Phospholipase C-η2 is activated by elevated intracellular Ca 2+ levels

Abstract

Phospholipase C-η2 (PLCη2) is a novel enzyme whose activity in a cellular context is largely uncharacterised. In this study the activity of PLCη2 was examined via [ 3H]inositol phosphate release in COS7 cells expressing the enzyme. PLCη2 activity increased approximately 5-fold in response to monensin, a Na +/H + antiporter. This was significantly inhibited by CGP-37157 which implies that the effect of monensin was due, at least in part, to mitochondrial Na +/Ca 2+-exchange. Direct activation of PLCη2 by < 1 μM Ca 2+ was confirmed in permeabilised transfected cells. The roles of the PH and C2 domains in controlling PLCη2 activity via membrane association were also investigated. A PH domain-lacking mutant exhibited no detectable activity in response to monensin or Ca 2+ due to an inability to associate with the cell membrane. Within the C2 domain, mutation of D920 to alanine at the predicted Ca 2+-binding site dramatically reduced enzyme activity highlighting an important regulatory role for this domain. Mutation of D861 to asparagine also influenced activity, most likely due to altered lipid selectivity. Of the C2 mutations investigated, none altered sensitivity to Ca 2+. This suggests that the C2 domain is not responsible for Ca 2+ activation. Collectively, this work highlights an important new component of the Ca 2+ signalling toolkit and given its sensitivity to Ca 2+, this enzyme is likely to facilitate the amplification of intracellular Ca 2+ transients and/or crosstalk between Ca 2+-storing compartments in vivo.