RACK1 is involved in endothelial barrier regulation via its two novel interacting partners

We identified two novel interacting partners of RACK1, namely, TGF-β inhibited membrane-associated protein, TIMAP, and farnesyl transferase. TIMAP is most abundant in endothelial cells where it is involved in the regulation of the barrier function. WD1-4 repeats of RACK1 were identified as critical regions of the interaction both with TIMAP and farnesyl transferase. Phosphorylation of TIMAP by activation of the cAMP/PKA pathway reduced the amount of TIMAP-RACK1 complex and enhanced translocation of TIMAP to the cell membrane in vascular endothelial cells. However, both membrane localization of TIMAP and transendothelial resistance were attenuated after RACK1 depletion. Farnesyl transferase, the enzyme responsible for prenylation and consequent membrane localization of TIMAP, is present in the RACK1-TIMAP complex in control cells, but it does not co-immunoprecipitate with TIMAP after RACK1 depletion.Transient parallel linkage of TIMAP and farnesyl transferase to RACK1 could ensure prenylation and transport of TIMAP to the plasma membrane where it may attend in maintaining the endothelial barrier as a phosphatase regulator.The vascular endothelium functions as a semi-permeable barrier between blood and the interstitium. Endothelial cell (EC) barrier regulation is under intense investigation, since EC barrier dysfunction is a well-known feature of acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) [1]. Barrier enhancing or protecting processes are not explored in details yet, however, several studies indicate the significance of dephosphorylation of key cytoskeletal/membrane associated targets by specific protein phosphatases [2]. TIMAP (TGF-β inhibited membrane-associated protein) protein has been considered as a member of the MYPT (myosin phosphatase targeting) family of the regulatory subunits of protein phosphatase 1 (PP1) based on its structural features [3]. TIMAP and MYPT3 are the most closely related members within the