The role of X/Y linker region and N-terminal EF-hand domain in nuclear translocation and Ca 2+ oscillation-inducing activities of phospholipase Cζ, a mammalian egg-activating factor

Abstract

Sperm-specific phospholipase C-zeta (PLCζ) causes intracellular Ca 2+ oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCζ, which consists of four EF-hand domains (EF1-EF4) in theNterminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLCζ mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374-381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg 376, Lys 377, Arg 378, Lys 379, or Lys 381 with glutamate, whereas Ca 2+ oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys 299 and/or Lys 301 in the C terminus of X domain, or Trp 13, Phe 14, or Val 18 in the N terminus of EF1. Ca 2+ oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter.Ashort sequence 374-383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296-309 or 1-19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2-4 or C2 domain. Thus, PLCζ is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLCδ1, seems to be required not only for enzymatic activity but also for nuclear translocation ability. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.