RESUMEN
The adaptor protein Src homology (SH)2 domain-containing and leukocyte-specific phosphoprotein of 76 kDa (SLP-76) is critical for signal transduction in multiple hematopoietic lineages. It links proximal and distal T cell receptor signaling events through its function as a molecular scaffold in the assembly of multimolecular signaling complexes. Here we studied the functional roles of sub-domains within the SLP-76 proline-rich region, specifically the Gads binding domain and the recently defined P1 domain. To gain a further understanding of the functions mediated by this region, we used three complementary approaches as follows: reconstitution of SLP-76-deficient cells with functional domain deletion mutants, blocking molecular associations through the expression of a dominant negative protein fragment, and directed localization of SLP-76 to assess the role of the domains in SLP-76 recruitment. We find the Gads binding domain and the P1 domain are both necessary for optimal SLP-76 function, and in the absence of these two regions, SLP-76 is functionally inert. Furthermore, we provide direct evidence that SLP-76 localization and, in turn, function are dependent upon association with Gads.
Asunto(s)
Fosfoproteínas/química , Prolina/química , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Alanina/química , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Arginina/química , Western Blotting , Calcio/metabolismo , Línea Celular , Linaje de la Célula , Citometría de Flujo , Eliminación de Gen , Genes Dominantes , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Jurkat , Lectinas Tipo C , Luciferasas/metabolismo , Activación de Linfocitos , Lisina/química , Microdominios de Membrana/metabolismo , Modelos Biológicos , Mutación , Fosfoproteínas/metabolismo , Plásmidos/metabolismo , Pruebas de Precipitina , Estructura Terciaria de Proteína , Transducción de Señal , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Transfección , Dominios Homologos srcRESUMEN
Fc epsilon RI activation of mast cells is thought to involve Lyn and Syk kinases proximal to the receptor and the signaling complex organized by the linker for activation of T cells (LAT). We report here that Fc epsilon RI also uses a Fyn kinase-dependent pathway that does not require Lyn kinase or the adapter LAT for its initiation, but is necessary for mast cell degranulation. Lyn-deficiency enhanced Fyn-dependent signals and degranulation, but inhibited the calcium response. Fyn-deficiency impaired degranulation, whereas Lyn-mediated signaling and calcium was normal. Thus, Fc epsilon RI-dependent mast cell degranulation involves cross-talk between Fyn and Lyn kinases.