Abstract
O-GlcNAcylation is a post-translational modification of a protein serine or threonine residue catalyzed by O-GlcNAc transferase (OGT) in the nucleus and cytoplasm. O-GlcNAcylation plays important roles in the cellular signaling that affect the different biological functions of cells, depending upon cell type. However, whether or not O-GlcNAcylation regulates cell adhesion and migration remains unclear. Here, we used the doxycycline-inducible short hairpin RNA (shRNA) system to establish an OGT knockdown (KD) HeLa cell line and found that O-GlcNAcylation is a key regulator for cell adhesion, migration, and focal adhesion (FA) complex formation. The expression levels of OGT and O-GlcNAcylation were remarkably suppressed 24 h after induction of doxycycline. Knockdown of OGT significantly promoted cell adhesion, but it suppressed the cell migration on fibronectin. The immunostaining with paxillin, a marker for FA plaque, clearly showed that the number of FAs was increased in the KD cells compared with that in the control cells. The O-GlcNAcylation levels of paxillin, talin, and focal adhesion kinase were down-regulated in KD cells. Interestingly, the complex formation between integrin β1, focal adhesion kinase, paxillin, and talin was greatly increased in KD cells. Consistently, levels of active integrin β1 were significantly enhanced in KD cells, whereas they were decreased in cells overexpressing OGT. The data suggest a novel regulatory mechanism for O-GlcNAcylation during FA complex formation, which thereby affects integrin activation and integrin-mediated functions such as cell adhesion and migration.
Highlights
O-GlcNAcylation is a post-translational modification of a protein serine or threonine residue catalyzed by O-GlcNAc transferase (OGT) in the nucleus and cytoplasm
Talin, FAK, and paxillin were labeled, which proved that they are O-GlcNAcylated proteins (Fig. 4, A–C). These results suggest that O-GlcNAcylation may affect both integrin 1–mediated complex formation and focal adhesion (FA) formation, which confirms this process as a regulator of cell adhesion and migration
We clearly showed that O-GlcNAcylation negatively regulates integrin-mediated cell adhesion and FA complex formation as well as integrin activation, which results in the control of cell migration on the extracellular matrix (ECM) (Fig. 7)
Summary
The data suggest a novel regulatory mechanism for O-GlcNAcylation during FA complex formation, which thereby affects integrin activation and integrin-mediated functions such as cell adhesion and migration. O-GlcNAcylation is controlled by OGT3 and is a specific type of post-translational modification that consists of the covalent attachment of single GlcNAc to the nucleus and cytoplasm of the serine or threonine residue of an extremely large family of target proteins [1, 2]. This post-translational modification is essential for cell survival and division [3], and aberrant. New insight into integrin-mediated cell migration and explain why O-GlcNAcylation is usually highly expressed in some malignant cancers
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