La proteína mal como componente de la maquinaria de transporte especializado en linfocito T

Resumen

T lymphocytes are specialized cells that can be activated in response to an specific antigen presented by an APC. The formation of the IS is a regulated process that involves the orderly, spatial-temporal, polarized redistribution of receptors, intracellular-signaling molecules and cytoskeletal proteins. Little is known about traffic processes which lead to the formation of the caraceristic structure of the IS, but the result is a highly ordered pattern which involves agregation and segregation of proteins and lipids. The formed structure, as leads to T cell activation, is known as supramolecular activation complex (SMAC). The MAL protein is a highly hydrophobic integral membrane protein consisting of four hydrophobic segments separated by short hydrophilic loops, that has been proposed as an essential component of the specialized machinery for apical targeting in epithelial cells. Its function is to reclute cargo proteins, as HA, and deliver them to the apical surface of MDCK cells (Puertollano et al., 1999). MAL protein is exclusively found in membranes rafts fractions in all the cell types in which is expressed. MAL cDNA is also expressed in Jurkat and primary T cells (Alonso and Weissman, 1987), but the role of the protein in these cells has remained elusive. The generation of a Jurkat cells mutants deficients in TCR signalling in combination with the use of siRNA silencing techiniques have been invaluable strategies for dissecting the T-cell signalling machinery. Here we used a TCRsignalling- defective mutant, named JTIM, that lacks MAL expression to investigate the role of MAL in T cells. JTIM cells was described as defective in the formation of a normal IS and induction of IL-2 production (Anton et al., 2008). One of the defects observed conparing Jurkat normal cells and JTIM cells was that the src kinase Lck was unusualy concentrated in the intracellular compartment in JTIM mutant. The abnormal distribution of Lck in JTIM cells lead us to think in a possible role of MAL in regulating Lck targeting to the plasma membrane. We show that Lck travels to the plasma membranne in specific transport carriers containing MAL. Coimmunoprecipitation experiments, indicated an association of MAL and Lck. This association seemed to facilitate the incoporation of Lck to lipid rafts. The forced expression of Lck at the plasma membrane in JTIM cells got partial correction of the latter defects observed in JTIM cells, but the complete correction only took place whith the exogenous expression of MAL. During the formation of the IS, a pool of MAL rapidly relocalices at the center of the contact (cSMAC). This localization seems to be dependent of the integrity of the las hidrophilic loop, as its disruption results in the missorting of the modified MAL molecule to the pSMAC. The presence the MAL at the pSMAC alters the SMAC in such a manner that Lck and LAT, both of them raft-associated proteins, and the GM1 ganglioside whic are normally distributed at the cSMAC, were mistargeted to the pSMAC. Moreover, ectopically-expressed HA was targeted to either the cSMAC or the pSMAC depending on the expression of intact or modified MAL, respectively. Based on these observations, we propose a MAL as an essential component of an specialized transport machinery in T cells and as a regulator of the membrane microdomains integrity, which are involved in several processes such as the SMAC formation.