The functional role of adaptor protein RACK1 in organization of microtubule and intermediate filaments in mast cells

Abstract

RACK1 (Receptor for Activated C Kinase 1) is an adaptor protein expressed in mammal cells and crucial for fundamental cellular activities. The RACK1 has a central role in the immune cells response, integrating signaling pathways, acting in calcium mobilization, and in components of the cytoskeleton. The mast cells are multifunctional effector cells of the immune system and their functions are directly related with cell activation and, subsequent, release of chemical mediators. However, until now, RACK1 had not been described in mast cells. Recent data from our laboratory show, for the first time, the expression of RACK1 in mast cells. By proteomic analysis, we demonstrated that RACK1 is present in lipid raft fractions, regions of lipid microdomains in the plasma membrane that act as a platform for the mast cell signaling elements during activation via the high affinity IgE receptor (FcepsilonRI). Our results show that the release of the preformed mediator beta-hexosaminidase was significantly increased in RACK1 knockdown cells stimulated or not via FcepsilonRI. The basal release of beta-hexosaminidase was also increased. Also, we observed that, in RACK1 knockdown mast cells, the granules are located in the cortical regions of the cells free of F-actin. The absence of actin filaments in these regions can facilitate the release of the mediators stored in the granule. In this project we propose to investigate if RACK1 has an influence on the arrangement of other cytoskeleton components, as the intermediate filaments and microtubules, which are also involved in some pathways of the cellular secretion. Furthermore, we will investigate if the possible changes of these cytoskeleton elements can affect the mast cell mediator release. RBL-2H3 mast cells transduced or not with shRNA for RACK1 and stimulated or not via FcepsilonRI will be immunolabeled with anti-alfa/beta-tubulin and vimentin antibodies and then analyzed by confocal microscopy. The relationship between cytoskeleton components and secretory granules will also be investigated. The role of RACK1 in microtubule dynamics will also be analyzed in vivo. The possible direct interaction between RACK1 and alfa/beta -tubulin or vimentin will be investigated by co-immunoprecipitation experiments. This additional information could contribute for the better understanding of the role of RACK1 in granule release in mast cells.