RESUMEN
In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Nucleares , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Proto-Oncogénicas pp60(c-src)/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Dominios Homologos src , Secuencia de Aminoácidos , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Secuencia de Bases , Calcio/metabolismo , Línea Celular , Clonación Molecular , ADN Complementario , Proteínas de Unión al ADN/genética , Humanos , Células Jurkat , Lectinas Tipo C , Datos de Secuencia Molecular , Ácido Mirístico/metabolismo , Factores de Transcripción NFATC , Fosforilación , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/inmunología , Homología de Secuencia de Aminoácido , Tetraciclina/farmacología , Transactivadores , Factores de Transcripción/genética , Activación Transcripcional , Tirosina/metabolismoRESUMEN
The condition of mucociliary system has been studied in 246 patients with chronic obstructive pulmonary diseases. It is stated that chronic obstructive pulmonary diseases patients observe the significant variation of bronchial clearance's tempos, imbalance in the system of lipids' peroxide oxidation and antioxidant defense during aggravation and remission. Dysfunction of mucociliary transport is associated with the changes of mucilaginous elastic characteristics of tracheal-bronchial secretion that leads to progression of bronchial obstruction.