High-throughput investigation of transglutaminase 2 kinase regulation using a novel cysteine-modified peptide array

Transglutaminase 2 (TGase2) kinase has emerged as an important regulator of apoptosis as well as chromatin structure and function; however, details about the pathophysiological functions of TGase2 kinase have been limited because of the lack of a suitable activity assay for systematic investigation of TGase2 kinase regulation in a high-throughput manner. Thus, we developed a novel on-chip TGase2 kinase activity assay using a cysteine-modified insulin-like growth factor-binding protein-3-derived peptide (CMI peptide) on an array platform. This peptide array-based activity assay was reproducible, with a detection limit of 2.127 μg/ml. We successfully applied this assay to investigate the effects of thiol-reactive compounds and divalent cations on TGase2 kinase by determining the half maximal inhibitory concentrations (IC₅₀). Thiol-reactive compounds inhibited TGase2 kinase activity in a concentration-dependent manner, with IC₅₀ values ranging from 0.125 to 5.550 mM. Divalent metal cations also showed a concentration-dependent inhibition, with IC₅₀ values ranging from 0.005 to 1.937 mM; however, Ca²⁺ had no effect on TGase2 kinase activity. Thus, this novel kinase activity assay using the CMI peptide array described here is suitable for systematic investigation of TGase2 kinase regulation and may be useful for investigating the roles of TGase2 kinase in pathogenesis of kinase-mediated diseases.