Phosphorylation of claudin-5 and occludin by Rho kinase in brain endothelial cells

Masaru Yamamoto, Servio H. Ramirez, Shinji Sato, Tomomi Kiyota, Ronald Cerny, Kozo Kaibuchi, Yuri Persidsky, Tsuneya Ikezu

Research output: Contribution to journalArticle

145 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)521-533
Number of pages13
JournalAmerican Journal of Pathology
Volume172
Issue number2
DOIs
StatePublished - Jan 1 2008

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Claudin-5
Occludin
rho-Associated Kinases
Endothelial Cells
Phosphorylation
Blood-Brain Barrier
Brain
Encephalitis
Tight Junction Proteins
HIV-1
Phospho-Specific Antibodies
Phosphopeptides
Tight Junctions
COS Cells
Post Translational Protein Processing
Monocytes
Anti-Idiotypic Antibodies
Maintenance
Staining and Labeling
Antibodies

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Phosphorylation of claudin-5 and occludin by Rho kinase in brain endothelial cells. / Yamamoto, Masaru; Ramirez, Servio H.; Sato, Shinji; Kiyota, Tomomi; Cerny, Ronald; Kaibuchi, Kozo; Persidsky, Yuri; Ikezu, Tsuneya.

In: American Journal of Pathology, Vol. 172, No. 2, 01.01.2008, p. 521-533.

Research output: Contribution to journalArticle

Yamamoto, M, Ramirez, SH, Sato, S, Kiyota, T, Cerny, R, Kaibuchi, K, Persidsky, Y & Ikezu, T 2008, 'Phosphorylation of claudin-5 and occludin by Rho kinase in brain endothelial cells', American Journal of Pathology, vol. 172, no. 2, pp. 521-533. https://doi.org/10.2353/ajpath.2008.070076
Yamamoto, Masaru ; Ramirez, Servio H. ; Sato, Shinji ; Kiyota, Tomomi ; Cerny, Ronald ; Kaibuchi, Kozo ; Persidsky, Yuri ; Ikezu, Tsuneya. / Phosphorylation of claudin-5 and occludin by Rho kinase in brain endothelial cells. In: American Journal of Pathology. 2008 ; Vol. 172, No. 2. pp. 521-533.
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