title: PKA compartmentalization via AKAP220 and AKAP12 contributes to endothelial barrier regulation.
abstract: cAMP-mediated PKA signaling is the main known pathway involved in maintenance of the endothelial barrier. Tight regulation of PKA function can be achieved by discrete compartmentalization of the enzyme via physical interaction with A-kinase anchoring proteins (AKAPs). Here, we investigated the role of AKAPs 220 and 12 in endothelial barrier regulation. Analysis of human and mouse microvascular endothelial cells as well as isolated rat mesenteric microvessels was performed using TAT-Ahx-AKAPis peptide, designed to competitively inhibit PKA-AKAP interaction. In vivo microvessel hydraulic conductivity and in vitro transendothelial electrical resistance measurements showed that this peptide destabilized endothelial barrier properties, and dampened the cAMP-mediated endothelial barrier stabilization induced by forskolin and rolipram. Immunofluorescence analysis revealed that TAT-Ahx-AKAPis led to both adherens junctions and actin cytoskeleton reorganization. Those effects were paralleled by redistribution of PKA and Rac1 from endothelial junctions and by Rac1 inactivation. Similarly, membrane localization of AKAP220 was also reduced. In addition, depletion of either AKAP12 or AKAP220 significantly impaired endothelial barrier function and AKAP12 was also shown to interfere with cAMP-mediated barrier enhancement. Furthermore, immunoprecipitation analysis demonstrated that AKAP220 interacts not only with PKA but also with VE-cadherin and ß-catenin. Taken together, these results indicate that AKAP-mediated PKA subcellular compartmentalization is involved in endothelial barrier regulation. More specifically, AKAP220 and AKAP12 contribute to endothelial barrier function and AKAP12 is required for cAMP-mediated barrier stabilization.
authors.affiliation: Institute of Anatomy and Cell Biology, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Mariya Y
authors.initials: MY
authors.lastname: Radeva
authors.affiliation: Institute of Anatomy and Cell Biology, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Kugelmann
authors.affiliation: Institute of Anatomy and Cell Biology, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Spindler
authors.affiliation: Institute of Anatomy and Cell Biology, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Waschke
mesh_terms: D054758:A Kinase Anchor Proteins / Q000235:genetics / Q000378:metabolism*; D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D022005:Adherens Junctions / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002199:Capillary Permeability / Q000187:drug effects*; D002451:Cell Compartmentation; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D020405:Dermis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D055806:Microvessels / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D020889:Rolipram / Q000494:pharmacology; D015398:Signal Transduction; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D054758:A Kinase Anchor Proteins; C099270:AKAP11 protein, human; C491162:AKAP12 protein, human; D018797:Cell Cycle Proteins; D010455:Peptides; C107953:RAC1 protein, human; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases; D020830:rac1 GTP-Binding Protein; D020889:Rolipram