Structure-function analysis of PLC-zeta
- Calcium Signals for Egg Activation in Mammals
A short form of PLCζ, s-PLCζ that lacks three EF-hand domains from the N-terminus is expressed in the mouse sperm, probably as a splicing variant. s-PLCζ has much less Ca2+ oscillation-inducing activity, compared with PLCζ: injection of RNA at 100-fold higher concentration barely induced Ca2+ oscillations after a delay of 3 h (18). This suggests that EF-hand domains which usually contain the Ca2+-binding site in a protein may be important for the enzymatic activity of PLCζ. Our molecular structure-function analysis showed that when 4 EF-hand domains (EF1 to EF4) from the Nterminus are deleted one-by-one, deletion of EF1 and EF2 of recombinant PLCζ causes marked reduction of the PIP2-hydrolyzing activity in vitro and loss of the Ca2+ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant PLCζ (20).
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 affects Ca2+-sensitivity of the PLC activity, while deletion of EF1 to EF3 caused 12-fold elevation of EC50 of Ca2+ concentration (20). Thus, EF1 and EF2 are important for the PLCζ activity, and EF3 is responsible for its high Ca2+ sensitivity. Deletion of EF1-4 or Cterminal C2 domain (Fig. 3A) results in complete loss of PLC activity (20), indicating that both regions are prerequisite for the PLCζ activity.
It seems that PLC activity of PLCζ may be derived from the highly coordinated structure of the EF-hand region and C2 domain rather than the primary sequence in the Ca2+-binding loop. According to crystal analysis of three dimensional structure, PLCδ1 is folded at the linker region between the X and Y catalytic domains in such a way that the C2 domain in the C-terminus makes extensive contact with EF-hand domains in the Nterminus and the catalytic domain, forming the catalytic core (21, 22). Considering PLCζ in analogy with PLCδ1, all domains have to be prepared to form the three dimensional active conformation. There may be Ca2+- dependent coordinating structural determinant(s) other than EF1-EF3 for the highly Ca2+-sensitive enzymatic activity.
C2 domain is known to play a significant role in Ca2+- dependent subcellular membrane targeting of several lipid-metabolizing enzymes such as PLCδ1 or cPLA2 (22). Screening of interaction between C2 domain and phosphoinositides has 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 (20). Interestingly, these phosphatidylinositol monophosphates interfers with the PI(4,5)P2-hydrolyzing activity of PLCζ in vitro (20). The interaction between C2 domain and PI(3)P may be significant for negative regulation of PLCζ.
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