PLC-gamma(1) and Syk to tyrosyl-phosphorylated CD22 was mediated by the NH2-terminal SH2 domain of PLC-gamma(1) and the COOH-terminal SH2 domain of Syk, respectively. These observations suggest that tyrosyl-phosphorylated CD22 may downmodulate the activity of this complex by dephosphorylation of CD22, Syk, and/or PLC-gamma(1). Transient expression of CD22 and a null mutant of PTP-1C (PTP-1CM) in COS cells resulted in an increase in tyrosyl phosphorylation of CD22 and its interaction with PTP-1CM. By contrast, CD22 was not tyrosyl phosphorylated or associated with PTP-1CM in the presence of wild-type PTP-1C. These results suggest that tyrosyl-phosphorylated CD22 may be a substrate for PTP-1C regulates tyrosyl phosphorylation of CD22.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/imunologia , Moléculas de Adesão Celular , Precursores Enzimáticos/metabolismo , Isoenzimas/metabolismo , Lectinas , Ativação Linfocitária , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fosfolipases Tipo C/metabolismo , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/genética , Sequência de Bases , Cálcio/metabolismo , Reagentes de Ligações Cruzadas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Fosfolipase C gama , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Transdução de Sinais , Quinase Syk , Transfecção , Células Tumorais Cultivadas , Domínios de Homologia de srcRESUMO
Activation of human platelets by cross-linking of the platelet low-affinity IgG receptor, the Fc gamma receptor IIA (Fc gamma-RIIA), or by collagen is associated with rapid phosphorylation on tyrosine of the non-receptor tyrosine kinase syk. Phosphorylation is still observed, albeit sometimes reduced, in the presence of a combination of a protein kinase C inhibitor, Ro 31-8220, and the intracellular calcium chelator, BAPTA-AM, demonstrating independence from phosphoinositide-specific phospholipase C (PLC) activity. In contrast, the combination of Ro 31-8220 and BAPTA-AM completely inhibits phosphorylation of syk in thrombin-stimulated platelets. Phosphorylation of syk increases its autophosphorylation activity measured in a kinase assay performed on syk immunoprecipitates. Fc gamma-RIIA also undergoes phosphorylation in syk immunoprecipitates from platelets activated by cross-linking of Fc gamma-RIIA but not by collagen, suggesting that it associates with the kinase. Consistent with this, tyrosine-phosphorylated Fc gamma-RIIA is precipitated by a glutathione S-transferase (GST) fusion protein containing the tandem src homology (SH2) domains of syk from Fc gamma-RIIA- but not collagen-activated cells. Two uncharacterized tyrosine-phosphorylated proteins of 40 and 65 kDa are uniquely precipitated by a GST fusion protein containing the tandem syk-SH2 domains in collagen-stimulated platelets. A peptide based on the antigen recognition activation motif (ARAM) of Fc gamma-RIIA, and phosphorylated on the two tyrosine residues found within this region, selectively binds syk from lysates of resting platelets; this interaction is not seen with a non-phosphorylated peptide. Kinase assays on Fc gamma-RIIA immunoprecipitates reveal the constitutive association of an unidentified kinase activity in resting cells which phosphorylates a 67 kDa protein. Syk is not detected in Fc gamma-RIIA immunoprecipitates from resting cells but associates with the receptor following activation and, together with Fc gamma-RIIA, is phosphorylated in the kinase assay in vitro. These results demonstrate that syk is activated by Fc gamma-RIIA cross-linking and collagen, independent of PLC, suggesting that it may have an important role in the early events associated with platelet activation. The association of syk with Fc gamma-RIIA appears to be mediated through the tandem SH2 domains in syk and the ARAM motif of Fc gamma-RIIA. A similar interaction may underlie the response to collagen, suggesting that its signalling receptor contains an ARAM motif.
