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

ABSTRACT: UNLABELLED: BACKGROUND: RACK1, receptor for activated protein kinase C, serves as an anchor in multiple signaling pathways. TIMAP, TGF-? inhibited membrane-associated protein, is most abundant in endothelial cells with a regulatory effect on the endothelial barrier function. The interaction of TIMAP with protein phosphatase 1 (PP1c?) was characterized, yet little is known about its further partners. RESULTS: We identified two novel interacting partners of RACK1, namely, TGF-? 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. CONCLUSIONS: 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.

SUBMITTER: Boratko A

PROVIDER: S-EPMC3560227 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Boratkó Anita A Gergely Pál P Csortos Csilla C

Cell communication and signaling : CCS 20130111 1

<h4>Unlabelled</h4><h4>Background</h4>RACK1, receptor for activated protein kinase C, serves as an anchor in multiple signaling pathways. TIMAP, TGF-β inhibited membrane-associated protein, is most abundant in endothelial cells with a regulatory effect on the endothelial barrier function. The interaction of TIMAP with protein phosphatase 1 (PP1cδ) was characterized, yet little is known about its further partners.<h4>Results</h4>We identified two novel interacting partners of RACK1, namely, TGF-β ...[more]

PMID: 23305203

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Project description:Blood vessels are covered with endothelial cells on their inner surfaces, forming a selective and semipermeable barrier between the blood and the underlying tissue. Many pathological processes, such as inflammation or cancer metastasis, are accompanied by an increased vascular permeability. Progress in live cell imaging techniques has recently revealed that the structure of endothelial cell contacts is constantly reorganized and that endothelial junctions display high heterogeneities at a subcellular level even within one cell. Although it is assumed that this dynamic remodeling is associated with a local change in endothelial barrier function, a direct proof is missing mainly because of a lack of appropriate experimental techniques. Here, we describe a new assay to dynamically measure local endothelial barrier function with a lateral resolution of ?15 ?m and a temporal resolution of 1 min. In this setup, fluorescence-labeled molecules are added to the apical compartment of an endothelial monolayer, and the penetration of molecules from the apical to the basal compartment is recorded by total internal reflection fluorescence microscopy utilizing the generated evanescent field. With this technique, we found a remarkable heterogeneity in the local permeability for albumin within confluent endothelial cell layers. In regions with low permeability, stimulation with the proinflammatory agent histamine results in a transient increase in paracellular permeability. The effect showed a high variability along the contact of one individual cell, indicating a local regulation of endothelial barrier function. In regions with high basal permeability, histamine had no obvious effect. In contrast, the barrier-enhancing drug forskolin reduces the permeability for albumin and dextran uniformly along the cell junctions. Because this new approach can be readily combined with other live cell imaging techniques, it will contribute to a better understanding of the mechanisms underlying subcellular junctional reorganization during wound healing, inflammation, and angiogenesis.

Dataset Information

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

Publications

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

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