Abstract
The epithelial cell adhesion molecule (EpCAM) is a transmembrane cell adhesion glycoprotein, which primarily contributes to stemness, proliferation, and metastasis properties of tumor cells. Regulated intramembrane proteolysis by ADAM proteases and γ-secretase cleaves EpCAM into an ∼27 kDa soluble extracellular and an ∼4 kDa cytoplasmic domain (EpICD). After the EpICD fragment is released inside the cell, the formation of a nuclear signaling complex with the FHL2 molecule is critical for exerting its regulatory role. Trop-2, a homologous protein of EpCAM, undergoes phosphorylation in its cytoplasmic domain (Trop-IC). The phosphorylation of Trop-2 is reported to be crucial for its function. This led us to ask the fundamental question if EpCAM does undergo similar post-translational modification(PTM) like its homologous protein to carry out its diverse biological function. Here, we identify a putative phosphorylation site at Tyr297 located in the cytoplasmic domain of EpCAM. Molecular dynamic simulation (MDS) of 90 ns was carried out to understand the biological/functional relevance of the putative phosphorylation. It was observed that this phosphorylation stabilizes the α-helical structure of the EpICD. Though Tyr297 does not affect the γ-secretase mediated cleavage of EpCAM, it affects the binding of EpICD to FHL2. Docking analysis revealed that phosphorylation mediated structural stability of EpICD positively impacts its binding affinity with FHL2, which was further validated using 100 ns MDS. Phosphorylated EpICD forms higher numbers of hydrogen bonds, salt bridges, and other non-bonded interactions with FHL2, leading to enhanced interactions. This in silico study reveals a potential PTM in the EpICD, providing the basis for future research in understanding the mechanism behind the diverse biological function of EpCAM.