Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M500629200 on July 6, 2005

J. Biol. Chem., Vol. 280, Issue 35, 31011-31018, September 2, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/35/31011    most recent
M500629200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nomikos, M.
Right arrow Articles by Lai, F. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nomikos, M.
Right arrow Articles by Lai, F. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Role of Phospholipase C-{zeta} Domains in Ca2+-dependent Phosphatidylinositol 4,5-Bisphosphate Hydrolysis and Cytoplasmic Ca2+ Oscillations*

Michail Nomikos{ddagger}, Lynda M. Blayney{ddagger}, Mark G. Larman{ddagger}§, Karen Campbell¶, Andreas Rossbach{ddagger}, Christopher M. Saunders{ddagger}, Karl Swann§, and F. Anthony Lai{ddagger}||

From the {ddagger}Cell Signalling Laboratory, Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff CF14 4XN, the §Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, and the Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom

Received for publication, January 18, 2005 , and in revised form, June 30, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The sperm-specific phospholipase C-{zeta} (PLC{zeta}) 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). PLC{zeta} may represent the physiological stimulus for egg activation and development at mammalian fertilization. PLC{zeta} 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 PLC{zeta} structure-function, we assessed the ability of PLC{zeta} 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. PLC{zeta} and the closely related PLC{delta}1 had similar Km values for phosphatidylinositol 4,5-bisphosphate, but PLC{zeta} was around 100 times more sensitive to Ca2+ than was PLC{delta}1. Notably, specific phosphatidylinositol 4,5-bisphosphate hydrolysis activity was retained in PLC{zeta} 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 PLC{zeta} regulation are essential for it to generate inositol 1,4,5-trisphosphate and Ca2+ oscillations in intact mouse eggs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A series of pre-programmed biochemical events are triggered during fertilization. The earliest signaling event in the activation of an egg by a sperm is a large, transient increase in intracellular free calcium ion concentration ([Ca2+]i) (1, 2). In many nonmammalian species, such as sea urchin, the observed Ca2+ increase in the egg comprises a single transient, but in mammals and some marine invertebrates there is a series of repetitive Ca2+ oscillations (3, 4). The frequency and duration of these Ca2+ oscillations vary between species (1). In response to this Ca2+ signal, the fertilized egg completes meiosis and initiates the process of embryonic development (5).

Several lines of evidence implicate the inositol 1,4,5-trisphosphate (IP3)1 signaling pathway (6) as the origin of the Ca2+ signals in mammalian eggs. IP3 is produced by hydrolysis of phosphatidylinositol 4,5-bisphosphate in a reaction that is catalyzed by phosphoinositide-specific phospholipase (PI-PLC) (6). Liberated IP3 then causes Ca2+ release by binding to IP3 receptors located on the endoplasmic reticulum of eggs and oocytes (7, 8). The essential role of IP3 and the IP3 receptor in fertilization is illustrated by studies in mouse and hamster eggs, where Ca2+ oscillations at fertilization can be inhibited by microinjection of antibodies that inhibit the IP3 receptor (9) or by down-regulation of IP3 receptors (10, 11). In addition, it has been shown that sustained injection of IP3, the repeated photorelease of caged IP3, or the microinjection of the IP3 analogue adenophostin can all lead to a series of Ca2+ oscillations in eggs (8, 12, 13). Hence, in mammalian eggs, IP3 is both necessary and potentially sufficient to explain the Ca2+ oscillations observed at fertilization. However, the precise mechanism employed by a sperm to generate an IP3 increase in the egg is not established.

In mammalian eggs there is evidence for the existence of different types of PI-PLCs. The stimulation of egg-derived PLC isoforms of the {beta} or {gamma} class by receptor tyrosine kinases (14) or by guanine nucleotide-binding proteins (G-proteins) (15) can lead to Ca2+ release in different species of eggs. However, there is evidence that neither the tyrosine kinase nor the G-protein pathways are necessary for Ca2+ release at fertilization in mouse eggs (2). In contrast to classical transmembrane receptor signaling, there is evidence that mammalian eggs can be activated by a sperm protein component that is introduced into the egg after gamete membrane fusion (1, 4). Egg microinjection of sperm extracts, or whole sperm, triggers Ca2+ oscillations similar to fertilization in a range of different species (17-23). The injection of such a sperm factor also activates the development of eggs and hence could represent the physiological agent that triggers egg activation and embryo development at fertilization (24).

We demonstrated that the ability of soluble mammalian sperm extracts to cause Ca2+ oscillations can be explained by the presence of a sperm-specific PLC activity (25, 26). This activity was shown to be due to a novel mammalian PI-PLC, phospholipase C{zeta} (PLC{zeta}), that we isolated from a spermatid cDNA library (27). Microinjection of cRNA encoding the mouse (27), human, and cynomolgus monkey PLC{zeta} (28) into mouse eggs triggered Ca2+ oscillations similar to those observed at fertilization. Furthermore, Ca2+ oscillations are abolished when PLC{zeta} is immunodepleted from native sperm extracts (27). A recent study showed that microinjection of recombinant PLC{zeta}, synthesized using a baculovirus expression system, could also trigger Ca2+ oscillations in mouse eggs (29). One unusual feature of PLC{zeta} is that it is effective at causing Ca2+ oscillations in eggs at very low concentrations (e.g. 10 fg/egg) (24, 27, 29). In contrast, other studies have shown that PLC isoforms of the {beta}, {gamma}, or {delta} class are either ineffective (25) or at least much less effective than PLC{zeta} at causing Ca2+ release when microinjected into eggs (2, 29, 30). The specific reasons why sperm PLC{zeta} is much more effective than other PLC isoforms at causing Ca2+ oscillations in eggs are currently unknown.

The unique functional features of PLC{zeta} may be attributable to its distinct domain structure. There are five subfamilies of PI-PLCs ({beta}, {gamma}, {delta}, {epsilon}, and {zeta}) classified on the basis of their sequence homology (27, 31). The regions of greatest sequence identity are the X and Y domains, which form the active site for enzymatic cleavage of PIP2 in all PI-PLCs. In addition, all isoforms have in common a C2 domain, which binds to lipids such as phosphatidylserine. In studies with PI-PLC{delta}1, the C2 domain has been suggested to orientate the enzyme to the membrane containing PIP2, and this association is Ca2+-dependent (31). PI-PLCs also contain EF hands, but the role of these Ca2+ binding domains in PLC isozymes is not clear. These domains usually bind one Ca2+ ion each, although variants that do not bind Ca2+ ions have been identified (31, 32). PLC{zeta} is most closely related to PLC{delta} but is distinct in that it lacks a PH domain (Fig. 1A), a common feature present in most PI-PLCs (27). The PH domain characteristically targets the PLC to phosphoinositides or other regulatory proteins such as G-protein {beta}{gamma} subunits in the plasma membrane (31-33).

