Dipeptidyl Peptidase 9: Novel Natural Substrates, Intracellular Localisation and its Mechanism of Regulating Cell Adhesion

Abstract

Dipeptidyl Peptidase 9 (DPP9) is intracellular, ubiquitously expressed and influences epidermal growth factor signalling. Mice lacking DPP9 proteolytic activity die as neonates. Super Resolution Microscopy revealed that DPP9-EGFP co-localises with microtubules. A long form of DPP9 was in the nucleus and active there. Cytoplasmic DPP9-short was visualised at the leading edge of migrating cells and co-localised with the focal adhesion proteins integrin-β1 and talin. DPP9 gene silencing or enzyme inhibition reduced cell adhesion and migration, the abundance of integrin-β1 and talin and the phosphorylation of focal adhesion kinase and paxillin. Gel-based proteomics, applied to embryonic fibroblasts derived from mice lacking DPP9 proteolytic activity, identified 111 proteins as novel DPP9 substrates. DPP9-mediated cleavage of nine substrates was confirmed by MALDI-TOF or immunoblotting. The dipeptide Xaa-Ala was identified as a site cleaved by DPP9 but not DPP8, which suggests different roles for DPP9 and DPP8. Collectively, this study underlines the biological significance of DPP9 in tumour cell adhesion and migration, which strengthens indications that DPP9 may become a therapeutic target for limiting tumour growth and metastasis. Moreover, these data represent the first identification of natural DPP9 substrates of the cell nucleus, and point to potential roles of DPP9 within different subcellular compartments.