Phosphatase 2A is involved in endothelial cell microtubule remodeling and barrier regulation.
We have recently shown that microtubule (MT) inhibitor, nocodazole (2-5 microM) 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.
Tar
Krisztina
K
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
Birukova
Anna A
AA
Csortos
Csilla
C
Bakó
Eva
E
Garcia
Joe G N
JG
Verin
Alexander D
AD
D000205:Actomyosin / Q000378:metabolism; D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D002417:Cattle; D002458:Cell Fractionation; D002460:Cell Line; D042783:Endothelial Cells / Q000166:cytology* / Q000201:enzymology* / Q000378:metabolism; D004727:Endothelium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D054648:Protein Phosphatase 2; D021381:Protein Transport; D016875:tau Proteins / Q000378:metabolism
D016875:tau Proteins; D019319:Okadaic Acid; D000205:Actomyosin; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D015739:Nocodazole