RESUMEN
Megasphaera cerevisiae is a Gram-negative obligate anaerobe that causes turbidity and off-flavour and aroma in beer. Seven isolates of M. cerevisiae were obtained worldwide, and their extractable surface antigens were focused upon to determine if there is more than one serogroup of this bacterium. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of ethylenediaminetetraacetic acid (EDTA) bacterial extracts revealed a predominant protein with apparent molecular weights of 46,000, 45,000, and 43,000 for three, two, and two isolates, respectively. When mouse anti-serum generated against any of the EDTA extracts was reacted with denatured bacterial proteins in immunoblots, all bacterial isolates exhibited extensive cross-reactivity involving three antigens, one being the major EDTA-extractable protein. In contrast, when the sera were tested for surface reactivity with intact bacteria, three cross-reactivity groups were observed, with the groups individually comprised of bacteria having the same size major EDTA-extractable surface protein. When BALB/c mice immunized with a bacterium from each of the three serogroups were used for monoclonal antibody (Mab) hybridoma production, bacterial surface-reactive Mabs were obtained whose reactivities parallel the three polyclonal antibody-defined serogroups. Through combining these surface-reactive Mabs, it will be possible to rapidly detect and identify beer contamination by M. cerevisiae belonging to any serogroup.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Cerveza/microbiología , Cocos Anaerobios Gramnegativos/clasificación , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Antígenos de Superficie , Ácido Edético , Electroforesis en Gel de Poliacrilamida , Fluoroinmunoensayo , Cocos Anaerobios Gramnegativos/inmunología , Hibridomas , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Peso Molecular , SerotipificaciónRESUMEN
Requirements for T cell activation are not fully established. One model is that receptor occupancy and down-regulation are essential for activation, and another, not necessarily mutually exclusive, model is that sustained signals are important. Here we examine the importance of signal duration in T cell activation. First, we demonstrate that immobilized, but not soluble cross-linked, Abs to CD3 stimulate degranulation by CTL. The cross-linked Abs are not deficient in their ability to signal since they stimulate the same tyrosine phosphorylation pattern as immobilized Ab, but it is very transient relative to that stimulated by immobilized Ab. Furthermore, novel decreased migratory forms of Lck occur to a significant extent only after stimulation with immobilized Abs. A dramatic difference in the duration of signals is very evident when mitogen-activated protein kinase (MAPK) activity is examined. Immobilized anti-CD3 stimulates very high levels of MAPK activation that is still detectable 1 h after stimulation. In contrast, cross-linked Ab stimulates only transient and incomplete activation of MAPK. Taken together, these results suggest that TCR engagement and induction of tyrosine phosphorylation alone are not sufficient for T cell activation and that the duration of TCR-stimulated signals is critical to attain a functional response.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Degranulación de la Célula/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/enzimología , Linfocitos T Citotóxicos/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Calcio/metabolismo , Células Clonales , Citoesqueleto/enzimología , Citoesqueleto/inmunología , Citoesqueleto/metabolismo , Activación Enzimática/inmunología , Líquido Intracelular/metabolismo , Activación de Linfocitos/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteína Quinasa C/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Solubilidad , Linfocitos T Citotóxicos/fisiología , Tirosina/metabolismoRESUMEN
Antibodies to either CD3 or CD45 have been shown to induce dramatic changes in cell morphology, increased tyrosine phosphorylation of cellular proteins, and the association of a subset of these proteins with the tyrosine kinase Lck. The current study was initiated to determine the identity of the tyrosine-phosphorylated 70-80 kDa protein that becomes Lck-associated after stimulation with anti-CD45 or anti-CD3. We demonstrate that the cytoskeletal protein paxillin becomes tyrosine-phosphorylated when cells are plated on immobilized antibodies specific for CD45 or CD3. Only tyrosine-phosphorylated paxillin is associated with Lck, suggesting that the association is through the SH2 domain of Lck. Consistent with this we demonstrate that the SH2 domain of Lck binds tyrosine-phosphorylated paxillin. In contrast, the association of paxillin with the FAK-related kinase Pyk2 was found to be constitutive and not altered by the phosphorylation of either protein. Finally, we establish that the phosphorylation of paxillin is dependent on the expression of Lck. Taken together, these results demonstrate that paxillin is physically associated with kinases from two different families in T cells and suggest that paxillin may function as an adaptor protein linking cellular signals with cytoskeletal changes during T cell activation.
