Role of phospholipase C-zeta domains in Ca2+-dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca2+ oscillations.
The sperm-specific phospholipase C-zeta (PLCzeta) elicits fertilization-like Ca2+ oscillations and activation of embryo development when microinjected into mammalian eggs (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development (Camb.) 129, 3533-3544; Cox, L. J., Larman, M. G., Saunders, C. M., Hashimoto, K., Swann, K., and Lai, F. A. (2002) Reproduction 124, 611-623). PLCzeta may represent the physiological stimulus for egg activation and development at mammalian fertilization. PLCzeta is the smallest known mammalian PLC isozyme, comprising two EF hand domains, a C2 domain, and the catalytic X and Y core domains. To gain insight into PLCzeta structure-function, we assessed the ability of PLCzeta and a series of domain-deletion constructs to cause phosphatidylinositol 4,5-bisphosphate hydrolysis in vitro and also to generate cytoplasmic Ca2+ changes in intact mouse eggs. PLCzeta and the closely related PLCdelta1 had similar K(m) values for phosphatidylinositol 4,5-bisphosphate, but PLCzeta was around 100 times more sensitive to Ca2+ than was PLCdelta1. Notably, specific phosphatidylinositol 4,5-bisphosphate hydrolysis activity was retained in PLCzeta constructs that had either EF hand domains or the C2 domain removed, or both. In contrast, Ca2+ sensitivity was greatly reduced when either one, or both, of the EF hand domains were absent, and the Hill coefficient was reduced upon deletion of the C2 domain. Microinjection into intact mouse eggs revealed that all domain-deletion constructs were ineffective at initiating Ca2+ oscillations. These data suggest that the exquisite Ca2+-dependent features of PLCzeta regulation are essential for it to generate inositol 1,4,5-trisphosphate and Ca2+ oscillations in intact mouse eggs.
Nomikos
Michail
M
Cell Signalling Laboratory, Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff CF14 4XN.
Blayney
Lynda M
LM
Larman
Mark G
MG
Campbell
Karen
K
Rossbach
Andreas
A
Saunders
Christopher M
CM
Swann
Karl
K
Lai
F Anthony
FA
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism*; D054801:Phosphoinositide Phospholipase C; D054803:Phospholipase C delta; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D010738:Type C Phospholipases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D011994:Recombinant Proteins; D010738:Type C Phospholipases; C518024:PLCD1 protein, human; C499235:PLCZ1 protein, human; D054801:Phosphoinositide Phospholipase C; D054803:Phospholipase C delta; C518025:Plcd1 protein, mouse; C518026:Plcd1 protein, rat; C499234:Plcz1 protein, mouse; D002118:Calcium