title: Filamin A is a phosphorylation target of membrane but not cytosolic adenylyl cyclase activity.
abstract: Transmembrane adenylyl cyclase (AC) generates a cAMP pool within the subplasma membrane compartment that strengthens the endothelial cell barrier. This cAMP signal is steered toward effectors that promote junctional integrity and is inactivated before it accesses microtubules, where the cAMP signal causes phosphorylation of tau, leading to microtubule disassembly and barrier disruption. During infection, Pseudomonas aeruginosa uses a type III secretion system to inject a soluble AC, ExoY, into the cytosol of pulmonary microvascular endothelial cells. ExoY generates a cAMP signal that disrupts the endothelial cell barrier. We tested the hypothesis that this ExoY-dependent cAMP signal causes phosphorylation of tau, without inducing phosphorylation of membrane effectors that strengthen endothelial barrier function. To approach this hypothesis, we first discerned the membrane compartment in which endogenous transmembrane AC6 resides. AC6 was resolved in caveolin-rich lipid raft fractions with calcium channel proteins and the cell adhesion molecules N-cadherin, E-cadherin, and activated leukocyte adhesion molecule. VE-cadherin was excluded from the caveolin-rich fractions and was detected in the bulk plasma membrane fractions. The actin binding protein, filamin A, was detected in all membrane fractions. Isoproterenol activation of ACs promoted filamin phosphorylation, whereas thrombin inhibition of AC6 reduced filamin phosphorylation within the membrane fraction. In contrast, ExoY produced a cAMP signal that did not cause filamin phosphorylation yet induced tau phosphorylation. Hence, our data indicate that cAMP signals are strictly compartmentalized; whereas cAMP emanating from transmembrane ACs activates barrier-enhancing targets, such as filamin, cAMP emanating from soluble ACs activates barrier-disrupting targets, such as tau.
authors.affiliation: Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, Alabama 36688, USA. ssayner@usouthal.edu
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Sayner
authors.forename: Ron
authors.initials: R
authors.lastname: Balczon
authors.forename: Dara W
authors.initials: DW
authors.lastname: Frank
authors.forename: Dermot M F
authors.initials: DM
authors.lastname: Cooper
authors.forename: Troy
authors.initials: T
authors.lastname: Stevens
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D051242:Caveolin 1 / Q000378:metabolism; D015815:Cell Adhesion Molecules; D002451:Cell Compartmentation / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D003285:Contractile Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D064448:Filamins; D005964:Glucosyltransferases / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D008168:Lung / Q000098:blood supply; D021962:Membrane Microdomains / Q000187:drug effects / Q000201:enzymology; D008840:Microfilament Proteins / Q000378:metabolism*; D055806:Microvessels / Q000166:cytology; D008954:Models, Biological; D000071740:ORAI1 Protein; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D050052:TRPC Cation Channels / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D016875:tau Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D015220:Calcium Channels; D051242:Caveolin 1; D015815:Cell Adhesion Molecules; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D000071740:ORAI1 Protein; C532493:Orai1 protein, rat; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; D016875:tau Proteins; D000242:Cyclic AMP; C111504:ExoY protein, bacteria; D005964:Glucosyltransferases; D013917:Thrombin; D000262:Adenylyl Cyclases; C121100:adenylyl cyclase 6; D007545:Isoproterenol