RESUMEN
Natural killer (NK) cells and NKT cells play critical early roles in host defense. Here we show that MIST, an adaptor protein belonging to the SLP-76 family, functions negatively in NK cells but positively in CD4(+)NKT cells. NK-cell receptor-mediated IFN-gamma production was enhanced in NK cells, whereas TCR- or NK-cell receptor-mediated cytokine production was reduced in CD4(+)NKT cells from MIST-deficient mice. These opposite effects of MIST paralleled the exclusive expression of the Src family kinase, Fgr, in NK cells between the 2 cell populations. We further demonstrated that interaction of MIST with Fgr, mediated by the C-terminal proline-rich region of MIST and the SH3 domain of Fgr, was required for the suppression of NK-cell receptor-induced IFN-gamma production. This functional interdependence of signaling molecules demonstrates a new mechanism by which adaptor proteins can act as molecular switches to control diverse responses in different cell populations.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Proteínas Portadoras/metabolismo , Interferón gamma/biosíntesis , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , ADN Complementario/genética , Femenino , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Metástasis de la Neoplasia/fisiopatología , Fenotipo , Fosfolipasa C gamma/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Dominios Homologos src , Familia-src Quinasas/química , Familia-src Quinasas/genéticaRESUMEN
SLP-76-related adaptor protein MIST (also called Clnk) is expressed in a variety of cytokine-dependent hematopoietic cell lines of myeloid and lymphoid origin as well as some cytokine-independent mast cell lines. To understand the molecular mechanisms underlying the MIST gene expression, we have characterized the 5'-flanking region of the mouse MIST gene. We have identified an enhancer region (-773 to -709), which is active in P815 mast cells expressing the endogenous MIST gene, but not in EL-4 T cells lacking MIST expression. Outside of this enhancer region, one STAT element present in the MIST promoter (-44 to -36) was found to bind STAT5A when IC-2 mast cells were stimulated with IL-3. Mutation of this STAT element did not affect basal MIST promoter activity in P815 mast cells, but was required for STAT5-mediated activation of the MIST promoter. Furthermore, endogenous MIST gene expression was induced in mast cells by a constitutively activated form of STAT5A, but not by an active mutant of c-Kit receptor. These findings suggest that STAT5 is involved in cytokine-mediated up-regulation of MIST gene expression, probably in collaboration with other lineage-specific transcription factors that promote basal MIST expression in mast cells.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Unión al ADN/metabolismo , Mastocitos/fisiología , Proteínas de la Leche/metabolismo , Fosfoproteínas/metabolismo , Transactivadores/metabolismo , Transcripción Genética/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Secuencia de Bases , Línea Celular , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , ARN Mensajero/genética , Mapeo Restrictivo , Factor de Transcripción STAT5 , Linfocitos T/fisiología , Activación TranscripcionalRESUMEN
MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST-SLAP-130-SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells.
