title: The role of EF-hand domains and C2 domain in regulation of enzymatic activity of phospholipase Czeta.
abstract: Sperm-specific phospholipase C-zeta (PLCzeta) induces Ca2+ oscillations and egg activation when injected into mouse eggs. PLCzeta has such a high Ca2+ sensitivity of PLC activity that the enzyme can be active in resting cells at approximately 100 nM Ca2+, suitable for a putative sperm factor to be introduced into the egg at fertilization (Kouchi, Z., Fukami, K., Shikano, T., Oda, S., Nakamura, Y., Takenawa, T., and Miyazaki, S. (2004) J. Biol. Chem. 279, 10408-10412). In the present structure-function analysis, deletion of EF1 and EF2 of the N-terminal four EF-hand domains caused marked reduction of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-hydrolyzing activity in vitro and loss of Ca2+ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant. However, deletion of EF1 and EF2 or mutation of EF1 or EF2 at the x and z positions of the putative Ca2+-binding loop little affected the Ca2+ sensitivity of the PLC activity, whereas deletion of EF1 to EF3 caused 12-fold elevation of the EC50 of Ca2+ concentration. Thus, EF1 and EF2 are important for the PLCzeta activity, and EF3 is responsible for its high Ca2+ sensitivity. Deletion of four EF-hand domains or the C-terminal C2 domain caused complete loss of PLC activity, indicating that both regions are prerequisites for PLCzeta activity. Screening of interactions between the C2 domain and phosphoinositides revealed that C2 has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P but not to PI(4,5)P2 or acidic phospholipids. PI(3)P and PI(5)P reduced PLCzeta activity in vitro, suggesting that the interaction could play a role for negative regulation of PLCzeta.
authors.affiliation: Department of Physiology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo 162-8666, Japan.
authors.forename: Zen
authors.initials: Z
authors.lastname: Kouchi
authors.forename: Tomohide
authors.initials: T
authors.lastname: Shikano
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Hideki
authors.initials: H
authors.lastname: Shirakawa
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Fukami
authors.forename: Shunichi
authors.initials: S
authors.lastname: Miyazaki
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D006868:Hydrolysis; D008081:Liposomes / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009991:Oscillometry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry; D054801:Phosphoinositide Phospholipase C; D010743:Phospholipids / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D013997:Time Factors; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008081:Liposomes; D019269:Phosphatidylinositol 4,5-Diphosphate; D010743:Phospholipids; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C499234:Plcz1 protein, mouse; D002118:Calcium