Asunto(s)
Complejo CD3/inmunología , Proteínas del Citoesqueleto/metabolismo , Antígenos Comunes de Leucocito/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T Citotóxicos/fisiología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Línea Celular , Citoesqueleto/fisiología , Quinasa 2 de Adhesión Focal , Ratones , Organofosfatos/farmacología , Paxillin , Fosforilación , Fosfotirosina/análisis , Pruebas de Precipitina , Dominios Homologos src/fisiologíaRESUMEN
Stimulation through the TCR is known to induce tyrosine phosphorylation of a number of proteins, which leads to functional activation of T cells. Identification of the substrates that become phosphorylated and defining their interactions with other signaling molecules will provide insight into the mechanisms controlling T cell activation. Focal adhesion kinase (FAK) and the recently described Pyk2 kinase are homologous members of a non-receptor protein tyrosine kinase family. FAK has been shown to become phosphorylated upon TCR stimulation, but its role, if any, in T cell activation remains to be defined. Although Pyk2 has been shown to play a role in neuronal cell activation stimulated through G-protein-coupled receptors, a role in T cell activation has not been described. In this study we show that FAK and Pyk2 are two of the major 115-to-120-kDa proteins that become tyrosine phosphorylated in T cells following TCR complex stimulation. Furthermore, coincident with the increase in tyrosine phosphorylation, we show an association of these kinases with the SH2 domain of the tyrosine kinase Lck in vivo. The increase in tyrosine phosphorylation of both FAK and Pyk2, however, occurs in Lck-deficient cells suggesting that phosphorylation of both of these kinases does not require Lck. Taken together, these results suggest that FAK and Pyk2, perhaps in coordination with Lck, play a role in T cell activation.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Tirosina/metabolismo , Animales , Línea Celular , Quinasa 1 de Adhesión Focal , Quinasa 2 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Activación de Linfocitos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Ratones , Ratones Endogámicos C57BL , Fosforilación , Linfocitos T/inmunología , Dominios Homologos srcRESUMEN
Fibronectin has been shown to stimulate tyrosine phosphorylation of a number of proteins in the 115-125 kDa range and facilitate degranulation by alloantigen-specific cytotoxic T lymphocyte (CTL) clones in response to substimulatory amounts of anti-CD3 or anti-T cell receptor (TCR). The current study was initiated to further characterize integrin expression and usage by these CTL clones. We demonstrate that vitronectin and fibrinogen, but not laminin or collagen, are also able to both facilitate degranulation in the presence of substimulatory anti-CD3 and stimulate tyrosine phosphorylation of these 115-125-kDa proteins, with a 115-kDa protein being the most prominently phosphorylated. These results implicate the expression and usage of the vitronectin receptor, alpha beta3 integrin, by these CTL clones. We demonstrate by both flow cytometry and immunoprecipitation that CTL clones do in fact express beta3 integrin. Immobilized antibody to beta3 stimulates the phosphorylation of the 115-125-kDa proteins, suggesting that engagement of beta3 transmits the same signal into these cells as fibronectin or vitronectin. The fibronectin and vitronectin-induced phosphorylation as well as adhesion to either fibronectin or vitronectin can be significantly inhibited with antibodies to beta3 integrins. Finally, we are able to immunoprecipitate 115-kDa proteins with antiserum to focal adhesion kinase and a related kinase, called PYK-2, that becomes phosphorylated in response to vitronectin or immobilized anti-beta3. Taken together, these results demonstrate that CTL express and use beta3-integrins as signaling molecules which can augment TCR-mediated stimulation.
Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Activación de Linfocitos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T Citotóxicos/metabolismo , Animales , Adhesión Celular , Degranulación de la Célula , Células Clonales , Fibrinógeno/metabolismo , Fibronectinas/metabolismo , Quinasa 1 de Adhesión Focal , Quinasa 2 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Inmunofenotipificación , Integrina beta3 , Ratones , Peso Molecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Transducción de Señal , Vitronectina/metabolismoRESUMEN
Purified intercellular adhesion molecule-1 (ICAM-1), a ligand of the LFA-1, was used to analyze the contribution of ICAM-1 to the activation of CTL. ICAM-1 facilitates degranulation when co-immobilized with substimulatory amounts of anti-CD3. This facilitated response is most likely mediated through LFA-1, since Abs to this molecule significantly inhibit the response, Interestingly, when ICAM-1 and anti-CD3 are immobilized on separate beads and presented to the CTL, no ICAM-1-enhanced degranulation is observed. The ICAM-1 and anti-CD3 must be immobilized on the same surface to augment the response, suggesting that ICAM-1 either does not transmit signals into the cell or it transmits a very localized signal, since the ICAM-1 and anti-CD3 must be juxtaposed. Consistent with this finding, we demonstrate that ICAM-1 does not induce tyrosine phosphorylation or a Ca(2+)-flux in the CTL clone, but does potentiate these responses when co-immobilized with substimulatory anti-CD3. Finally ICAM-1 and anti-CD3 must be immobilized on the same bead for stable adhesion of CTL to ICAM-1. When ICAM-1 and anti-CD3 are immobilized on separate beads, there is only a transient, low level of adhesion to the ICAM-1 beads. Taken together, these results suggest that LFA-1 is acting principally as an adhesion molecule, with respect to ICAM-1, in CTL and that this adhesion is regulated through the TCR complex.