Papel de las GTPasas Ral en la función linfoide y la citotoxicidad celular

Resumen

Ral GTPases are small monomeric G proteins that belong to the Ras superfamily. The two known isoforms, RalA and RalB, are widely distributed in mammals. Both isoforms are anchored to the plasma membrane and to intracellular vesicles. Ral GTPases are thought to be implicated in a broad spectrum of processes that comprise exo- and endocytosis, cell migration and transformation. Ral signalling is considered one of the three most important Ras effector pathways, together with the PI3K and Raf routes. Ras signalling is essential in the immune system, since it regulates T and B cell development as well as processes such as T cell activation after antigen-MHC recognition. GTP-bound Ras recruits and activates Ral guanine exchange factors including RalGDS or Rlf, which activate Ral; this suggests Ral involvement in immune response regulation. Here we discuss the implication of RalGTPases in T cell activation via the T cell receptor (TCR). We show that Ral activity increases after TCR stimulation, and these GTPases relocalize to the immunological synapse. Alterations in Ral activity or expression by mutation or shRNA transfection lead to impaired activation of the Jurkat CD4+ T cell line, as measured by membrane expression levels of CD69. We also studied the effect of Ral activity downregulation in RalGDS - /- mice, which showed no obvious defect either in T cell development or in activation ability after TCR stimulation Ral GTPases regulate vesicular trafficking and polarized granule secretion in several cell types. The second part of our study focused on Ral regulation of polarized secretion of lytic granules by lytic cells. Using a human NK cell line, we show that both RalA and RalB isoforms are activated rapidly after target cell recognition. Silencing of RalA and RalB impaired NK cell cytotoxicity. RalA regulated granule polarization towards the immunological synapse and subsequent degranulation, whereas RalB regulated degranulation but not polarization of lytic granules. Analysis of the molecular mechanism indicated that Ral activation in NK cells leads to assembly of the exocyst, an octameric protein complex involved in polarized secretion and vesicle tethering. The assembly of this complex is required for degranulation, as interference with expression of the exocyst component Sec5 led to reduced degranulation and impaired cytotoxicity in NK cells, although it did not affect polarization. Our results thus identify a role for Ral in cellmediated cytotoxicity, implicating these GTPases in lymphocyte function.