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

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.Epithelial tight junctions (TJs)² form a selective barrier to the diffusion of toxins, allergens, and pathogens from the external environment into the tissues in the gastrointestinal tract, lung, liver, and kidney (1). Disruption of TJs is associated with the gastrointestinal diseases such as inflammatory bowel disease, celiac disease, infectious enterocolitis, and colon cancer (2–4) as well as in diseases of lung and kidney (5, 6). Numerous inflammatory mediators such as tumor necrosis factor α, interferon γ, and oxidative stress (7–12) are known to disrupt the epithelial TJs and the barrier function. Several studies have indicated that hydrogen peroxide disrupts the TJs in intestinal epithelium by a tyrosine kinase-dependent mechanism (11, 12).Four types of integral proteins, occludin, claudins, junctional adhesion molecules, and tricellulin are associated with TJs. Occludin, claudins, and tricellulin are tetraspan proteins, and their extracellular domains interact with homotypic domains of the adjacent cells (1, 2, 13). The intracellular domains of these proteins interact with a variety of soluble proteins such as ZO-1, ZO-2, ZO-3, 7H6, cingulin, and symplekin (14–23); this protein complex interacts with the perijunctional actomyosin ring. The interactions among TJ proteins are essential for the assembly and the maintenance of TJs. Therefore, regulation of the interactions among TJ proteins may regulate the TJ integrity. A significant body of evidence indicates that numerous signaling molecules are associated with the TJs. Protein kinases and protein phosphatases such as protein kinase Cζ (PKCζ), PKCι/λ (24), c-Src (25), c-Yes (26, 27), mitogen-activated protein kinase (28), PP2A, and PP1 (29) interact with TJs, indicating that TJs are dynamically regulated by intracellular signal transduction involving protein phosphorylation. Additionally, other signaling molecules such as calcium (30), phosphatidylinositol 3-kinase (31), Rho (32), and Rac (33) are involved in the regulation of TJs.Occludin, a ～65-kDa protein, has been well characterized to be assembled into the TJs. Although occludin knock-out mice showed the formation of intact TJs in different epithelia (34), numerous studies have emphasized that it plays an important role in the regulation of TJ integrity. Occludin spans the membrane four times to form two extracellular loops and one intracellular loop, and the N-terminal and C-terminal domains hang into the intracellular compartment (35–37). In epithelium with intact TJs, occludin is highly phosphorylated on Ser and Thr residues (38), whereas Tyr phosphorylation is undetectable. However, the disruption of TJs in Caco-2 cell monolayers by oxidative stress and acetaldehyde leads to Tyr phosphorylation of occludin; the tyrosine kinase inhibitors attenuate the disruption of TJs (39, 40). Furthermore, a previous in vitro study demonstrated that Tyr phosphorylation of the C-terminal domain of occludin leads to the loss of its interaction with ZO-1 and ZO-3 (25).In the present study we identified the Tyr residues in occludin that are phosphorylated by c-Src and determined their role in regulated interaction between occludin and ZO-1 and its assembly into the TJs. Results show that 1) Tyr-379 and Tyr-383 in chicken occludin and Tyr-398 and Tyr-402 in human occludin are the exclusive sites of phosphorylation by c-Src, and these Tyr residues are located in a highly conserved sequence of occludin, YET-DYTT, 2) deletion of YEDTYTT or point mutation of Tyr-398 and Tyr-402 in human occludin attenuates the phosphorylation-dependent regulation of ZO-1 binding, 3) Y398D/Y402D mutation of human occludin leads to loss of ZO-1 binding and prevents its translocation to the plasma membrane and cell-cell contact sites in Rat-1 cells, 4) Y398D/Y402D mutation of occludin delays its assembly into the intercellular junctions during the calcium-induced assembly of TJs, and 5) expression of Y398D/Y402D mutant occludin sensitizes cell monolayers for hydrogen peroxide-induced disruption of barrier function.