The aim of this study is to examine the importance of the EF hand and C2 domains with respect to PLC{zeta} enzymatic activity in vitro and their involvement in initiating Ca2+ oscillations in eggs. A series of recombinant PLC{zeta} domain-deletion constructs were prepared, together with the well characterized PLC{delta}1 (34, 35) for comparison, and were tested by using an in vitro PLC activity assay (hydrolysis of PIP2) and also an in situ assay by microinjection into eggs of the corresponding cRNA constructs (Ca2+ oscillations). Our studies show that although the XY catalytic domain is minimally sufficient for in vitro enzymatic hydrolysis of PIP2, all of the disparate domains, i.e. EF hands and C2 domains, are required for PLC{zeta} to initiate the trademark Ca2+ oscillations observed in mammalian eggs upon fertilization by sperm.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of PLC{zeta}, PLC{delta}1, and Deletion Constructs—Mouse PLC{zeta} and a series of domain-deletion constructs were amplified by PCR from the original cDNA clone (GenBankTM accession number AF435950 [GenBank] ), using Pfu polymerase and the appropriate primers to incorporate a 5'-EcoRI site and a 3'-SalI site. PCR products were cloned into pGEX-5X2 (Amersham Biosciences). The following primers were used for each construct: for full-length PLC{zeta}, 5'-AATCGAATTCTCATGGAAAGCCAACTTCATGAG-3' (forward) and 5'-AATGGTCGACATGCGTCACTCTCTGAAGTA-3' (reverse); for {zeta}{Delta}EF1, 5'-ACCGGAATTCATATTTTTAAGGAAAATGAC-3' (forward) and for {zeta}{Delta}EF1,2, 5'-ACCGGATTCGATACATGTTTTCATCAGAATGT-3'(forward). For the latter two constructs, the reverse primer was the same as that of full-length PLC{zeta} stated above. For {zeta}{Delta}C2, the forward primer was the same as for full-length PLC{zeta} and 5'-GATGGTCGACATATTCTGGTTAATTGGGGT-3' (reverse); and for {zeta}XY, the primers were ACCGGATTCGATACATGTTTTCATCAGAATGT-3' (forward) and 5'-GATGGTCGACATATTCTGGTTAATTGGGGT-3' (reverse).

The rat PLC{delta}1 clone (GenBankTM accession number M20637 [GenBank] ) was kindly provided by M. Katan (Cancer Research UK, Centre for Cell and Molecular Biology, London, UK). Appropriate primers were designed to incorporate a 5'-SalI site and a 3'-NotI site at the ends of PLC{delta}1. PLC{delta}1 was amplified by PCR and cloned into pGEX-5X2 by using these restriction sites. The following primers were used for PLC{delta}1: 5'-CTTCGTCGACCATGGACTCGGGTAGGGAC-3' (forward) and 5'-CACCGCGGCCGCTTAGTCCTGGATGGAGATCTTC-3' (reverse); for {Delta}PH{delta}1 the forward primer was 5'-TTCGTCGACTGGGCTCCATGGACCAGCGGCAGAAGC-3'(forward) and reverse primer was the same as for full-length PLC{delta}1. For {Delta}PH-PLC{delta}1 the primers incorporated a 5'-EcoRI site, TTCAGAATTCCCATGGACTCGGGTAGGGACTT-3' (forward), and a 3'-SalI site, 5'-TGTCGACCAGCCTTTCGCAAGCAGGAGTGAAT-3' (reverse). Each of the above expression vector constructs was confirmed by dideoxynucleotide sequencing (Prism Big Dye kit; ABI Prism® 3100 Genetic Analyzer, Applied Biosystems, Warrington, UK).

For quantitation of expression of microinjected cRNAs in mouse oocytes, PLC{delta}1, PLC{zeta}, and various deletion constructs were tagged via their C termini with firefly luciferase. PCR amplification of the PLCs was performed with the original templates, described above, using Pfu polymerase. PLC{zeta} constructs were amplified using a 5' primer incorporating an EcoRI site and a 3' primer that ablated the stop codon and created a NotI site. PLC{delta}1 was amplified using a 5' primer containing a SalI site and a 3' primer that removed the stop codon and incorporated a NotI site. PCR products were cloned into pCR3 (Invitrogen) via the appropriate restriction sites. The luciferase open reading frame was amplified from pGL2 (Promega) with primers incorporating NotI sites, and the product was cloned into the NotI site of the pCR3-PLC vector. Each construct was confirmed by dideoxynucleotide sequencing, as described above. cRNA was generated as described below, using the mMessage mMachine T7 kit (Ambion) and polyadenylated using the poly(A) tailing kit (Ambion), as per the manufacturer's instructions.

Protein Expression and Purification—For bacterial expression of GST fusion proteins, Escherichia coli (BL21 or Rosetta cells, Novagen) was transformed with the appropriate pGEX-5X-2 construct and cultured at 30 °C until the A600 reached 0.5, and then protein expression was induced for 4 h at 25°C with 0.5 mM isopropyl {beta}-D-thiogalactopyranoside (Promega). Cells were harvested by centrifugation at 6000 x g for 10 min, resuspended in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4·7H2O, 1.4 mM KH2PO4, pH 7.4) containing 20% glycerol, 2 mM dithiothreitol, and protease inhibitor mixture (Roche Applied Science), and then sonicated three times for 20 s on ice. After 15 min of centrifugation at 15,000 x g, 4 °C, soluble GST fusion proteins were purified by affinity chromatography using glutathione-SepharoseTM 4B (GS4B) beads following standard procedures (Amersham Biosciences). Eluted proteins were dialyzed overnight (Pierce; SnakeSkin 10,000 molecular weight cut-off) at 4 °C against 4 liters of PBS, then concentrated with centrifugal concentrators, and stored at -80 °C in buffer containing 40% glycerol, 2 mM dithiothreitol, and EDTA-free protease inhibitors (Roche Applied Science).

Assay of PLC Activity—PIP2 hydrolytic activity of recombinant PLC{delta}1, PLC{zeta}, and their deletion constructs was assayed as described previously (36), with some modifications. The final volume of the assay mixture was 50 µl containing 100 mM NaCl, 0.4% sodium cholate (w/v), 2 mM CaCl2, 4 mM EGTA, 20 µg of bovine serum albumin, 5 mM 2-mercaptoethanol, and 20 mM Tris-HCl buffer, pH 6.8. The final concentration of PIP2 in the reaction mixture was 220 µM, containing 0.05 µCi of [3H]PIP2. The assay conditions were optimized for linearity, requiring a 10-min incubation of 20 pmol of PLC protein sample at 25 °C. Reactions were stopped by addition of 0.25 ml of chloroform/methanol/concentrated HCl (100:100:0.6 v/v) followed by 0.075 ml of concentrated HCl. The mixture was vortexed and centrifuged at 2000 x g for 2 min, and then 0.2 ml of the upper aqueous phase was removed and added to 10 ml of Optiphase "Hisafe 3" scintillation mixture (Wallac), and the radioactivity was determined by liquid scintillation spectrofluorimetry (Packard Tri-Carb 2100TR). In assays to determine dependence on PIP2 concentration, 0.05 µCi of [3H]PIP2 was mixed with cold PIP2 to give the appropriate final concentration. In assays examining the Ca2+ sensitivity, Ca2+ buffers were prepared by EGTA/CaCl2 admixture, as described previously (37). In the assays to monitor pH dependence of the PLCs, Tris-HCl buffers were prepared over the range of pH 5.2-8.6.

cRNA Synthesis and in Vitro Translation—Full-length mouse PLC{zeta} and the domain-deletion constructs used for RNA synthesis were amplified by PCR from the original clone cDNA using appropriate primers and Pfu polymerase, as described above. PCR products were cloned into pCR-Blunt-TOPO (Invitrogen), sequence-verified, and then subcloned into the pTarget vector (Promega). cRNAs encoding the wild type PLC and deletion constructs were produced from the linearized pTarget constructs using the Ribomax RNA synthesis kit (Promega) in the presence of 3 mM m7G(5'ppp(5')G (37 °C, 2 h). Synthesized cRNA products were analyzed by agarose gel electrophoresis, isopropyl alcohol-precipitated, and resuspended in diethyl pyrocarbonate-treated water containing 4 units of RNasin µl-1 (Promega). Integrity of complementary RNAs were assayed by in vitro expression of proteins (Reticulocyte Lysate System, Promega) in the presence of [35S]methionine (Amersham Biosciences) and analyzed by 8% SDS-PAGE and autoradiography, as described previously (27).

