PKC is expressed predominantly in the epithelial tissues; however, its role in the regulation of epithelial tight junctions (TJs) is unknown. We present evidence that PKC phosphorylates occludin on threonine residues (T403 and T404) and plays a crucial role in the assembly and/or maintenance of TJs in Caco-2 and MDCK cell monolayers. Inhibition of PKC by specific pseudo substrate inhibitor or knockdown of PKC by specific shRNA disrupts the junctional distribution of occludin and ZO-1 and compromises the epithelial barrier function. Expression of dominant negative, PKC K394R disrupts the TJ and barrier function, whereas wild-type PKC and constitutively active PKC A161E enhance the TJ integrity. Inhibition and knockdown of PKC or expression of PKC K394R induce dephosphorylation of occludin on threonine residues, whereas active PKC elevates occludin phosphorylation. PKC directly interacts with the C-terminal domain of occludin and phosphorylates it on highly conserved T403 and T404. T403/404A mutations result in the loss of occludin's ability to localize at the TJs, whereas T403/404D mutations attenuates the PKC inhibitormediated redistribution of occludin from the intercellular junctions. These results reveal an important mechanism of epithelial TJ regulation by PKC .differentiation ͉ epithelium ͉ protein kinase T he epithelial tight junctions (TJs) on one hand determine the cell polarity by forming a fence between the apical and basolateral membranes (1), and on the other hand, it prevents the diffusion of toxins, allergens, and pathogens from the lumen into the tissue (2). Additionally, TJs play essential roles in the regulation of cell-cell adhesion and the epithelium-tomesenchymal transition (3). Dysfunctional TJs are associated with the pathogenesis of inflammatory diseases (2) and tumor metastasis (3). Therefore, understanding the molecular structure of TJs and the regulatory mechanisms that control the integrity of TJs is essential to advance our knowledge in epithelial homeostasis in health and disease.The assembly of TJs involves at least 3 types of transmembrane proteins, occludin, claudins, and junctional adhesion molecule (4). The intracellular domains of occludin and claudins interact with the plaque proteins such as ZO-1, ZO-2, and ZO-3, which form the platforms for recruitment of scaffold proteins such as cingulin, Par-3, Par-6, etc.; this TJ protein complex is anchored into the perijunctional actomyosin ring. Although occludin knockout mice showed the formation of intact TJs in different epithelia (5), several studies indicated that occludin does play an important role in the regulation of TJ integrity (6, 7).Protein kinases (8-10) and protein phosphatases (11) are either localized at the TJs or interact directly with the TJ proteins. Whereas atypical PKCs (PKC and PKC / ) directly interact with the TJs (10), PKC and PKC␤I may indirectly regulate the integrity of TJs (12). PKC , a novel PKC isoform, is predominantly expressed in epithelial tissues (13). The function of PKC in the epithelial tissues...

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.

The failure of anti-CD20 antibody (Rituximab) as therapy for lupus may be attributed to the transient and incomplete B cell depletion achieved in clinical trials. Here, using an alternative approach, we report that complete and sustained CD19+ B cell depletion is a highly effective therapy in lupus models. CD8+ T cells expressing CD19-targeted chimeric antigen receptors (CARs) persistently depleted CD19+ B cells, eliminated autoantibody production, reversed disease manifestations in target organs, and extended life spans well beyond normal in the (NZB × NZW) F1 and MRLfas/fas mouse models of lupus. CAR T cells were active for 1 year in vivo and were enriched in the CD44+CD62L+ T cell subset. Adoptively transferred splenic T cells from CAR T cell–treated mice depleted CD19+ B cells and reduced disease in naive autoimmune mice, indicating that disease control was cell-mediated. Sustained B cell depletion with CD19-targeted CAR T cell immunotherapy is a stable and effective strategy to treat murine lupus, and its effectiveness should be explored in clinical trials for lupus.

Interactions between E-cadherin, beta-catenin and PTP1B (protein tyrosine phosphatase 1B) are crucial for the organization of AJs (adherens junctions) and epithelial cell-cell adhesion. In the present study, the effect of acetaldehyde on the AJs and on the interactions between E-cadherin, beta-catenin and PTP1B was determined in Caco-2 cell monolayers. Treatment of cell monolayers with acetaldehyde induced redistribution of E-cadherin and beta-catenin from the intercellular junctions by a tyrosine phosphorylation-dependent mechanism. The PTPase activity associated with E-cadherin and beta-catenin was significantly reduced and the interaction of PTP1B with E-cadherin and beta-catenin was attenuated by acetaldehyde. Acetaldehyde treatment resulted in phosphorylation of beta-catenin on tyrosine residues, and abolished the interaction of beta-catenin with E-cadherin by a tyrosine kinase-dependent mechanism. Protein binding studies showed that the treatment of cells with acetaldehyde reduced the binding of beta-catenin to the C-terminal region of E-cadherin. Pairwise binding studies using purified proteins indicated that the direct interaction between E-cadherin and beta-catenin was reduced by tyrosine phosphorylation of beta-catenin, but was unaffected by tyrosine phosphorylation of E-cadherin-C. Treatment of cells with acetaldehyde also reduced the binding of E-cadherin to GST (glutathione S-transferase)-PTP1B. The pairwise binding study showed that GST-E-cadherin-C binds to recombinant PTP1B, but this binding was significantly reduced by tyrosine phosphorylation of E-cadherin. Acetaldehyde increased the phosphorylation of beta-catenin on Tyr-331, Tyr-333, Tyr-654 and Tyr-670. These results show that acetaldehyde induces disruption of interactions between E-cadherin, beta-catenin and PTP1B by a phosphorylation-dependent mechanism.