Vesicle fusion protein, YKT6, is a novel regulator of epithelial cell‐matrix adhesion and migration

Epithelial cell migration is a key regulatory element of a variety of biological and pathological processes, including embryonic morphogenesis, tissue repair, and tumor metastasis. This is a multistage process involving epithelial cell adhesion to the extracellular matrix (ECM) and remodeling of the cortical cytoskeleton. Trafficking of ECM adhesion proteins and other membrane and signaling proteins to the migrating cellular edge plays an essential role in regulating cell motility. YKT6 is a key component of the vesicle fusion machinery that regulates ER‐Golgi trafficking, however its functions in cell migration remain uninvestigated. In this study, we found that siRNA‐mediated knockdown of YKT6 in DU145 and p69 human prostate epithelial cells significantly accelerated planar cell migration in a wound closure assay and 3‐D cell invasion into Matrigel. These effects were accompanied by a significant increase in cell adhesion to ECM and cell spreading. Immunoblotting analysis of different focal adhesion proteins revealed two vivid effects of YKT6 knockdown. One effect was defective Golgi‐dependent glycosylation of β1‐integrin while the other involved decreased levels of phosphorylated paxillin. However, subsequent experiments demonstrated that neither of these effects is responsible for the increased motility of YKT6‐depleted cells. We also investigated the effects of YKT6 knockdown on the expression of cell‐cell adhesion proteins and observed a marked and selective increase in the expression of Junctional Adhesion Molecule A (JAM‐A) protein. Given the known pro‐migratory role of JAM‐A in different cell types, we probed JAM‐A upregulation to determine if it could mediate the increased motility of YKT6‐depleted cells. Co‐knockdown of JAM‐A and YKT6 reversed the accelerated migration of DU145 cells caused by YKT6 depletion. Since JAM‐A can promote epithelial cell migration by activating Rap1 small GTPase, we next investigated if Rap1 activity mediates the increased motility of YKT‐6 depleted epithelial cells. We observed that pharmacological Rap1 inhibitor, GGTI 298, reversed the accelerated wound healing in YKT6‐depleted epithelial cell monolayers. These results reveal a novel role for YKT6 in limiting epithelial cell migration via mechanisms involving the suppression of the JAM‐A‐Rap1 signaling nexus. Support or Funding Information Supported by NIH grants DK083968 and DK084953 to AII and by the Crohn's and Colitis Foundation of America grant 254881 to NGN.