Phosphorylation of Tyr-398 and Tyr-402 in Occludin Prevents Its Interaction with ZO-1 and Destabilizes Its Assembly at the Tight Junctions

• Published in: The Journal of biological chemistry Vol. 284; no. 3; pp. 1559 - 1569
• Main Authors: Elias, Bertha C., Suzuki, Takuya, Seth, Ankur, Giorgianni, Francesco, Kale, Gautam, Shen, Le, Turner, Jerrold R., Naren, Anjaparavanda, Desiderio, Dominic M., Rao, Radhakrishna
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.01.2009; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Caco-2 Cells; Chickens; CSK Tyrosine-Protein Kinase; Dogs; Humans; Hydrogen Peroxide - pharmacology; Mass Spectrometry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular Basis of Cell and Developmental Biology; Mutation, Missense; Occludin; Oxidants - pharmacology; Phosphoproteins - genetics; Phosphoproteins - metabolism; Phosphorylation - drug effects; Phosphorylation - physiology; Protein Binding - drug effects; Protein Binding - physiology; Protein Structure, Tertiary - drug effects; Protein Structure, Tertiary - physiology; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - metabolism; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Rats; src-Family Kinases; Tight Junctions - genetics; Tight Junctions - metabolism; Tyrosine - genetics; Tyrosine - metabolism; Zonula Occludens-1 Protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M804783200

Occludin is phosphorylated on tyrosine residues during the oxidative stress-induced disruption of tight junction, and in vitro phosphorylation of occludin by c-Src attenuates its binding to ZO-1. In the present study mass spectrometric analyses of C-terminal domain of occludin identified Tyr-379 and Tyr-383 in chicken occludin as the phosphorylation sites, which are located in a highly conserved sequence of occludin, YETDYTT; Tyr-398 and Tyr-402 are the corresponding residues in human occludin. Deletion of YETDYTT motif abolished the c-Src-mediated phosphorylation of occludin and the regulation of ZO-1 binding. Y398A and Y402A mutations in human occludin also abolished the c-Src-mediated phosphorylation and regulation of ZO-1 binding. Y398D/Y402D mutation resulted in a dramatic reduction in ZO-1 binding even in the absence of c-Src. Similar to wild type occludin, its Y398A/Y402A mutant was localized at the plasma membrane and cell-cell contact sites in Rat-1 cells. However, Y398D/Y402D mutants of occludin failed to localize at the cell-cell contacts. Calcium-induced reassembly of Y398D/Y402D mutant occludin in Madin-Darby canine kidney cells was significantly delayed compared with that of wild type occludin or its T398A/T402A mutant. Furthermore, expression of Y398D/Y402D mutant of occludin sensitized MDCK cells for hydrogen peroxide-induced barrier disruption. This study reveals a unique motif in the occludin sequence that is involved in the regulation of ZO-1 binding by reversible phosphorylation of specific Tyr residues.