Preparation and Handling of Gametes—Experiments were carried out with mouse eggs in Hepes-buffered saline (H-KSOM) as described previously (38). All compounds were from Sigma unless stated otherwise. Female mice were superovulated by injection of human chorionic gonadotrophin (Intervet). Eggs were collected 13.5-14.5 h later (5) and maintained in 100-µl droplets of H-KSOM under mineral oil at 37 °C. Microinjection of the eggs was carried out 14.5-15.5 h after human chorionic gonadotrophin injection (27).

Microinjection and Measurement of Intracellular Ca2+ and Luciferase Expression—Mouse eggs were washed in H-KSOM and microinjected as described previously (27) with cRNA diluted in injection buffer (120 mM KCl, 20 mM Hepes, pH 7.4). The volume injected was estimated from the diameter of cytoplasmic displacement caused by the bolus injection. All injections were 3-5% of the egg volume. In experiments with untagged PLC{zeta}, Ca2+ changes were monitored with a CCD-based imaging system using a Zeiss Axiovert 100 microscope, with illumination from a monochromator (Photonics) controlled by MetaFluor version 4.0 (Universal Imaging Corp.). Eggs were loaded for 10 min with 4 µM Fura Red-AM (Molecular Probes) and dissolved in Me2SO + 5% (w/v) pluronic acid, and the loading medium was supplemented with sulfinpyrazone, which helps prevent compartmentalization and extrusion of the dye (5).

For experiments with luciferase-tagged PLC{zeta}, eggs were microinjected with the appropriate cRNA mixed with an equal volume of 1 mM Oregon Green BAPTA dextran (Molecular Probes) in KCl Hepes buffer. Eggs were then maintained in H-KSOM with 100 µM luciferin and imaged on a Nikon TE2000 or Zeiss Axiovert 100 microscope equipped with a cooled intensified CCD camera (Photek Ltd., UK). Ca2+ was monitored in these eggs for 4 h after injection by measuring the Oregon Green BAPTA dextran fluorescence with low level excitation light from a halogen lamp. At the end of Ca2+ measurements, the same set of eggs was then monitored for luminescence by integrating light emission (in the absence of fluorescence excitation) for 20 min using the same intensified CCD camera. The fluorescence signals were typically 10-100 times greater than the luminescence signals. Ca2+ measurements for an egg were considered valid only if the same egg was also luminescent. Groups of eggs verified as being luminous were then collected and placed in a test tube containing PBS with 1 mM MgATP + 100 µM luciferin, which was held in a custom-made luminometer equipped with a cooled S20 photomultiplier tube (Electron Tubes Ltd., UK). The eggs were then lysed with 0.5% Triton X-100, and the steady state light was compared with that emitted from calibrated amounts of recombinant firefly luciferase (Sigma). The amount of luciferase activity measured for each group of eggs was then divided by the number of luminous eggs to obtain the mean value for protein expression of each type of luc-PLC{zeta}.

SDS-PAGE and Western Blotting—Recombinant proteins were separated by SDS-PAGE as described previously (27). Separated proteins were transferred onto polyvinylidene difluoride membrane, incubated overnight at 4 °C in Tris-buffered saline, 0.1% Tween 20 (TBS-T) containing 5% nonfat milk powder, and probed with anti-GST polyclonal antibody (1:5000 dilution). Detection of horseradish peroxidase-coupled secondary antibody was achieved using Super Signal West Dura (Pierce) and a Bio-Rad ChemiDoc gel documentation system for image capture.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and Enzymatic Characterization of Recombinant PLC{zeta}—PLC{delta}1 and PLC{zeta} were expressed in E. coli BL21 cells and purified by GST affinity chromatography, as described under "Experimental Procedures." Empirically determined, optimal protein production required maintaining bacterial cultures at 37 °C until A600 of 0.5-0.6, followed by induction of protein expression upon addition of 0.5 mM isopropyl 1-thio-{beta}-D-galactopyranoside and 4 h of vigorous shaking at 25 °C. Fig. 1A schematically depicts the distinct domains of PLC{delta}1 and PLC{zeta} and illustrates their close similarity because of common structural features, specifically the two EF hands, catalytic X and Y domains, and C2 domain, with the notable exception of an N-terminal PH domain absent from PLC{zeta} (27). Fig. 1B shows the GST affinity-purified recombinant proteins analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining and immunoblot detection using an anti-GST antibody. The predicted molecular mass, including the GST tag (26 kDa), for GST-PLC{delta}1 and GST-PLC{zeta} was 111 and 100 kDa, respectively. The corresponding proteins with appropriate molecular mass were observed as the top band in both Coomassie Brilliant Blue staining and on the immunoblot (Fig. 1B). The lowest band of 26 kDa is consistent with cleaved GST, which along with the much fainter intermediate molecular mass bands detected by the anti-GST antibody are probably the result of protein degradation occurring through the bacterial expression and purification processes. Hydrolysis of [3H]PIP2 to [3H]IP3 to monitor the enzymatic activities of recombinant PLC{delta}1 and PLC{zeta} (36) was optimized for PLC activity by varying a series of parameters, including incubation time, reaction temperature, and protein concentrations. Linearity of [3H]PIP2 cleavage was obtained with 20 pmol of recombinant protein incubated with 220 µM [3H]PIP2 for 10 min at 25 °C (data not shown).



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 1.
Expression of recombinant PLC{delta}1 and PLC{zeta}. A, schematic linear representation of PLC{delta}1 and PLC{zeta} showing the major structural domains; the pleckstrin homology domain (PH), the tandem potential Ca2+ binding motifs (EF hands), the central catalytic domains (X and Y), and the potential lipid binding domain (C2). Note the absence of a PH domain in PLC{zeta} (27). B, SDS-PAGE of recombinant PLC{delta}1 and PLC{zeta} expressed in E. coli BL21 Rosetta cells. Affinity-purified GST-PLC{delta}1 and GST-PLC{zeta} (1 µg) were analyzed by 8% SDS-PAGE and then either Coomassie Brilliant Blue staining (left panel) or immunoblot analysis using anti-GST polyclonal antibody (right panel).

