Although RACK1 is known to act as a signaling hub in immune cells, its presence and role in mast cells (MCs) is undetermined. MC activation via antigen stimulation results in mediator release and is preceded by cytoskeleton reorganization and calcium mobilization. In this study RACK1 was distributed throughout the MC cytoplasm both in vivo and in vitro. After RACK1 knockdown (KD), MCs cells were rounded, and the cortical F-actin was fragmented. Following antigen stimulation, in RACK1 KD MCs there was a reduction in cortical F-actin, an increase in monomeric G-actin, and a failure to organize F-actin. RACK1 KD also increased and accelerated degranulation. CD63+-secretory granules were localized in F-actin-free cortical regions in non-stimulated RACK1 KD MCs. Additionally, RACK1 KD increased antigen-stimulated Ca2+ mobilization, but attenuated antigen-stimulated depletion of ER Ca2+-stores and thapsigargin-induced Ca2+ entry. Following MC activation there was also an increase in interaction of RACK1 with Orai1 Ca2+-channels, β-actin and the actin binding proteins vinculin and MyoVa. These results show that RACK1 is a critical regulator of actin dynamics affecting mediator secretion, and calcium signaling in MCs.

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