El adaptador de membrana latanálisis funcional de motivos conservados en la transmisión de señales intracelulares asociadas a tcr/cd3

Resumen

LAT adapter is a membrane protein essential for distribution of the TCR-mediated signals. Once phosphorylated, LAT tyrosines generate binding sites for proteins that are critical for the activation of multiple downstream pathways. In order to prevent immunological responses against selfantigens or foreign harmless substances, the immune system has developed mechanisms aimed at reducing the activity of components of the TCR signalling cassette. LAT has been proposed as negative regulator of TCR-mediated signals, since mice harbouring a mutation on its tyrosine 136 develop a lymphoproliferative disorder . Interestingly, non tyrosine-based motifs of LAT have been proposed as negative regulators of T cell activation. Thus, phosphorylation of a threonine residue of human LAT reduces its ability to recruit cytosolic activatory elements leading to attenuation of downstream signalling events . Also, cleavage of proteins belonging to the TCR signalling cassette has been proposed as a mechanism to negatively control T cell activation In this report we analyse the function of non-tyrosine based motifs of LAT as potential regulators of activatory signals triggered by the TCR. This report has been divided in two parts. Study 1: Here we show that mutation of serines 38, 40, 106, 164, and 180 reduces the ability of LAT for associating to PLC#-1 and SLP-76 adapter. According with these data, we have found a deficient cytoplasmic calcium influx in these cells. Study 2: In this study we show that upon Fas engagement, LAT is cleaved in both mature T lymphocytes and thymocytes, and also upon TCR crosslinking. We have found that the human form of LAT is cleaved at aspartic acid residues 126, 138 and 186, since this cleavage is abrogated when these aminoacids are mutated to alanines. We also show that LAT tyrosine phosphorylation, mediated by pervanadate or H2O2, negatively modulate LAT cleavage. In addition, we have found LAT is basally cleaved in anergic human primary T cells, which suggest cleavage may act as a regulatory mechanism in TCR-mediated activation of T cells.