Essential role of the EF-hand domain in targeting sperm phospholipase Cζ to membrane phosphatidylinositol 4,5-bisphosphate (PIP2)

Ondertitel:Essential role of the EF-hand domain in targeting sperm phospholipase C zeta to membrane phosphatidylinositol 4,5-bisphosphate (PIP2)

Sperm-specific phospholipase C-zeta (PLC zeta) is widely considered to be the physiological stimulus that triggers intracellular Ca2+ oscillations and egg activation during mammalian fertilization. Although PLC zeta is structurally similar to PLC delta 1, it lacks a pleckstrin homology domain, and it remains unclear how PLC zeta targets its phosphatidylinositol 4,5-bisphosphate (PIP2) membrane substrate. Recently, the PLC delta 1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLC delta 1 are notably conserved in PLC zeta. We investigated the potential role of these conserved cationic residues in PLC zeta by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLC zeta EF-hand domain. Microinjection of the PLC zeta EF-hand mutants into mouse eggs enabled their Ca2+ oscillation inducing activities to be compared with wild-type PLC zeta. Furthermore, the mutant proteins were purified, and the in vitro PIP2 hydrolysis and binding properties were monitored. Our analysis suggests that PLC zeta binds significantly to PIP2, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLC zeta significantly alters in vivo Ca2+ oscillation inducing activity and in vitro interaction with PIP2 without affecting its Ca2+ sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca2+ frequency and the binding ability of different PLC zeta mutants to PIP2. Moreover, a PLC zeta mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLC zeta to PIP2, leading to complete abolishment of its Ca2+ oscillation inducing activity.

Jaar van publicatie:2015