DOWN REGULATION OF PHOSPOLIPASE C EPSILON ACTIVITY BY TRANSGLUTAMINASE 2 IS INDEPENDENT OF ITS CROSS‐LINKING ACTIVITY.

Abstract

PLCε and transglutaminases (TG2) are both multidomain and multifunctional Ca2+ dependent enzymes involved in signal transduction. The present work shows for the first time that these two enzymes are functionally linked. In an in vitro assay, the phosphodiesterase activity of PLCε was inhibited by TG2/GαH in a concentration and time dependent manner. When compared to the different isoforms of TG (HRBC > GPL > CRBC >FXIIIrA2) human erythrocyte TG2/GαH is the most potent inhibitor. The inhibitory activity of TG2/GαH is exhibited only at low concentrations of substrate (Phosphatidylinositol, below Km), suggesting that the mechanism involves a change in Km, and is not due to competition or sequestering of substrate. The inhibition of PLCε activity by TG2/GαH was independent of the catalytic activity of the latter, i.e. protein crosslinking by Nε (g-glutamyl)lysine side bridges, and did not require GTP/Mg2+. ELISA technique was employed to demonstrate the binding of PLCε to TG2/GαH with high affinity with a dissociation constant of approximately 7x10^-7 M. In vivo studies, by co-expressing TG2 and PLCε in TSA-201 cells, demonstrated the down regulation of PLCε activity by TG2. By expressing different domains of PLCε in E.Coli and by Far-Western analysis we have identified the binding region for TG2 on PLCε as its CDC25 domain. In TG2 stably transfected cells, NIH-3T3 cells, cotransfection with PLCε, TG2 is translocated to membranes mediated by Ca2+. Thus our data suggest that free Ca2+ generated by phosphodiesterase activity leads the G protein TG2 to act as a negative feedback regulator of PLCε.