 
Fig. 2A shows the effect of total PIP2 concentration on PLC{delta}1 and PLC{zeta} enzyme activity. For both PLC{delta}1 and PLC{zeta}, the maximum hydrolytic enzyme activity was obtained at 660 µM PIP2, with specific activity values of 1884 and 770 nmol/min/mg measured, respectively. The Michaelis-Menten constant, Km, calculated by a Lineweaver-Burk reciprocal plot for both recombinant proteins was very similar, with PLC{zeta} having a Km value of 87 µM in comparison to 75 µM for PLC{delta}1 (Fig. 2B). Although PLC{delta}1 and PLC{zeta} had common enzymatic properties with regard to PIP2, the Ca2+ dependence of their activities was markedly different (Fig. 3A). PLC{zeta} was activated between 0.01 and 0.1 µM Ca2+, whereas the threshold for PLC{delta}1 was 0.1 µM, with maximum activity at about 100 µM. The EC50 was 82 nM (Hill constant, 4.3) for PLC{zeta} and 6 µM (Hill constant, 1.5) for PLC{delta}1 (calculated from Fig. 3A). PLC{zeta} showed maximal activity over a broad pH range, varying between 5.2 and 6.0, in contrast with PLC{delta}1, which displayed an optimum pH at 6.0 (Fig. 3B).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 2.
Enzymatic characterization of recombinant PLC{delta}1 and PLC{zeta}. A, [3H]PIP2 hydrolysis assay (see "Experimental Procedures") of PLC{delta}1 and PLC{zeta} activities as a function of PIP2 concentration. B, Lineweaver-Burk reciprocal plots for determination of the Km value for PIP2, yielding values of 75 and 87 µM for PLC{delta}1 and PLC{zeta}, respectively. A, n = 2 ± S.E., using two different batches of recombinant proteins, and each experiment was performed in duplicate.

 



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 3.
Calcium and pH dependence of recombinant PLC{delta}1 and PLC{zeta}. A, effect of [Ca2+] on the PIP2 hydrolysis activity of PLC{delta}1 and PLC{zeta}. Enzyme assays were performed in different free [Ca2+] ranging from 0.1 mM to 0.1 nM, as outlined under "Experimental Procedures." B, effect of pH on enzyme activity of PLC{delta}1 and PLC{zeta}. The pH of the reaction was varied between pH5 and pH 9, as outlined under "Experimental Procedures." For all assays, n = 2 ± S.E., using two different batches of recombinant proteins, and each experiment was performed in duplicate.

 



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 4.
Effect of PLC{zeta} domain deletion on PIP2 hydrolysis. A, schematic representation of the domain-deletion constructs of PLC{zeta}, expressed as GST fusion proteins. B, affinity-purified, truncated PLC{zeta} proteins (1 µg) analyzed by 8% SDS-PAGE and then either Coomassie Brilliant Blue staining (left panel) or immunoblot analysis using anti-GST polyclonal antibody (right panel). Lanes 1-4 show {zeta}{Delta}EF1, {zeta}{Delta}EF1,2, {zeta}{Delta}C2, and {zeta}XY, respectively. C, PIP2 hydrolysis activity of the PLC{zeta} domain-deletion constructs (20 pmol) using the standard [3H]PIP2 cleavage assay as described under "Experimental Procedures," n = 3 ± S.E., using three different batches of recombinant protein, and each experiment was performed in duplicate.

 
Enzymatic Analysis of Domain-truncated PLC{zeta} in Vitro—To examine the role of the distinct structural domains on enzymatic activity, four domain-deletion constructs of PLC{zeta} were expressed and purified as GST fusion proteins. Fig. 4A schematically illustrates the full-length PLC{zeta} and the various domain-truncated PLC{zeta} versions that have one or both EF hands removed ({zeta}{Delta}EF1 and {zeta}{Delta}EF1,2, respectively), the C2 domain deleted ({zeta}{Delta}C2) or all the above domains absent ({zeta}XY), and their specific amino acid sequence coordinates. Following expression in E. coli and purification by affinity chromatography, samples of each protein were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining and immunoblotting using anti-GST polyclonal antibody. Fig. 4B shows that the major protein band, with mobility corresponding to the predicted molecular mass for each construct, was present for all four of the domain-truncated proteins (left panel), and these major bands were also recognized by the corresponding anti-GST immunoblot (right panel), confirming the appropriate expression of all domain-truncated PLC{zeta} proteins. Enzyme activity assays performed for each of the recombinant proteins, determined by using the standard [3H]PIP2 hydrolysis assay, showed that every domain-deletion construct retained some of the enzymatic activity present with the full-length PLC{zeta}. The histogram of Fig. 4C plots the enzyme specific activity values obtained for each protein and reveals that the PLC{zeta} proteins lacking either one or both EF hand domains or the C2 domain retained about 70% of the activity of the full-length PLC{zeta} protein. Even the XY catalytic domain alone exhibited well over half of the activity of the full-length PLC{zeta}. These data suggest that the PLC{zeta} catalytic site alone, comprising the X and Y domains, is capable of binding and hydrolyzing PIP2 and that the C2 and EF hand domains are not essential for enzymatic activity in vitro.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 5.
Ca2+ changes in eggs injected with full-length and domain deletions of PLC{zeta}. A, schematic representation of the PLC{zeta} domain-deletion constructs used for cRNA microinjections into eggs. B, changes in cytoplasmic [Ca2+] in mouse eggs were measured by fluorescence excitation ratio measurements using intracellular Fura Red as described under "Experimental Procedures." Recordings were obtained for over 3 h after microinjection of cRNA into eggs for each PLC{zeta} construct. Full-length PLC{zeta}, used as a positive control, triggered Ca2+ oscillations ~18-22 min after cRNA microinjection into the eggs (n = 10 ± S.E.). 100% of the PLC{zeta} control eggs produced Ca2+ oscillations; one representative trace is shown. Microinjection of cRNA for {Delta}EF1-PLC{zeta}, {Delta}EF1,2-PLC{zeta}, or {Delta}C2-PLC{zeta} failed to cause any Ca2+ oscillations in any of the eggs (n = 10 for each cRNA). A representative trace is shown for each domain deletion. The extensively deleted PLC{zeta}-XY also did not cause any changes in cytoplasmic [Ca2+] in mouse eggs (traces not shown). WT, wild type.

 
Activity Analysis of PLC{zeta} Domain Deletions Expressed in Mouse Eggs—Because the recombinant deletion constructs of PLC{zeta} displayed robust enzymatic activity in vitro, further experiments were conducted to determine whether this is matched by their ability to trigger Ca2+ oscillations when the corresponding cRNA is injected into mouse eggs (Fig. 5A). Injection of 0.2 mg/ml cRNA encoding the full-length PLC{zeta} (WT in Fig. 5) was used as the positive control, and this caused a robust series of Ca2+ oscillations in all eggs, as reported previously (27, 28). The Ca2+ oscillations started about 20 min after injection, and spiking behavior persisted at an interval of about 3 min for over 2 h. In contrast, when the cRNAs for {Delta}EF1-PLC{zeta}, {Delta}EF1,2-PLC{zeta}, or {Delta}C2-PLC{zeta} were microinjected into eggs under the same conditions, there were no Ca2+ oscillations observed in any of the injected eggs (Fig. 5B). All the cRNAs used in these experiments were synthesized in parallel with batches of control (wild type PLC{zeta}) cRNA, and as demonstrated previously (27, 28), the cRNA used for these microinjection experiments was able to generate proteins of the correct size when expressed in an in vitro reticulocyte lysate expression system (data not shown). Consequently, these data suggest that the absence of either of the EF hand domains or the C2 domain of PLC{zeta} prevents it from being able to trigger Ca2+ oscillations in intact eggs.



