RESUMO
The adapter protein ADAP regulates T lymphocyte adhesion and activation. We present evidence for a previously unrecognized function for ADAP in regulating T cell receptor (TCR)-mediated activation of the transcription factor NF-kappaB. Stimulation of ADAP-deficient mouse T cells with antibodies to CD3 and CD28 resulted in impaired nuclear translocation of NF-kappaB, a reduced DNA binding, and delayed degradation and decreased phosphorylation of IkappaB (inhibitor of NF-kappaB). TCR-stimulated assembly of the CARMA1-BCL-10-MALT1 complex was substantially impaired in the absence of ADAP. We further identified a region of ADAP that is required for association with the CARMA1 adapter and NF-kappaB activation but is not required for ADAP-dependent regulation of adhesion. These findings provide new insights into ADAP function and the mechanism by which CARMA1 regulates NF-kappaB activation in T cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Transcrição RelA/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína 10 de Linfoma CCL de Células B , Antígenos CD28/imunologia , Complexo CD3/imunologia , Caspases/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Isoenzimas/metabolismo , Células Jurkat , Ativação Linfocitária , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Mutação , Proteínas de Neoplasias/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C-theta , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Nuclear translocation of NF-kappaB initiates transcription of numerous genes, many of which are critical to host defense. Fluorescent image-based methods that quantify this event have historically utilized adherent cells with large cytoplasm-to-nuclear area ratios. However, many immunologically relevant cells are naturally non-adherent and have small cytoplasm-to-nuclear area ratios. Using the ImageStream imaging flow cytometer, we have developed a novel method that measures nuclear translocation in large populations using cross-correlation analysis of nuclear and NF-kappaB images from each cell. This approach accurately measures NF-kappaB translocation in cells with small cytoplasmic areas in dose- and time-dependent manners. Further, NF-kappaB translocation was accurately measured in a subset of cells contained in a mixed population and the technique was successfully employed to measure IRF-7 translocation in plasmacytoid dendritic cells (PDC) obtained from human peripheral blood. The techniques described here provide an objective and statistically robust method for measuring cytoplasmic to nuclear molecular translocation events in a variety of immunologically relevant cell types with characteristically low cytoplasm-to-nuclear area ratios.
