Journal of Cellular Biochemistry 92:534–546 (2004) Phosphatase 2A Is Involved in Endothelial Cell Microtubule Remodeling and Barrier Regulation Krisztina Tar, 1,2 Anna A. Birukova, 1 Csilla Csortos, 2 E ´ va Bako ´, 2 Joe G.N. Garcia, 1 and Alexander D. Verin 1 * 1 Department of Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224 2 Department of Medical Chemistry, University of Debrecen, Medical and Health Science Center, H-4026 Debrecen, Hungary Abstract We have recently shown that microtubule (MT) inhibitor, nocodazole (2–5 mM) significantly increases endothelial cells (EC) actomyosin contraction and permeability indicating the importance of MT in maintaining the EC barrier (Verin et al. [2001]: Cell Mol Physiol 281:L565 – L574). Okadaic acid (OA, 2 – 5 nM), a powerful inhibitor of protein phosphatase 2A (PP2A), significantly potentiates the effect of submaximal concentrations of nocodazole (50 – 200 nM) on transendothelial electrical resistance (TER) suggesting the involvement of PP2A activity in the MT-mediated EC barrier regulation. Immunofluorescent staining of EC revealed that in control cells PP2A distributes in a pattern similar to MT. Consistent with these results, we demonstrated that significant amounts of PP2A were present in MT-enriched EC fractions indicating tight association of PP2A with MT in endothelium. Treatment of EC with OA leads to disappearance of MT-like PP2A staining suggesting dissociation of PP2A from the MT network. Next, we examined the effect of PP2A inhibition on phosphorylation status of MT-associated protein tau, which in its unphosphorylated form promotes MT assembly. OA caused significant increases in tau phosphorylation confirming that tau is a substrate for PP2A in endothelium. Immunofluorescent experiments demonstrated that the OA-induced increases in tau phosphorylation strongly correlated with translocation of phospho-tau to cell periphery and disassembly of peripheral MT. These results suggest the involvement of PP2A-mediated tau dephosphorylation in alteration of EC MT structure and highlight the potential importance of PP2A in the regulation of EC the MT cytoskeleton and barrier function. J. Cell. Biochem. 92: 534–546, 2004. ß 2004 Wiley-Liss, Inc. Key words: endothelium; phosphatases; permeability; microtubules; okadaic acid; nocodazole; endothelial cytoskeleton A major function of the vascular endothelial cell (EC) monolayer is to serve as a selective barrier to fluid and solute flux across the blood vessel wall. Increased endothelial permeability is a prominent characteristic of acute inflam- matory lung syndrome and is the result of intercellular gap formation evoked by bioactive agents [Garcia et al., 1986, 1995]. EC barrier integrity is administrated by a dynamic equili- brium between competing contractile and tethering forces. The equilibrium is dependent on the coordinated functioning of the compo- nents of the cytoskeleton and is regulated via reversible phosphorylation of numerous cytos- keletal proteins [for review see Dudek and Garcia, 2001]. Acto-myosin interaction and cell contractility depends upon the phosphorylation state of myosin light chains (MLC) [Garcia et al., 1986, 1995]. Increased levels of MLC phos- phorylation are followed by actin redistribution, F-actin filament formation, and EC barrier dysfunction. We have previously shown that protein phosphatase 1 (PP1) associates with myosin filaments and is directly involved in EC ß 2004 Wiley-Liss, Inc. Grant sponsor: National Heart, Lung, and Blood Institutes; Grant numbers: HL67307, HL68062, HL58064; Grant sponsor: American Heart Association; Grant sponsor: Hungarian Science Research Fund; Grant number: OTKA T043133. *Correspondence to: Alexander D. Verin, PhD, 5200 Eastern Avenue, Center Tower, MFL Building, 6th Floor, Baltimore, MD 21224. E-mail: averin1@jhmi.edu Received 1 October 2003; Accepted 17 December 2003 DOI 10.1002/jcb.20036