Assuntos
Plaquetas/metabolismo , Colágeno/farmacologia , Precursores Enzimáticos/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Receptores de IgG/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Quelantes/farmacologia , Primers do DNA/química , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/química , Humanos , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Coelhos , Receptores de IgG/química , Quinase Syk , Fosfolipases Tipo C/metabolismoRESUMO
Baby hamster kidney cells transfected with murine CD22 (mCD22) mediate adhesion to B- and T-lineage cells. To further characterize mCD22-mediated cell adhesion, we generated a panel of recombinant globulins (Rg) consisting of different extracellular Ig-like (Ig) domains of mCD22. FACS analysis using these mCD22.Rgs revealed that ligands for mCD22 are expressed on both B and T cell lines and also normal B and T cells. In B-lineage cells, the expression of mCD22 ligands began on sIgM- pre-B cells in bone marrow. The ligand-binding site of mCD22 for ligands was mapped to Ig domains 1 and 2: mCD22.Rgs containing Ig domains 1 and 2 bound target cells and immunoprecipitated sets of glycoproteins similar to Rgs containing Ig domains 1 to 3 or all 7 CD22 Ig domains, whereas Rgs containing Ig domains 2 to 3 or 3 to 7 did not bind either B or T cells. Furthermore, B cells apparently expressed higher levels of mCD22 ligands than that of T cells, suggesting a potential competition for CD22 binding between ligands expressed on the same B cell and those expressed on another B cell or T cells. Immunoprecipitation experiments using the mCD22.Rgs identified mCD22 itself and the B cell-specific isoform of mCD45RA (B220) as two of the mCD22 ligands expressed on B cells. Thus, mCD22 may potentially regulate B cell activation through interactions with itself or mCD45RA/B220.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos B/imunologia , Imunoglobulinas/imunologia , Lectinas , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/química , Antígenos de Diferenciação de Linfócitos B/genética , Sequência de Bases , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Células Cultivadas , Cricetinae , Primers do DNA , Técnicas de Transferência de Genes , Imunoglobulinas/química , Imunoglobulinas/genética , Ligantes , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Análise de Sequência , Lectina 2 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
The 72-kDa spleen tyrosine kinase (Syk) and Src-family kinase p53/56Lyn (Lyn) contribute to signaling via the B-cell antigen receptor complex. Here we show that Syk and Lyn from human B lymphocytes can interact directly. Syk and Lyn coimmunoprecipitated from mature and activated B-cell lines, and gel-purified Syk and Lyn reassociated in vitro, demonstrating their direct interaction. This Syk-Lyn interaction may be dependent on the stage of B-cell differentiation, since Syk-Lyn associations were not detected in pre-B and myeloma cell lines and Syk from an immature B-cell line did not reassociate with Lyn in vitro. Serine/threonine kinase activity was also associated with Syk. Crosslinking of cell surface IgM led to rapid activation of both tyrosine and serine/threonine protein kinase activities that resulted in phosphorylation in vitro of proteins coprecipitating with Syk--in particular, a serine/threonine phosphorylated protein 120 kDa in size (pp120). Several phosphoproteins, including one of 72 kDa and one of 120 kDa, coprecipitated with phospholipase C-gamma 1 (PLC gamma 1). Sequential immunoprecipitation identified the 72-kDa protein associated with PLC gamma 1 as Syk. The 120-kDa serine/threonine phosphorylated protein that coprecipitated with PLC gamma 1 resembled the Syk-associated pp120 by several criteria. Thus, pp120 may serve as a link between Syk and PLC gamma 1, coupling the B-cell antigen receptor to the phosphatidylinositol pathway.
Assuntos
Linfócitos B/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Baço/enzimologia , Sequência de Aminoácidos , Linhagem Celular , Precursores Enzimáticos/metabolismo , Humanos , Imunoglobulina M/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Fosfolipase C gama , Testes de Precipitina , Ligação Proteica , Baço/citologia , Quinase Syk , Fosfolipases Tipo C/metabolismo , Quinases da Família src/metabolismoRESUMO
Hallux valgus is one of the most common fore-foot problem in civilized populations. 49 feet of 33 cases, 8 males and 25 females with an average age of 50 years old, were treated surgically from 1987 to 1992 for hallux valgus and followed up for an average of 47.8 months, comprising 20 feet with McBride's soft tissue procedure and 29 feet of Mitchell metatarsal osteotomy. The clinical criteria for follow up included pain relief, cosmetic out look, shoe wearing comfort, metatarsophalangeal motion etc. 87.5% of the cases in the McBride's group and 95% of the cases in the Mitchell group were satisfied with the clinical results. The immediate post-operative metatarsophalangeal (MP) angle and intermetatarsal (IM) angle were both well corrected, but there was a significantly higher recurrence rate of hallux valgus in the group with the McBride's procedure (P < 0.05). One case in the McBride's group had the complication of hallux varus and one case from the Mitchell group had delayed union. The Mitchell bony procedure has a more stable result compared to the McBride's soft tissue procedure for the correction of moderate hallux valgus, and the modified Mitchell osteotomy is less invasive, easier to perform, easier to care for, and has more satisfactory long-term results.
Assuntos
Hallux Valgus/cirurgia , Adolescente , Adulto , Idoso , Feminino , Seguimentos , Hallux Valgus/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Osteotomia/métodos , Complicações Pós-Operatórias , RadiografiaRESUMO
Accessory molecules play an important role in the regulation of lymphocyte activation mediated by the B-cell antigen receptor (BCR). CD22 is one such accessory molecule expressed on B-lineage cells. Here, Che-Leung Law and colleagues review current knowledge on the structure-function relationship between CD22 and the BCR, discuss the role of CD22 as a cell-adhesion molecule and suggest models for potential in vivo functions of CD22.