Protein kinase A phosphorylation of tau-serine 214 reorganizes microtubules and disrupts the endothelial cell barrier

Abstract

compartmentalized to near membrane domains in endothelium, where it strengthens endothelial cell barrier function. Phosphodiesterase 4D4 (PDE4D4) interacts with the spectrin membrane skeleton and prevents cAMP from accessing microtubules. Expression of a dominant-negative PDE4D4 peptide enables cAMP to access microtubules, where it results in phosphorylation of the nonneuronal microtubule-associated protein tau at serine 214. Presently, we sought to determine whether PKA is responsible for tau-Ser214 phosphorylation and furthermore whether PKA phosphorylation of tau-Ser214 is sufficient to reorganize microtubules and induce endothelial cell gaps. In cells expressing the dominant-negative PDE4D4 peptide, forskolin activated transmembrane adenylyl cyclases, increased cAMP, and induced tau-Ser214 phosphorylation that was accompanied by microtubule reorganization. PKA catalytic and regulatory I subunits, but not the regulatory II subunit, coassociated with reorganized microtubules. To determine the functional consequence of tau-Ser214 phosphorylation, wild-type human tau40 and tau40 engineered to possess an alanine point mutation (S214A) were stably expressed in endothelium. In cells expressing the dominant-negative PDE4D4 peptide and tau-S214A, PKA-dependent phosphorylation of both the endogenous and heterologously expressed tau were abolished. Expression of tau-S214A prevented forskolin from depolymerizing microtubules, inducing intercellular gaps, and increasing macromolecular permeability. These findings therefore identify nonneuronal tau as a critical cAMP-responsive microtubule-associated protein that controls microtubule architecture and endothelial cell barrier function. adenylyl cyclase; cytoskeleton; phosphodiesterase ENDOTHELIAL CELLS FORM A RESTRICTIVE barrier that limits fluid, solute, and macromolecule access to the interstitium. The microtubule network plays a key role in establishing endothelial cell shape and hence barrier integrity (17, 41). Whereas stabilizing microtubules enhances endothelial barrier function, disrupting microtubules induces interendothelial cell gap formation that increases permeability (33,47).Tau and other microtubule-associated proteins bind to and stabilize microtubules (3,45). Although tau is a relatively small protein, it shares a conserved 18-amino acid microtubule-binding repeat in common with larger microtubule-associated proteins (3, 44). Tau isoforms vary in their number of repeat domains (20), with the longest tau40 isoform having 4 microtubule-binding repeats at residues 256 -273, 287-304, 318 -335, and 350 -367.Tau40 possesses multiple consensus phosphorylation sites (3, 25); 20% of the tau40 amino acid sequence comprises serine, threonine, and tyrosine residues that can potentially be phosphorylated (25). At least two types of kinase phosphorylation sites exist in tau proteins: proline-direct kinase sites, which possess a threonine-proline (TP)-serine-proline (SP) sequence, and nonproline-directed sites. Most of the TP-SP sites are located in either the NH 2 -or C...

Pseudomonas aeruginosa Exotoxin Y Is a Promiscuous Cyclase That Increases Endothelial Tau Phosphorylation and Permeability

“…In future studies it will be imperative to clone and sequence the endothelial Tau. (25,46,47). We found that the increased levels of cAMP and cGMP that paralleled gap formation in P. aeruginosa ExoY ϩ -infected pulmonary microvascular endothelial cells resulted in increased Tau Ser-214 phosphorylation, whereas cells infected with P. aeruginosa ExoY K81M did not display increased Tau Ser-214 phosphorylation (Fig.…”

“…Previous studies from our group (25,46,47) and others (48) have resolved that Tau or a Tau-like protein is expressed in pulmonary microvascular endothelial cells. It is not clear, however, whether endothelial Tau is similar to those Tau isoforms found in the central nervous system.…”

Urokinase-type Plasminogen Activator (uPA) Induces Pulmonary Microvascular Endothelial Permeability through Low Density Lipoprotein Receptor-related Protein (LRP)-dependent Activation of Endothelial Nitric-oxide Synthase

“…In addition, membrane-associated cAMP protects endothelial barrier function by activating the exchange protein directly activated by cAMP (EPAC), which forms a ternary complex with PKA and PDE4D4 and subsequently stabilizes VE-cadherin-based adherens junctions (84). However, if PDE4D4 is inhibited, cAMP diffuses into the cytosol and activates cytosolyic PKA, which initiates a reorganization of the microtubules through phosphorylation of tau-Ser 214 , generating intercellular gaps that increase endothelial permeability (85). Additional studies will be needed to determine whether these or alternative mechanisms are responsible for the uPA-PAI-1-induced PKA-dependent increase in PMV permeability that we observed.…”

Emerging themes of cAMP regulation of the pulmonary endothelial barrier

“…Interestingly, these effects were analogous to the barrier-disruptive effects of soluble ExoY or sACI/II adenylyl cyclase activity. When tau serine 214 is mutated to alanine, loss of phosphodiesterase D4D activity failed to reorganize microtubules and disrupt the barrier (179). Thus, in pulmonary endothelium, phosphodiesterase 4D4 localizes to the subplasma membrane space to spatially limit migration of the cAMP signal into the cytosol.…”

“…Thus, in pulmonary endothelium, phosphodiesterase 4D4 localizes to the subplasma membrane space to spatially limit migration of the cAMP signal into the cytosol. Furthermore, cytosolic cAMP leads to phosphorylation of tau and decreased endothelial barrier integrity (30,179).…”