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 6.
Eggs injected with luciferase-tagged PLC{zeta}. The fluorescence of eggs microinjected with Oregon Green BAPTA-dextran was used to monitor Ca2+ as described under "Experimental Procedures." Eggs were monitored in medium containing 100 µM luciferin. A shows a representative fluorescence trace of Ca2+ from an egg injected with the wild type (WT) PLC{zeta}-luciferase cRNA. B shows an integrated image of the luciferase luminescence from the eggs injected with this construct. C shows a sample Ca2+ recording from an egg injected with {Delta}EF1-PLC{zeta}-luc cRNA. D is a sample image of luciferase luminescence. E and F show a sample fluorescence recording of Ca2+ and integrated luminescence of luciferase from eggs injected with {Delta}C2-PLC{zeta}-luc cRNA. The y axes on the fluorescence traces are in arbitrary units, and each x axis starts between 5 and 20 min after the injection of eggs. The luminescence images are taken from eggs emitting an average of 5, 21, and 52 photons/s for B, D, and F, respectively.

 
Quantitation of PLC{zeta} Expression in Mouse Eggs—To verify that the domain-deleted versions of PLC{zeta} were faithfully expressed as proteins in cRNA-microinjected eggs, we generated luciferase-tagged versions of these constructs to enable quantitation of relative protein expression. Fig. 6A shows that eggs injected with cRNA encoding the wild type PLC{zeta}-luciferase (PLC{zeta}-luc) fusion construct caused a series of Ca2+ oscillations in eggs. This indicates that a fusion tag at the C terminus of PLC{zeta} can also retain the ability to generate Ca2+ oscillations, as has been previously shown for N-terminal tags (27). At the end of 4 h of monitoring the changes in Ca2+, we measured the light emitted from the same set of eggs (in the absence of fluorescence excitation), and we found that they were luminescent (Fig. 6B). Every mouse egg injected with PLC{zeta}-luc cRNA that showed clear expression of luciferase activity after 4 h (n = 19) had also exhibited prior robust Ca2+ oscillations. When eggs were injected with {Delta}EF1-PLC{zeta}-luc cRNA, however, none of the eggs showed any Ca2+ increase (n = 26), although they were visibly expressing luciferase activity, as confirmed by the intense luminescence detected at the end of the experiment (Fig. 6, C and D). Similarly, upon injection of {Delta}C2-PLC{zeta}-luc cRNA, all 25 eggs failed to show any Ca2+ oscillations, although they exhibited strong luciferase luminescence (Fig. 6, E and F). The exact level of luminescence in cells can depend upon the amount of luciferase protein, the intracellular pH, and the ATP concentration (39). Consequently, we quantified the relative expression of the luciferase fusion protein by lysing the mouse eggs in a buffer with a fixed concentration of ATP. Upon lysing the eggs in a luminometer, we determined that at the end of the experiment (4 h), a mean value of 0.19 pg of wild type PLC{zeta}-luc protein was expressed per egg (n = 19). With {Delta}EF1-PLC{zeta}-luc, a mean of 0.98 pg of protein was expressed per egg (n = 26), and with {Delta}C2-PLC{zeta}-luc a mean of 2.7 pg of protein was expressed per egg (n = 25). These data show that the two PLC{zeta}-luc domain-deletion constructs did not cause any Ca2+ oscillations but were demonstrably being expressed in the eggs that were injected with cRNA. The {Delta}EF1-PLC{zeta}-luc and {Delta}C2-PLC{zeta}-luc were expressed at levels that were 5- and 14-fold that of PLC{zeta}-luc, respectively. This high level of expression of domain-deletion constructs more than compensates for the reduced specific activity of these proteins (~70%) relative to full-length PLC{zeta} (Fig. 4). Because the threshold for PLC{zeta} to cause a Ca2+ oscillation is around 50 fg (27), the two domain-deletion constructs were expressed at levels that are 20-50 times the amount required to cause Ca2+ oscillations with PLC{zeta}.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 7.
Ca2+-dependent enzyme activity of full-length PLC{zeta} and PLC{zeta} domain deletions. A, effect of varying [Ca2+] from 0.1 mM to 0.1 nM on the specific [3H]PIP2 hydrolysis activity of domain-deletion constructs of PLC{zeta}. B, effect of [Ca2+] on the normalized % activity of deletion constructs. For these assays n = 2 ± S.E., using two different batches of recombinant proteins, and each experiment was performed in duplicate.

 
Effect of [Ca2+] on the Activity of PLC{zeta} Domain Deletions—The activity assays described above suggest that the X and Y catalytic domains alone are sufficient for in vitro enzymatic activity (Fig. 4), although all domains appear to be essential for Ca2+ oscillations activity in intact eggs (Figs. 5 and 6). To examine the role of selected domains on the Ca2+ sensitivity of PLC{zeta} activity, we tested the ability of the domain-deletion constructs of PLC{zeta} to hydrolyze [3H]PIP at different Ca2+ concentrations ranging from 0.1 mM to 0.1 nM (Fig. 7). Fig. 7A illustrates the Ca2+ dependence of specific PIP2 hydrolytic activity for the full-length PLC{zeta} and each of the truncated proteins, and these are shown normalized to the maximum specific activity in Fig. 7B. Table I summarizes the EC50 and Hill coefficients of PLC{delta}1, PLC{zeta}, and domain-deletion constructs. Deletion of EF1 increased the EC50 by 10-fold and reduced the Hill coefficient from 4.135 to 2.204. Deletion of both EF hands led to a dramatic increase of the EC50 of PLC{zeta} from 82 nM to 30 µM and a decrease of the Hill coefficient from 4.315 to 0.642. Deletion of the C2 domain did not change the EC50 of PLC{zeta} but reduced the Hill coefficient from 4.315 to 1.139. Finally, deletion of both EF hands and C2 domain (PLC{zeta}-XY) drastically changed the EC50 and Hill coefficient (62 and 0.327 µM, respectively). These results suggest the EF hands may play a direct modulatory role in the Ca2+ regulation of PLC{zeta} activity.


View this table:
[in this window]
[in a new window]
  		TABLE I
Summary of the EC50 and Hill coefficients of Ca2+-dependent enzyme activity determined for the full-length PLC{delta}1, PLC{zeta}, and the PLC{zeta} domain deletions (see Figs. 4A and 7)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
PLC{zeta} was identified as a sperm-specific PLC that is highly effective in causing Ca2+ oscillations and activation in mouse eggs (27). It appears to be responsible for the previously described PLC activity and Ca2+-releasing activity present in sperm extracts (27). PLC{zeta} has also been identified as the protein factor that is responsible for causing Ca2+ oscillations after injection of whole sperm into mouse eggs (24). This "sperm factor" is proposed to enter the egg upon fusion with the sperm at fertilization and to be responsible for activating embryo development in mammals (27, 28). Despite its significance in cell signaling at fertilization, the regulation of PLC{zeta} activity and the mechanism by which PLC{zeta} locates and targets the substrate in eggs are currently unknown (40). The aims of this study were to compare the enzymatic properties of the bacterially expressed, full-length PLC{zeta} and PLC{delta}1 in vitro. We then determined which structural domains within the PLC{zeta} sequence are essential for its enzymatic function and whether this correlates with its unique ability to cause sustained Ca2+ oscillations in mouse eggs.

