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Published online before print January 10, 2008
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Copyright © 2008 American Society for Investigative Pathology
American Journal of Pathology, doi:10.2353/ajpath.2008.070076

Accepted for publication October 16, 2007.

Article

Phosphorylation of Claudin-5 and Occludin by Rho Kinase in Brain Endothelial Cells

Masaru Yamamoto*{dagger}, Servio H. Ramirez*{dagger}{ddagger}, Shinji Sato*{dagger}, Tomomi Kiyota*{dagger}, Ronald L. Cerny{sect}, Kozo Kaibuchi, Yuri Persidsky*{dagger}{ddagger}, and Tsuneya Ikezu*{dagger}@

From the Departments of Pharmacology and Experimental Neuroscience,* and Pathology and Microbiology,{ddagger} and the Center for Neurovirology and Neurodegenerative Disorders,{dagger} University of Nebraska Medical Center, Omaha, Nebraska; the Department of Chemistry,{sect} University of Nebraska, Lincoln, Nebraska; and the Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Japan

@ To whom correspondence should be addressed. E-mail: tikezu{at}unmc.edu.


  Abstract

Critical to the proper maintenance of blood-brain-barrier (BBB) integrity are the endothelial tight junctions (TJs). Posttranslational modifications of essential endothelial TJ proteins, occludin and claudin-5, contribute and possibly disrupt BBB integrity. Our previous work has shown that Rho kinase (RhoK) activation mediates occludin and claudin-5 phosphorylation resulting in diminished barrier tightness and enhanced monocyte migration across BBB in the setting of human immunodeficiency virus-1 encephalitis (HIVE). To determine whether RhoK can directly phosphorylate TJ proteins, we examined phosphorylation of cytoplasmic domains of recombinant claudin-5 and occludin by RhoK. We found that RhoK predominately phosphorylated two sites on occludin (T382 and S507) and one site on claudin-5 (T207). Specific anti-phosphopeptide antibodies were developed for these sites, allowing the detection of phosphorylated occludin at T382 and S507, and claudin-5 at T207 from full-length recombinant occludin and claudin-5 transiently expressed in COS-7 cells and mouse brain microvascular endothelial cells. Finally, these phosphospecific antibodies demonstrated enhanced staining of brain endothelial cells in the mouse model for HIVE and human HIVE brains featuring mononuclear cell infiltration across disrupted BBB. Our results demonstrated the direct phosphorylation of occludin and claudin-5 by RhoK at specific sites, which was increased in encephalitic brain tissue. These antibodies could be useful reagents for monitoring BBB dysfunction in vivo.




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