A Canonical EF-Loop Directs Ca2+-Sensitivity in Phospholipase C-η2

Phospholipase C- (PLC) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLC2 can sense Ca2+ (stimulated by approximate to 1 mu M free Ca2+) suggesting that it can amplify transient Ca2+ signals. PLC enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca2+-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLC2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[H-1,H-1] TOCSY NMR. Both PLC2 EF-loop peptides bound Ca2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLC2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca2+-binding. To determine whether the EF-hand is responsible for Ca2+-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLC2 proteins. Addition of 70 mu M monensin (a Na+/H+ antiporter that increases intracellular Ca2+) induced a 4- to 7-fold increase in wild-type PLC2 activity. In permeabilized cells, PLC2 exhibited a approximate to 4-fold increase in activity in the presence of 1 mu M free Ca2+. The D256A (EF-loop1) mutant exhibited a approximate to 10-fold reduction in Ca2+-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca2+-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A, and E304A mutants. Interestingly, the monensin responses and Ca2+-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca2+-sensing. J. Cell. Biochem. 115: 557-565, 2014. (c) 2013 Wiley Periodicals, Inc.