The dependence of PLC{zeta} and PLC{delta}1 enzyme activity on PIP2 concentration indicated that the Km values for these closely related isoforms were very similar (Fig. 2) and in reasonable agreement with the Km value obtained for recombinant PLC{delta}3 (142 µM) in another study (41). This suggests that the enzymes have similar affinity for their substrate PIP2, which is consistent with the presence of highly conserved X and Y active site domains found throughout the PI-PLC family.

To determine the role of different parts of PLC{zeta} in PIP2 hydrolyzing activity, we generated a series of truncated proteins with deletions of selected structural domains (Fig. 4). Our data showed that the recombinant X + Y catalytic domains alone are able to retain considerable enzyme activity (~70%) compared with that of the full-length PLC{zeta}. Furthermore, the proteins with deletions of either the EF hands or the C2 domains also showed a similar PLC activity in vitro compared with that of full-length PLC{zeta}. Thus, the absence of both EF hands and the C2 domain is unable to ablate PLC{zeta} enzyme activity when the X + Y catalytic domains are presented with PIP2 in micellar form. These data are consistent with experiments on other PLC isoforms (34).

In order to assess the relevance of PLC{zeta} structural domains in triggering Ca2+ oscillations in intact cells, various truncated forms of PLC{zeta} were prepared, and individual mouse eggs were microinjected with cRNA corresponding to each domain-deletion construct (Fig. 5). Microinjection of PLC{zeta} cRNA into eggs is used in preference to recombinant PLC{zeta} protein because it avoids the problems of maintaining protein activity and solubility and to negate potential cross-contamination from bacterial proteins during the purification process. In previous work with recombinant PLCs, we have found that "purified" preparations can be also contaminated with IP3, and this can give the misleading impression that microinjection of a purified PLC directly triggers Ca2+ oscillations in intact eggs (25). In contrast, the use of microinjected cRNA ensures the sample delivered into the egg is free from such contamination; moreover, it is a robust and reliable method because we have demonstrated previously that the mouse egg is capable of efficiently translating the cRNA encoding the active sperm PLC{zeta} protein (27, 28, 42, 43). By using cRNA injection, we found that expression of the PLC{zeta} domain-deletion constructs in mouse eggs did not lead to the generation of any Ca2+ oscillations (Fig. 5). Because the in vitro assays demonstrated that these shortened versions possess PLC enzyme activity (Fig. 4), we can infer that the absence of either of the EF hands or the C2 domain of PLC{zeta} causes it to be unable to hydrolyze PIP2 under the conditions found in intact cells.

One issue that remains to be addressed with the use of microinjected cRNA samples into eggs is the level of expression achieved with each of the various PLC{zeta} truncations. For example, if the full-length PLC{zeta} is expressed with much greater efficiency than the PLC{zeta} domain deletions, then microinjection of equivalent amounts of cRNA would not be directly comparable. Thus, to examine whether expression of specific domain-deletion constructs was selectively impaired relative to full-length PLC{zeta}, a series of luciferase-tagged fusion constructs for the domain deletions was microinjected into eggs. Although at levels of luciferase expression for the two domain deletions that were significantly higher than that of the full-length PLC{zeta}-luc (Fig. 6), no Ca2+ oscillations were produced by any of the domain-deleted PLC{zeta}-luc constructs. It was notable that the Ca2+ oscillations observed upon injection of the full-length PLC{zeta}-luc cRNA occurred about 1 h after injection, and at the end of the experiment (4 h) the level of expressed PLC{zeta}-luc protein was determined to be 190 fg/egg. If we assume a linear increase in PLC{zeta}-luc protein during the 4 h of recording, then we can estimate that the amount of PLC{zeta}-luc required to initiate Ca2+ oscillations is around 50 fg. This value is similar to our previous estimate that 20-50 fg of PLC{zeta} is required to initiate Ca2+ oscillations in eggs (27), as well as the estimate that 10-40 fg of venusGFP-PLC{zeta} is required to initiate Ca2+ release (44). These data therefore suggest that the PLC{zeta}-luc has a similar efficiency is generating IP3 in eggs to other N-terminally tagged PLC{zeta} fusion proteins.

One potential reason for the striking difference between the activity observed in vitro and in intact eggs could be the way in which Ca2+ regulates PLCs. Most mammalian PLCs show some stimulation of activity with increasing Ca2+ concentration, but PLCs are often only stimulated by Ca2+ concentrations that are much higher than those found in resting cells. This is even true for PLC{delta}1, which has been reported previously to be one of the most Ca2+-sensitive of the PLC isozymes, with marked stimulation by Ca2+ concentrations in the micro-molar range (45). Our assays of PLC{delta}1 are consistent with this earlier work, because we found the EC50 value for Ca2+ stimulation of PLC{delta}1 to be about 6 µM (Fig. 3). In contrast to PLC{delta}1, PLC{zeta} is about 100 times more sensitive to Ca2+ with an EC50 of 50-80 nM, which is well within the range of reported resting Ca2+ concentration in eggs (3, 4). The difference between these isoforms means that PLC{zeta} is not only likely to show significant activity at resting Ca2+ levels, but it will be maximally active at 1 µM Ca2+, although PLC{delta}1 will not be fully activated until Ca2+ reaches 30 µM. As well as the high Ca2+ sensitivity, we found that the dependence of PLC{zeta} activity on Ca2+ had a Hill coefficient of 4.3, suggesting the binding of 4 Ca2+ molecules/protein. This is greater than the previous value of 0.9 observed by Kouchi et al. (29) in their study of PLC{zeta} expressed in insect cells. The reason for the difference is unclear, but it is not likely to be due to systematic differences in the assay, because the calculated EC50 values and the Hill coefficients for the Ca2+ dependence of PLC{delta}1 were very similar between the present data and those of Kouchi et al. (1.5 and 1.7, respectively). The difference in Ca2+ sensitivity and Hill coefficient between PLC{delta}1 and PLC{zeta} is of interest because it could be one source of explanation for why PLC{zeta} is so effective at causing Ca2+ oscillations in mouse eggs, and yet PLC{delta}1 is reported to be either ineffective (25) or at least much less effective (29). This idea is supported by our current observations of the behavior of the PLC{zeta} domain-deletion constructs in eggs.

