Regulation And Mechanism Of The Interaction Between P120 And E-cadherin By O-GlcNAc

Perturbation of E-cadherin-mediated cell adhesion is a key event in most of the epithelial cell carcinoma, interferes with tissue integrity and allows malignant cells to disseminate from the primary tumor thereby initiating cancer metastasis, p120 plays a role as an essential regulator of E-cadherin adhesion complex. It is responsible for stabilizing cadherin-catenin complexes and controlling the balance of E-cadherin turnover. Uncoupling of the p120-E-cadherin interaction or a reduction in p120 protein levels in cultured cells significantly increases levels of E-cadherin internalization and reduces cell-cell adhesion, resulting in an increase of the tumor malignancy. Therefore, the study of the molecular mechanism of the interaction between p120 and E-cadherin is very important. Previous studies showed that the post-translational modification of p120 protein is involved in the functional regulation of E-cadherin complex. The phosphorylation of multiple tyrosine, serine and threonine residues in p120 is implicated in the regulation of E-cadherin function.O-GlcNAc, the modification of Ser and Thr residues of nuclear and cytoplasmic proteins with O-linked β-N-acetylglucosamine, is one of a growing number of posttranslational modifications of proteins thought to modulate the function/activity of proteins in cells, which is involved in protein localization, protein-protein interactions and protein degradation, etc. Abnormal levels of O-GlcNAc involve in a variety of human diseases. Our previous studies showed that O-GlcNAc played an important regulatory role in breast cancer, lung cancer, and colon cancer. Increased global levels of O-GlcNAc inhibited the membrane localization of E-cadherin in mouse breast cancer cells 4T1, in turn reduced cell-cell adhesion. Meanwhile, we found that the p120 protein was modified by O-GlcNAc in 4T1 cells. Based on the above results, we speculated that the O-GlcNAc modification of p120 could be involved in this process and thus affect cell-cell adhesion. Therefore, we further studied the underlying molecular mechanism of that O-GlcNAc modification and the O-GlcNAc transferase (OGT) regulate the interaction between p120 and E-cadherin.In previous studies, we found that the increasing global O-GlcNAc levels impaired the cell adhesion in 4T1 cells. In this study, we examined the regulation of O-GlcNAc in the p120/E-cadherin complex in several other cells (HEK293T cells, lung carcinoma A549 cells and prostate cancer BPH-1 cells). We found that increased O-GlcNAc inhibited the cytoskeleton-associated p120 and E-cadherin, as well as,the interaction between p120 and E-cadherin in all of the detected cells.We already found that p120 is O-GlcNAcylated in mouse breast cancer 4T1 cells. In this study, we found that p120 could also be modified by O-GlcNAc in several human cells (embryonic kidney cells HEK293T, non-small cell lung cancer cell H1299 and adenocarcinoma alveolar basal epithelial cells A549). We established the cell model with decreased O-GlcNAc through silencing OGT gene expression. Meanwhile, we constructed the cell model with elevated O-GlcNAc by OGT overexpression or OGA inhibitor (Thiamet G) treatment. O-GlcNAc-specific antibody (RL2) recognition, sWGA Pulldown assay, efficient and specific Metabolic Labeling and Enzyme Labeling methods were performed to detect the O-GlcNAc modification of p120. The results showed that both endogenous and exogenous p120 were O-GlcNAcylated in HEK293T, H1299 and A549 cells. OGT silencing could not only reduce the global O-GlcNAc levels but also significantly inhibit the O-GlcNAc modification of p120; in contrast, both OGT overexpression and Thiamet G treatment could promote O-GlcNAc modification of p120 followed. We demonstrated that there is a universal O-GlcNAc modification of p120 in different cell lines, and the O-GlcNAc level of p120 varied with the changes of the global O-GlcNAc levels.In order to elucidate the regulatory role of p120 O-GlcNAc modification in p120/E-cadherin complex, we extracted cytoskeleton-associated and unassociated E-cadherin adhesion complex from HEK293T cells and compared the O-GlcNAc levels of p120 in these two parts. O-GlcNAc modification assay demonstrated that the O-GlcNAc level of either endogenous or exogenous p120 binding to E-cadherin adhesion complex is lower than that of the soluble p120. In addition, we also certified that the O-GlcNAc of p120 played a negative regulatory role in the interaction between p120 and E-cadherin through Flag-pulldwon assay between Flag-p120 with different O-GlcNAc modification levels and GST-ECD (ECD, the cytoplasmic region of E-cadherin) in vitro.To determine the mechanism of that the O-GlcNAc modification of p120 inhibits the interaction between p120 and E-cadherin, we splited full-length p120 into four truncated p120 mutants:the N-terminal domain, phosphorylation regulatory domain, the Armadillo domain and the C-terminal domain. We detected the O-GlcNAc level of these four mutants using metabolic labeling assay and determined the main O-GlcNAc modification domain that is the Armadillo repeat region that just is E-cadherin binding region. These results indicated that O-GlcNAc inhibits the interaction of p120 with E-cadherin probably through modifying p120 Armadillo domain.Finally, we detected whether OGT could bind to p120 directly. Co-immunoprecipitation and Pulldown assay demonstrated that OGT could directly bind p120 through the phosphorylated regulatory domain or the Armadillo domain, while OGT could combine neither with E-cadherin nor to p120/E-cadherin complex. Protein binding assy indicated that that OGT inhibited the interaction of p120 with E-cadherin in a dose dependent manner in vitro; additionally, we found OGT could also inhibit the formation of E-cadherin adhesion complex independent of its catalytic activity through extracting cytoskeleton-associated proteins from H1299 cells with transfected wild-type OGT or mutant OGT (H558A) without catalytic activity. These findings provide a novel regulatory mechanism for the interaction between p120 and E-cadherin and thus E-cadherin-mediated cell-cell adhesion.In conclusion, this paper demonstrates the universality of the O-GlcNAc modification of p120 in different cells for the first time, and the O-GlcNAc modification of p120 inhibits the interplay with E-cadherin, which could be the potential molecular mechanism of O-GlcNAc reducing cell adhesion and promoting tumor development. Moreover, OGT inhibits the formation of E-cadherin adhesion complex independent of its catalytic activity possibly partly through binding p120 Armadillo domain to block the interaction between p120 and E-cadherin. This study not only provides a novel mechanism for the regulation of E-cadherin adhesion complex, but also opens up the idea for the molecular mechanism underlying O-GlcNAc modulates the metastasis of tumor. Moreover, it also provides important inspiration and theoretical basis for the targeted therapies in cancer.