It is clear from our data that PLC{zeta} is not effective in stimulating Ca2+ oscillations when it lacks one or both of its EF hand domains (Figs. 5 and 6). This result is consistent with previous observations with a short form of PLC{zeta} that lacks the first 110 amino acids at the N terminus (29), which corresponds to removal of the first EF hand and part of the second (Fig. 4A). The basic ability to hydrolyze PIP2 in vitro would be preserved for these deletion constructs, so the lack of Ca2+ oscillation-inducing activity in eggs injected with EF hand domain deletions of PLC{zeta} may be explained by their differential response to Ca2+ regulation. EF hands appear to play a vital role in the Ca2+ sensitivity of PLC{zeta} activity, because deletion of both EF hands dramatically changed the EC50 of PLC{zeta} from 82 nM to 30 µM. The N-terminal truncation of both EF hands would ablate the ability of this domain deletion to generate IP3 in an intact cell with a Ca2+ level of around 100 nM. Even deletion of the first EF hand domain raised the EC50 for Ca2+ to >700 nM, which is well above the resting Ca2+ level in an egg. The Hill coefficients were also decreased by deletion of one or both of the EF hand domains. The Hill coefficient describes the minimum number of interacting active sites required for enzyme function, suggesting that upon removal of the EF hands the minimum number of sites is reduced from ~4 to ~1. This in turn suggests that Ca2+ binding to the EF hands is important for the interaction of the X-Y domain with PIP2 substrate and thus for PLC{zeta} enzyme activity.

It is of interest that the loss of the C2 domain from PLC{zeta} also leads to the inability to cause Ca2+ oscillations in intact eggs, although the EC50 value for Ca2+ stimulation was unchanged. This result suggests two potential explanations. One possibility is linked to the significant change in the Hill coefficient, as the C2 domain removal caused a marked reduction in the Hill coefficient for Ca2+ stimulation from ~4 to 1. This loss of cooperativity in Ca2+ stimulation could be important for generating Ca2+ oscillations (see below). The other possibility is that the C2 domain plays an important role in targeting PLC{zeta} to the correct subcellular source of PIP2 in eggs. C2 domains display functional diversity and can be involved in binding to lipids, or proteins, in a way that can be Ca2+-dependent (46).

The precise physiological mechanism by which IP3 production leads to the exquisite pattern of Ca2+ oscillations at fertilization in mammalian eggs is unknown. By analogy with mechanisms proposed in somatic cells, the positive and delayed negative feedback effect of Ca2+ on the IP3 receptor may be an important part of the mechanism for generating regular Ca2+ oscillations (6). The observation that Ca2+ oscillations similar, but not identical, to those occurring at fertilization can be stimulated by the sustained introduction of IP3 alone into eggs supports this idea (12, 13). However, because of the high Ca2+ dependence of PLC{zeta} activity, it is plausible that some Ca2+- induced IP3 formation occurs in eggs after the introduction of PLC{zeta}. Such a positive feedback loop of Ca2+ release and IP3 production has been suggested to play an important role in generating Ca2+ oscillations (47, 48). This feedback phenomenon could provide an explanation for the enhanced sensitivity of the egg to Ca2+-induced Ca2+ release, which occurs after fertilization, or sperm factor injection (12, 16). The nonlinear dependence of PLC{zeta}-induced IP production on Ca2+ levels could also help generate the rapid rising phase of Ca2+ release that is a specific feature of fertilized eggs (3, 45). Consequently, it is possible that a combination of Ca2+-stimulated PLC{zeta} activity and the properties of the IP3 receptor combine to generate the Ca2+ oscillations at fertilization in mammals.

Addendum—A similar PLC{zeta} study showing the importance of the EF and C2 domains has been reported (49) in overall agreement with our data.


   FOOTNOTES
 
* This work was supported by a School of Medicine Ph.D. studentship (to M. N.), an Education and Learning Wales Knowledge Exploitation Fund grant and Cardiff Partnership Fund grant (to F. A. L.), and by a Wellcome Trust grant (to K. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. Tel.: 44-29-2074-2338; Fax: 44-29-2074-3500; E-mail: lait{at}cf.ac.uk.

1 The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PI-PLC, phosphoinositide-specific phospholipase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; GST, glutathione S-transferase; PH, pleckstrin homology. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Matilda Katan for the gift of the PLC{delta}1 plasmid.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Stricker, S. A. (1999) Dev. Biol. 211, 157-176[CrossRef][Medline] [Order article via Infotrieve]
  2. Runft, L. L., Jaffe, L. A., and Mehlmann L. M. (2002) Dev. Biol. 245, 237-254[CrossRef][Medline] [Order article via Infotrieve]
  3. Miyazaki, S., Shirakawa, H., Nakada, K., and Honda, Y. (1993) Dev. Biol. 58, 62-78
  4. Swann, K., and Ozil, J. P. (1994) Int. Rev. Cytol. 152, 183-222[Medline] [Order article via Infotrieve]
  5. Lawrence, Y., Ozil, J. P., and Swann, K. (1998) Biochem. J. 335, 335-342
  6. Berridge, M. J. (1993) Nature 361, 315-325[CrossRef][Medline] [Order article via Infotrieve]
  7. Rice, A., Parrington, J., Jones, K., and Swann, K. (2000) Dev. Biol. 228, 125-135[CrossRef][Medline] [Order article via Infotrieve]
  8. Wu, H., Smyth, J., Luzzi, V., Fukami, K., Takenawa, T., Black, S., Allbritton, N., and Fissore, R. (2001) Biol. Reprod. 64, 1338-1349[Abstract/Free Full Text]
  9. Miyazaki, S., Yuzaki, M., Nakada, K., Shirakawa, H., Nakanishi, S., Nakade, S., and Mikoshiba, K. (1992) Science 257, 251-255[Abstract/Free Full Text]
  10. Parrington, J., Brind, S., De Smedt, H., Gangeswaran, R., Lai, F. A., Wojcikiewicz, R., and Carroll, J. (1998) Dev. Biol. 203, 451-461[CrossRef][Medline] [Order article via Infotrieve]
  11. Jellerette, T., He, C. L., Wu, H., Parys, J. B., and Fissore, R. A. (2000) Dev. Biol. 223, 238-250[CrossRef][Medline] [Order article via Infotrieve]
  12. Swann, K. (1994) Cell Calcium 15, 331-339[CrossRef][Medline] [Order article via Infotrieve]
  13. Jones, K. T., and Nixon, V. L. (2000) Dev. Biol. 225, 1-12[CrossRef][Medline] [Order article via Infotrieve]
  14. Kim, H. K., Kim, J. W., Zilberstein, A., Margolis, B., Kim, J. G., Schelssinger, J., and Rhee, S. G. (1991) Cell 65, 435-441[CrossRef][Medline] [Order article via Infotrieve]
  15. Smrcka, A. V., Hepler, J. R., Brown, K. O., and Sternweis, P. C. (1991) Science 251, 804-807[Abstract/Free Full Text]
  16. Igusa, Y., and Miyazaki, S. (1983) J. Physiol. (Lond.) 340, 611-632[Abstract/Free Full Text]
  17. Swann, K. (1990) Development (Camb.) 110, 1295-1302[Abstract/Free Full Text]
  18. Nakano, Y., Shirakawa, H., Mitsuhashi, N., Kuwubara, Y., and Miyazaki, S. (1997) Mol. Hum. Reprod. 3, 1087-1093[Abstract/Free Full Text]
  19. Stricker, S. (1997) Dev. Biol. 186, 185-201[CrossRef][Medline] [Order article via Infotrieve]
  20. Wu, H., He, C. L., and Fissore, R. A. (1997) Mol. Reprod. Dev. 46, 176-189[CrossRef][Medline] [Order article via Infotrieve]
  21. Kyozuka, K., Deguchi, R., Mohri, T., and Miyazaki, S. (1998) Development (Camb.) 125, 4099-4105[Abstract]
  22. Wu, H., He, C. L., Jehn, B., Black, S. J., and Fissore, R. A. (1998) Dev. Biol. 203, 369-381[CrossRef][Medline] [Order article via Infotrieve]
  23. Tang, T. S., Dong, J. B., Huang, X. Y., and Sun, F. Z. (2000) Development (Camb.) 127, 1141-1150[Abstract]
  24. Fujimoto, S., Yoshida, N., Fukui, T., Amanai, M., Isobe, T., Itagaki, C., Izumi, T., and Perry, A. F. C. (2004) Dev. Biol. 274, 370-383[CrossRef][Medline] [Order article via Infotrieve]
  25. Jones, K. T., Matsuda, M., Parrington, J., Katan, M., and Swann, K. (2000) Biochem. J. 346, 743-749
  26. Parrington, J., Jones, M. L., Tunwell, R., Davader, C., Katan, M., and Swann, K. (2002) Reproduction 123, 31-39[Abstract]
  27. 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[Abstract/Free Full Text]
  28. Cox, L. J., Larman, M. G., Saunders, C. M., Hashimoto, K., Swann, K., and Lai, F. A. (2002) Reproduction 124, 611-623[Abstract]
  29. Kouchi, Z., Fukami, K., Shikano, T., Oda, S., Nakamura, Y., Takenawa, T., and Miyazaki, S. (2004) J. Biol. Chem. 279, 10408-10412[Abstract/Free Full Text]
  30. Mehlmann, L. M., Chattopadhyay, A., Carpenter, G., and Jaffe, L. A. (2001) Dev. Biol. 236, 492-501[CrossRef][Medline] [Order article via Infotrieve]
  31. Rhee, S. (2001) Annu. Rev. Biochem. 70, 281-312[CrossRef][Medline] [Order article via Infotrieve]
  32. Katan, M. (1998) Biochim. Biophys. Acta 1436, 5-17[Medline] [Order article via Infotrieve]
  33. Essen, L. O., Perisic, O., Cheung, R., Katan, M., and Williams, R. L. (1996) Nature 380, 595-602[CrossRef][Medline] [Order article via Infotrieve]
  34. Katan, M., and Williams, R. L. (1997) Semin. Cell Dev. Biol. 8, 287-296[CrossRef][Medline] [Order article via Infotrieve]
  35. Williams, R. L. (1999) Biochim. Biophys. Acta 1441, 255-267[Medline] [Order article via Infotrieve]
  36. Katan, M., and Parker, J. (1987) Eur. J. Biochem. 168, 413-418[Medline] [Order article via Infotrieve]
  37. Fabiato, A. (1981) J. Gen. Physiol. 78, 457-497[Abstract/Free Full Text]
  38. Summers, M. C., McGinnis, L. K. M., Lawitts, J. A., Raffin, M., and Biggers, J. D. (2000) Hum. Reprod. 15, 1791-1801[Abstract/Free Full Text]
  39. Allue, I., Gandelman, O., Dementieva, E., Ugarova, N., and Cobbold, P. (1996) Biochem. J. 319, 463-469
  40. Swann, K., Larman, M. G., Saunders, C. M., and Lai, F. A. (2004) Reproduction 127, 431-439[Abstract/Free Full Text]
  41. Pawelczyc, T., and Matecki, A. (1997) Acta Biochim. Pol. 44, 221-230[Medline] [Order article via Infotrieve]
  42. Larman, M. G., Saunders, C. M., Carrol, J., Lai, F. A., and Swann, K. (2004) J. Cell Sci. 117, 2513-2521[Abstract/Free Full Text]
  43. Rogers, N., Hobson, E., Pickering, S., Lai, F. A., Braude, P., and Swann, K. (2004) Reproduction 128, 697-702[Abstract/Free Full Text]
  44. Yoda, A., Oda, S., Shikano, T., Kouchi, Z., Awaji, T., Shirakawa, H., Konoshita, K., and Miyazaki, S. (2004) Dev. Biol. 268, 245-257[CrossRef][Medline] [Order article via Infotrieve]
  45. Allen, V., Swigert, P., Cheung, R., Cockroft, S., and Katan, M. (1997) Biochem. J. 327, 545-552
  46. Nalefski, E. A., Wisner, M. A., Chen, J. Z., Sprang, S. R., Fukuda, M., Mikoshiba, K., and Falke, J. J. (2001) Biochemistry 40, 3089-3100[CrossRef][Medline] [Order article via Infotrieve]
  47. De Young, G., and Keizer, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9895-9899[Abstract/Free Full Text]
  48. Hirose, K., Kadowaki, S., Tanabe, M., Takeshima, H., and Iino, M. (1999) Science 284, 1527-1530[Abstract/Free Full Text]
  49. Kouchi, Z., Shikano, T., Nakamura, Y., Shirakawa, H., Fukami, K., and Miyazaki, S. (2005) J. Biol. Chem. 280, 21015-21021[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Phil Trans R Soc BHome page
C. M Freisinger, I. Schneider, T. A Westfall, and D. C Slusarski
Calcium dynamics integrated into signalling pathways that influence vertebrate axial patterning
Phil Trans R Soc B, April 12, 2008; 363(1495): 1377 - 1385.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
Y. Yu, C.M. Saunders, F.A. Lai, and K. Swann
Preimplantation development of mouse oocytes activated by different levels of human phospholipase C zeta
Hum. Reprod., February 1, 2008; 23(2): 365 - 373.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Nomikos, A. Mulgrew-Nesbitt, P. Pallavi, G. Mihalyne, I. Zaitseva, K. Swann, F. A. Lai, D. Murray, and S. McLaughlin
Binding of Phosphoinositide-specific Phospholipase C-{zeta} (PLC-{zeta}) to Phospholipid Membranes: POTENTIAL ROLE OF AN UNSTRUCTURED CLUSTER OF BASIC RESIDUES
J. Biol. Chem., June 1, 2007; 282(22): 16644 - 16653.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
B. Lee, E. Vermassen, S.-Y. Yoon, V. Vanderheyden, J. Ito, D. Alfandari, H. De Smedt, J. B. Parys, and R. A. Fissore
Phosphorylation of IP3R1 and the regulation of [Ca2+]i responses at fertilization: a role for the MAP kinase pathway
Development, November 1, 2006; 133(21): 4355 - 4365.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Kuroda, M. Ito, T. Shikano, T. Awaji, A. Yoda, H. Takeuchi, K. Kinoshita, and S. Miyazaki
The Role of X/Y Linker Region and N-terminal EF-hand Domain in Nuclear Translocation and Ca2+ Oscillation-inducing Activities of Phospholipase C{zeta}, a Mammalian Egg-activating Factor
J. Biol. Chem., September 22, 2006; 281(38): 27794 - 27805.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
K. T Jones
Mammalian egg activation: from Ca2+ spiking to cell cycle progression
Reproduction, December 1, 2005; 130(6): 813 - 823.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/35/31011    most recent
M500629200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nomikos, M.
Right arrow Articles by Lai, F. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nomikos, M.
Right arrow Articles by Lai, F. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement