Regulation of Intracellular pH by p90Rsk-dependent Activation of an Na+/H+ Exchanger in Starfish Oocytes*

Starfish oocytes arrest at metaphase of the first meiotic division (MI arrest) in the ovary and resume meiosis after spawning into seawater. MI arrest is maintained by lower intracellular pH (pHi) and release from arrest by cellular alkalization. To elucidate pHi regulation in oocytes, we cloned the starfish (Asterina pectinifera) Na+/H+ exchanger 3 (ApNHE3) expressed in the plasma membrane of oocytes. The cytoplasmic domain of ApNHE3 contains p90 ribosomal S6 kinase (p90Rsk) phosphorylation sites, and injection of a constitutively active p90Rsk and the upstream regulator Mos to immature oocytes, stimulated an increase in pHi. This increase was blocked by 5-(N-ethyl-N-isopropyl)-amiloride, a NHE inhibitor, and SL0101, a specific Rsk inhibitor. The MAPK kinase (MEK) inhibitor U0126 blocked the Mos-induced, but not the p90Rsk-induced, pHi increase, suggesting that the Mos-MEK-MAPK-p90Rsk pathway promotes ApNHE3 activation. In a cell-free extract, the Mos-MEK-MAPK-p90Rsk pathway phosphorylates ApNHE3 at Ser-590, -606, and -673. When p90Rsk-dependent ApNHE3 phosphorylation was blocked by a dominant-negative C-terminal fragment, or neutralizing antibody, the p90Rsk-induced pHi increase was suppressed in immature oocytes. However, ApNHE3 is up-regulated via the upstream phosphatidylinositol 3-kinase pathway before MAPK activation and the active state is maintained until spawning, suggesting that the p90Rsk-dependent ApNHE3 phosphorylation is unlikely to be the primary regulatory mechanism involved in MI arrest exit. After meiosis is completed, unfertilized eggs maintain their elevated pHi (∼7.4) until the onset of apoptosis. We suggest that the p90Rsk/ApNHE3-dependent elevation of pHi increases fertilization success by delaying apoptosis initiation.

During oocyte maturation, the occurrence of an arrest at metaphase of the first (MI) or second (MII) meiotic division is a widespread phenomenon in the animal kingdom. Although MI arrest is frequently observed in invertebrates (1)(2)(3), the molecular mechanism has been poorly understood. Starfish oocytes arrested in prophase of meiosis I are released from arrest by the maturation inducing hormone 1-methyladenine (1-MeAde) 3 (4). Cytoplasmic alkalization takes place shortly after 1-MeAde administration, which is driven by G protein ␤␥ subunits and PI3K signaling (5,6). The increase of pH i continues through prophase I to metaphase I (0 -40 min) reaching ϳ7.4. After germinal vesicle breakdown (GVBD), MAPK is activated by a newly synthesized starfish homolog of Mos (7). When the consecutive meiotic divisions are completed, unfertilized eggs are arrested in GI where DNA synthesis is blocked by MAPK-induced p90Rsk activity (8). Thereafter, elevated pH i is maintained for the remainder of the cell cycle.
In normal procedures, full-grown GI-arrested oocytes are isolated and placed in seawater, and then treated with 1-MeAde (in vitro maturation). Meiosis is then completed without MI or MII arrest. However, under more physiological conditions where females are injected with 1-MeAde into the body cavity, ovarian oocytes simultaneously commit meiosis resumption followed by MI arrest in the ovary (6). Because elevation of pH from 7.0 to 7.2 in maturing extracts causes cyclin B destruction (9), we speculated that the MI arrest of ovarian oocytes is maintained by suppressing pH i below 7.0. Furthermore, when pH i was measured in oocytes immediately after spawning, pH i of ovarian oocytes was estimated at Ͻ7.0 (6). Thus, pH homeostasis of ovarian oocytes plays a pivotal role in MI arrest.
Recently, we found that in MI-arrested ovarian oocytes, MAPK remains inactive, and subsequently becomes activated 5 min after spawning (10). Because MAPK activation is coincident with the onset of cytoplasmic alkalization in spawned oocytes, we first hypothesized that the MAPK-dependent pH i increase mechanism may be present, and if so, may be involved in release from MI arrest.
To understand the molecular mechanism of pH i regulation during meiosis, we cloned the starfish Na ϩ /H ϩ exchanger (NHE) located in the plasma membrane of oocytes. Starfish NHE is similar to human NHE3 and its C-terminal cytoplasmic * This work was supported by grants from the Human Frontier Science Pro-domain contains potential phosphorylation sites for multiple kinases such as MAPK and p90Rsk. Experiments with in vitro and in vivo assays suggest that starfish NHE is activated by phosphorylation through the Mos-MEK-MAPK-p90Rsk pathway. However, the increase in pH i at spawning is thought to occur mainly due to PI3K-dependent NHE activation, suggesting that p90Rsk-dependent NHE activation does not participate in the release from MI arrest.
Animals and Oocytes-Starfish (Asterina pectinifera) were collected on the Pacific coast of Honshu, Japan, and kept in laboratory aquaria supplied with circulating seawater at 15°C. Microinjection of starfish oocytes and quantification of injection volumes were carried out as previously described (11,12). The experiments with living oocytes were at 20°C.
Measurement of NHE Activity-The measurement of pH i was performed as previously described (6). For most experiments, artificial seawater (20 mM HEPES, 480 mM NaCl, 10 mM KCl, 29 mM MgSO 4 , 27 mM MgCl 2 , 2 mM NaHCO 3 , 10 mM CaCl 2 , pH 8.0) was used, and modified seawaters were prepared by replacing MOPS for HEPES (for low pH seawaters) or choline-Cl for NaCl (for low Na ϩ seawaters). For measurement of NHE activity, BCECF-loaded oocytes immobilized in the injection chamber were placed in artificial seawater containing 4.8 mM Na ϩ (1% NaSW, i.e. 1 part NaSW and 99 parts choline-Cl SW). After baseline recordings, oocytes were placed in 1% NaSW containing 1 M 1-MeAde for the desired period (usually 5 min) followed by an extensive wash with 1% NaSW. Thereafter, oocytes were placed in artificial seawater containing 48 mM Na ϩ (10% NaSW, i.e. 1 part NaSW and 9 parts choline-Cl SW) at the indicated time points. The rate of the pH i increase after Na ϩ recovery (an initial increase of 5 min) was calculated by averaging three to six independent experiments.
Phylogenetic Analysis and Motif Search-The protein sequence of full-length starfish NHE was aligned with other eukaryotic NHEs using ClustalW. Based on multiple align-ments, a conserved motif was found in the N-terminal twothirds encompassing amino acid residues 93-527. This sequence is extended to the C terminus from the Na ϩ /H ϩ exchanger motif (Pfam00999). An unrooted tree was prepared by the neighbor-joining method using PAUP 4.0b10. The prediction of kinase-specific phosphorylation sites was performed by using the data base (NetPhosK 1.0 Server).
Preparation of Egg Ghosts and Cell-free Extract-Immature GV-stage oocytes isolated from ovaries were placed in Ca 2ϩfree seawater to remove follicle cells. These oocytes were homogenized gently in chilled homogenization buffer (100 mM KCl, 100 M leupeptin, 10 mM Pipes, pH 6.8). After extensive washing with the homogenization buffer, egg ghosts were recovered by centrifugation (200 ϫ g, 2 min), suspended in homogenization buffer, and stored at Ϫ20°C. The cell-free extract was made as previously described (12).
Immunofluorescence Microscopy-Follicle cell-free GV oocytes were treated with or without 0.05% Actinase E for 30 min at room temperature. After washing with Ca 2ϩ -free seawater, oocytes were fixed with 2% paraformaldehyde in 80% artificial seawater for 3 h. The specimens were transferred to phosphatebuffered saline containing 0.1% Triton X-100 and blocked with 3% bovine serum albumin in phosphate-buffered saline overnight with an end-over-end rotator. Thereafter, the oocytes were incubated with an anti-C-terminal-ApNHE3 antibody (1:50 dilution) for 1 h, followed by incubation with TRITClabeled goat anti-rabbit IgG (1:100 dilution) for 1 h. The fluorescence images were viewed by confocal microscopy with a G-excitation filter set.
In Vitro Phosphorylation-Immature and maturing cell-free extracts at pH 7.0 were used. The reaction was initiated by addition of 0.6 mg/ml of ApNHE3-(489 -755 or 568 -755) followed by 37 kBq/ml of [␥-32 P]ATP to the cell-free extracts. After a 10-min incubation, the samples were boiled in 30 volumes of 1ϫ sample buffer and subjected to 7.5% PAGE. The gels were dried under vacuum and processed for autoradiography using BAS Imaging Plate and FLA-2000 Imaging Analyzer (Fuji Film).

NHE in the Plasma Membrane of Starfish
Oocytes Is the Homolog of Mammalian NHE3-A full-length NHE clone was obtained from a starfish ovary cDNA library. The deduced amino acid sequence of starfish NHE revealed a conserved N-terminal two-thirds including 12 transmembrane domains and an amiloride-binding region (Fig. 1A). The C-terminal cytoplasmic domain contains the conserved phosphorylation sites for MAPK and p90Rsk as well as for serum-and glucocorticoid-induced kinase 1 (SGK1). By phylogenetic analysis with the conserved transmembrane domains (93-527 amino acids), starfish NHE was found to be a member of eukaryotic NHE subfamilies (14) including human NHE1 and NHE3 ( Fig. 1B and supplemental Fig. S1). Subsequently, a search against the Conserved Domain Data base (CCD version 2.17) identified sodium/hydrogen exchanger 3 (TIGR00840) with a highest E-value of 4e-104. Here, the cloned NHE from oocytes of the starfish A. pectinifera is referred to as ApNHE3. Further screening by reverse transcription-PCR with degenerate primers specific to the human NHE1 isoform failed to amplify a starfish homolog of human NHE1 from ovarian cDNAs. In addition, genome annotation of sea urchin (Strongylocentrotus purpuratus), a sister group in Echinoderms found only the predicted isoform similar to NHE3, but not the other eight somatic mammalian NHE isoforms was found (data not shown). Accordingly, NHE3, but not NHE1, was found in sea urchin eggs by Western blotting (15). Taken together, these data suggest that starfish oocytes express the ApNHE3 isoform as the sole plasma membrane Na ϩ /H ϩ exchanger.
A polyclonal antibody against the cytoplasmic domain of ApNHE3 recognized an 87.5-kDa protein in the egg ghost preparation (Fig. 1C). The calculated molecular mass of ApNHE3 is 84.1-kDa, which is in good agreement with the result from immunoblotting. ApNHE3 appeared in the extract from muscle (tube feet) where mammalian NHE3 is absent, suggesting that tissue distribution of ApNHE3 is different from that of mammalian NHE3 (supplemental Fig. S2). Immunofluorescence staining was performed with immature oocytes with and without pretreatment of actinase E. Actinase E was used to remove the jelly coat and the vitelline layer, and therefore the anti-C-terminal NHE antibody was able to cross the permeabilized cell membrane. The antibody stained the surface of oocytes predominantly only when they were pretreated with actinase E (Fig. 1D).
Biochemical and Pharmacological Characteristics of ApNHE3-To examine which mammalian NHE isoform is most related functionally to starfish ApNHE3, we first used pharmacological antagonists. Cimetidine is known to be a potent inhibitor of rat NHE1, whereas it has little effect on the rat NHE3 activity (16). By contrast, clonidine inhibits rat NHE3 about 3-fold greater than rat NHE1 (16). Immature starfish oocytes were treated with either cimetidine or clonidine for 10 min at a concentration of 1 mM, then treated with 1-MeAde. We found that clonidine, but not cimetidine, blocked the 1-MeAde-induced pH i increase (supplemental Fig. S3, A and B). Another difference between mammalian NHE1 and NHE3 is their sensitivity to amiloride and its derivatives such as EIPA. NHE3 is more resistant to amiloride than NHE1 (17). EIPA blocks NHE1 and NHE3 more effectively than amiloride (17). Therefore, we tested the sensitivity to these drugs. A dose-dependent inhibition of the 1-MeAde-induced pH i increase was observed by EIPA but not by amiloride (supplemental Fig. S3C). Because the determinants for these drug sensitivities reside within membrane-spanning domains (18), the data indicate that the biochemical characteristics of the transmembrane segments of ApNHE3 are similar to mammalian NHE3.
We next investigated functional properties of the C-terminal cytoplasmic domain. Because acute activation of mammalian NHE3 is regulated by membrane trafficking in the intestine and kidney, and the cytoplasmic domain of mammalian NHE3 contains several motifs that interact with regulatory proteins responsible for endocytotic and exocytotic pathways (19), we investigated if ApNHE3 activation is associated with membrane trafficking. Depolymerization of actin cytoskeleton by cytochalasin D either decreases or increases NHE3 activity due to different effects on its intrinsic activity or protein transport from the endosomal pool to the cell surface (20,21). More recent studies suggest that lipid rafts are involved in NHE3 activity and trafficking (22). Immature starfish oocytes incubated with 50 M cytocharasin D or 50 mM 2-hydroxylpropyl-␤-cyclodextrin were either treated with 1-MeAde or injected with CA-Rsk (see below). Both 1-MeAde-and CA-Rsk-induced pH i increases of treated oocytes were the same as that of untreated oocytes (supplemental Fig. S4). Accordingly, motif analysis within the C-terminal region revealed that domains known to interact with ezrin, an actin-binding protein (19), and NHERF1/2, ezrin binding scaffold proteins (23,24) are either absence or mutated (supplemental Fig. S5). From these results, it is unlikely that trafficking regulates ApNHE3 activity during meiosis of the starfish oocyte.
The Mos-MEK-MAPK-p90Rsk Pathway Stimulates Cytoplasmic Alkalization of Immature Starfish Oocytes-To investigate the signal pathway that leads to cytoplasmic alkalization of maturing oocytes, we hypothesized that the Mos-MEK-MAPK-p90Rsk pathway is involved. We first injected 10 g/ml of GST-Mos into prophase-arrested oocytes. GST-Mos increased pH i in a sigmoidal curve, reaching a plateau within 40 min (Fig. 2D, GST-Mos). This Mos-induced pH i increase was blocked completely by a 1-h pretreatment with either 150 M EIPA, an inhibitor of NHE, or 10 M U0126, an inhibitor of MEK (Fig. 2B). Mos-induced MAPK activation was confirmed by Western blotting (Fig. 2A). In addition, a specific p90Rsk inhibitor, SL0101 (25), also blocked the pH i increase at a concentration of 100 M. In control, injections of GST alone had no effect on pH i . These results suggested that it is p90Rsk-dependent pH i Regulation the Mos-MEK-MAPK-p90Rsk pathway that promotes cytoplasmic alkalization.
Next, a constitutively active form of p90Rsk (CA-Rsk) at a final concentration of 10 g/ml was injected into immature oocytes in the presence of various inhibitors. CA-Rsk, but not a kinase-inactive form of p90Rsk (KI-Rsk) promoted a pH i increase (Fig. 2C). Similar to the Mos-induced pH i increase, EIPA inhibited the CA-Rsk-induced pH i increase more effec- In the C-terminal cytoplasmic domain, potential phosphorylation sites for p90Rsk, SGK, and MAPK are marked. B, multiple alignment (ClustalW) of the NHE family from diverse organisms (humans, sea urchins, fruit flies, mosquitoes, and nematodes) identified a conserved domain in the N-terminal two-thirds of starfish NHE (amino acids 93-527). Phylogenetic analysis of the conserved domain in the eukaryotic NHE gene family revealed that starfish NHE belongs to the recycling cluster of the plasma membrane subfamily (14). The phylogenetic tree is represented as an unrooted phylogram using the neighbor-joining method. C, by Western blot analysis, a polyclonal antibody raised against the cytoplasmic domain of starfish NHE recognized an 87.5-kDa protein in the egg ghost preparation, where the cell surface complex is enriched and cytosolic proteins are largely eliminated. D, immunostaining of prophase-arrested oocytes shows starfish NHE localizes to the cell surface. The oocytes were treated with (ϩactn. E) or without (Ϫactn. E) Actinase E. After fixation, oocytes were treated with 0.1% Triton X-100 and then blocked with 3% bovine serum albumin. The primary antibody used was an anti-NHE IgG (1:500 dilution). As negative controls, a preimmune serum (1:500 dilution) and no primary antibody were also tested. Representative images were photographed by confocal microscopy. Scale bar, 30 m.

p90Rsk-dependent pH i Regulation
tively than SL0101. However, pretreatment of oocytes with 10 M U0126 did not block the CA-Rsk-induced pH i increase, suggesting that p90Rsk is a main downstream effector of the Mos-MEK-MAPK pathway to activate Na ϩ /H ϩ exchange. Kinetic studies revealed that oocytes increased pH i shortly after injection with CA-Rsk (Fig. 2D, CA-Rsk), however, there was a ϳ10min delay before initiation of the pH i increase in oocytes injected with GST-Mos (Fig. 2D, GST-Mos). This delay is not due to the amount being injected (data not shown), but rather is accounted for by the time required for sequential activation of the Mos-MEK-MAPK-p90Rsk pathway.
p90Rsk Phosphorylates ApNHE3-Phosphorylation of the C-terminal domain of ApNHE3 might modulate Na ϩ /H ϩ exchange activity, as reported for mammalian NHEs (26 -28). Cell-free extracts prepared from maturing (maturing extract) and immature (immature extract) oocytes (12) were used in an in vitro phosphorylation assay with a recombinant GST-tagged C-terminal domain of ApNHE3 (NHE-⌬N WT ). In the maturing extract where MAPK is active, NHE-⌬N WT is highly phosphorylated after a 10-min incubation (Fig. 3A, first lane). Conversely, in the immature extract where MAPK is inactive, no significant phosphorylation of NHE-⌬N WT was observed (Fig.  3A, third lane). To verify that this phosphorylation resulted from the MAPK pathway, MAPK was inactivated by incubation with 3 M U0126 (Fig. 3B, first lane), and thereafter NHE-⌬N WT was added. We found that NHE-⌬N WT phosphorylation was significantly lower in the presence of U0126 as compared with the inactive analog U0124 (Fig. 3, C and D). These data, in conjunction with in vivo experiments (Fig. 2), support the hypothesis that the Mos-MEK-MAPK pathway promotes ApNHE3 phosphorylation that up-regulates Na ϩ /H ϩ exchange activity.
To investigate what kinase(s) is responsible for direct phosphorylation of ApNHE3 under control of Mos signaling, putative phosphorylation sites were mutated. First, when the putative MAPK phosphorylation sites (Thr-620 and Thr-633) were mutated to Gly (NHE-⌬N ⌬MAPK ), the level of phosphorylation in NHE-⌬N ⌬MAPK was similar to that in the NHE-⌬N WT construct (Fig. 3E), suggesting that MAPK does not directly phosphorylate ApNHE3. Next, all three putative Rsk phosphorylation sites (the RXXS motif at Ser-590, Ser-606, and Ser-673) were mutated to Ala (NHE-⌬N ⌬Rsk ). Phosphorylation of NHE-⌬N ⌬Rsk by GST-Mos was completely abolished (Fig. 3F). The level of phosphorylation of NHE-⌬N ⌬Rsk was similar to that of NHE-⌬N WT by GST. CA-Rsk phosphorylated NHE-⌬N WT , whereas the level of phosphorylation of NHE-⌬N ⌬Rsk by CA-Rsk was as low as that in NHE-⌬N WT by KI-Rsk (Fig. 3G). All possible double alanine mutants in three potential p90Rsk phosphorylation sites showed a similar ␥-32 P incorporation (supplemental Fig. S6). These results indicate that all three p90Rsk phosphorylation sites were equally phosphorylated in vitro.
Endogenous ApNHE3 Is Phosphorylated in Accordance with p90Rsk Activation-In an in vitro maturation system, the Mos-MEK-MAPK-p90Rsk pathway is activated shortly after onset of GVBD (8). To examine whether phosphorylation of endogenous ApNHE3 occurs by this pathway, we generated a polyclonal antibody specific to phosphoserine at residue 606 (one of the p90Rsk phosphorylation sites) in the cytoplasmic tail of ApNHE3. By enzyme-linked immunosorbent assay analysis, an

p90Rsk-dependent pH i Regulation
affinity purified antibody was found to react with a phosphorylated synthetic peptide, but not with the non-phosphorylated form of the peptide (Fig. 4A, ELISA). By Western blot analysis with maturing oocytes, a single band corresponding to ϳ90 kDa was detected (Fig. 4A, WB). With this anti-phospho-ApNHE3 (Ser-606) antibody, we found that ApNHE3 was phosphorylated at basal levels in prophase-arrested oocytes, resulting in a ϳ3-fold increase in Ser-606 phosphorylation synchronously with p90Rsk activation (Fig.  4, B and C).
ApNHE3 Phosphorylation by p90Rsk Up-regulates Na ϩ /H ϩ Exchange Activity-To examine whether phosphorylation of ApNHE3 by p90Rsk up-regulates Na ϩ /H ϩ exchange activity, we produced a recombinant ApNHE3 99-amino acid fragment (581-679) as a pseudo-substrate for p90Rsk (NHEfrag WT ) and its triple mutant lacking Rsk phosphorylation sites (NHEfrag ⌬Rsk ). The CA-Rsk-induced pH i increase was completely abolished by the prior injection of NHE-frag WT , presumably due to competitive blocking of phosphorylation of endogenous ApNHE3 (Fig. 5A). In contrast, the CA-Rsk-induced pH i increase occurred normally when a non-competitor, NHE-frag ⌬Rsk , was injected. In addition, the 1-MeAde-induced increase in pH i , promoted by the PI3K pathway (6), occurred normally in NHE-frag WT -injected oocytes. These data suggest that NHE phosphorylation by p90Rsk is directly coupled to Na ϩ /H ϩ exchange activity.
In another approach for inferring a functional relationship between NHE phosphorylation and its exchange activity, we used a neutralizing antibody to block phosphorylation sites. A polyclonal antibody that reacts with a 15-mer peptide harboring the second RXXS motif (residues 603-606) of ApNHE3 was injected into immature oocytes. Injection of the antibody did not cause any change in the BCECF ratio for the following 20 min (Fig.  5B, traces 2 and 4). Thereafter, when CA-Rsk was injected into these oocytes, the pH i increase that normally occurs continuously over 30 min was not observed (trace 1, 3, and 4). In contrast, the 1-MeAde-induced pH i increase was observed in antibody-injected oocytes (trace 4), suggesting that the neutralizing effect of the antibody is specific to p90Rsk-dependent ApNHE3 activation. was phosphorylated in the maturing extract, but not in the immature extract. B and C, the maturing extracts were treated with 3 M U0126, 3 M U0124 or dimethyl sulfoxide (DMSO) at 0°C for 30 min, followed by incubation at 20°C for 1 h. The immature extract was treated with DMSO as well. One part of these samples was subjected to 12.5% SDS-PAGE followed by Western blotting with anti-ERK1/2 (extracellular signal-regulated kinase) antibody. The other part of the samples was incubated with [␥-32 P]ATP and NHE-⌬N WT at 20°C for 10 min. The samples were subjected to 10% SDS-PAGE and autoradiographed. D, the time course of NHE-⌬N WT phosphorylation was examined. The relative phosphorylation levels in the maturing extract with U0126 (OE), U0124 (Ⅺ), or DMSO (F), and in the immature extract with DMSO (E) are shown. The data represent the mean Ϯ S.E. obtained from three independent experiments. E and F, the immature extracts were incubated with GST-Mos or GST at 0°C for 1 h, following by 20°C for 1 h. Thereafter, they were added with NHE-⌬N WT , NHE-⌬N ⌬MAPK (E), or NHE-⌬N ⌬Rsk (F) with [␥-32 P]ATP at 20°C for 20 min. G, CA-Rsk, or the KI-Rsk, was incubated with the immature extract at 20°C for 1 h. Thereafter, NHE-⌬N WT or NHE-⌬N ⌬Rsk was added with [␥-32 P]ATP, followed by a 10-min incubation at 20°C. CA-Rsk requires phosphorylation at Ser-227 in the activation loop, which is catalyzed by 3-phosphoinositide-dependent protein kinase-1 (PDK1) (58). For this reason, cell-free extracts that contain active PDK1 were used. The percentage of phosphorylation of the recombinant proteins was expressed as described earlier and the data represented as the mean Ϯ S.E. obtained from three independent experiments. The p values by analysis of variance followed by the Tukey post hoc analysis are shown on top of the graphs (E-G), i.e. **, p Ͻ 0.005; ***, p Ͻ 0.001. JULY 30, 2010 • VOLUME 285 • NUMBER 31

MI-arrested Ovarian Oocytes Maintain NHE in an Active
State at the Time of Spawning-Previously, we showed that an initial pH i increase after 1-MeAde treatment occurs within 10 min, followed by the onset of GVBD in the next 10 min (6). In parallel with this, a Western blot with anti-ApNHE3 showed an apparent upward shift of the band following treatment with 1-MeAde for 10 min (Fig. 4B, bottom), which precedes ApNHE3 (Ser-606) phosphorylation concomitantly with GVBD (Fig. 4B, middle). Therefore, we speculated that ApNHE3 is activated through at least two cas-cades, i.e. the upstream G␤␥-PI3K cascade (6) and the downstream Mos-MEK-MAPK-p90Rsk cascade.
To examine whether or not the robust pH i increase seen at spawning is mainly due to ApNHE3 activation by the G␤␥- was subjected to 7.5% SDS-PAGE followed by Western blotting with affinity-purified anti-phospho-NHE (Ser-606) antibody ("Experimental Procedures"). With the same blot, preimmune serum was tested as a negative control. Enzyme-linked immunosorbent assay (ELISA) was performed with a 15-mer peptide (TRGDSYFDSIRRR) and its phosphorylated derivative (TRGD-phospho-SYFDSIRRR) as antigens. An anti-non-phospho-ApNHE3 antibody was prepared as described. The data presented are: 15-mer peptide/anti-phospho-Ser-606 ApNHE3 antibody (F), phosphorylated peptide/anti-phospho-Ser-606 ApNHE3 antibody (f), 15-mer peptide/anti-nonphospho-ApNHE3 antibody (E), and phosphorylated peptide/anti-nonphospho-ApNHE3 antibody (Ⅺ). B, prophase-arrested oocytes were treated with 1-MeAde in seawater at pH 8.0. The samples were aliquoted at different time points, subjected to 7.5% SDS-PAGE followed by Western blotting (WB). Primary antibodies used were anti-phospho-Thr-573 p90Rsk, anti-phospho-Ser-606 ApNHE3, and anti-non-phospho-ApNHE3. Asterisks indicate two unknown minor bands that appear transiently during meiosis reinitiation. C, the intensity of each band corresponding to the phosphorylated forms of p90Rsk (E) and ApNHE3 (F) shown in B was quantified and expressed relative to the initial value (0 min) of 1.

FIGURE 5. ApNHE3 phosphorylation by p90Rsk is coupled with its function.
A, the cytoplasmic fragment of ApNHE3 that contains all three potential Rsk phosphorylation sites (Ser-590, -606, and -673) was expressed with a GST tag (NHE-frag WT ). A mutant recombinant fragment in which all three serines were substituted by alanines (NHE-frag ⌬Rsk ) was also expressed. Affinity purified NHEfrag WT and NHE-frag ⌬Rsk at final concentrations of 50 mg/ml, indicated as "Competitor," were microinjected to GV oocytes and incubated for 30 min at 20°C. Injection of these recombinant fragments does not cause any significant changes in the BCECF ratio for up to 1 h (last two columns). Oocytes pre-injected with NHE-frag ⌬Rsk or control injection buffer increased in pH i after CA-Rsk injection. However, this CA-Rsk-induced pH i increase was completely abolished by injection of NHE-frag WT . In contrast, the 1-MeAde-induced pH i increase that occurs independently of p90Rsk activation was observed in NHE-frag WT -injected oocytes. The data represente the amount of change (n ϭ 3) in the BCECF ratio (⌬BCECF ratio/h) after injecting or treating with stimulators indicated as "Trigger." B, representative time course traces for the relative BCECF fluorescence intensity (FI) with various treatments. Anti-non-phospho-ApNHE3 antibody (␣-ApNHE3), anti-GST antibody (␣-GST), or CA-Rsk was injected into immature oocytes at the time points indicated by arrows. In some experiments, oocytes were treated with 1-MeAde. CA-Rsk induced an increase in pH i (trace 1). This increase is blocked by prior injection of ␣-ApNHE3 (trace 4), but not ␣-GST (trace 3). These antibodies did not block the 1-MeAde-induced pH i increase (trace 2 and 4).

p90Rsk-dependent pH i Regulation
PI3K cascade, we developed an assay for Na ϩ /H ϩ exchange activity that distinguishes between the stimulated state and the basal state (Fig. 6A). Prophase-arrested oocytes placed in 4.8 mM Na ϩ -containing seawater (1% NaSW, see "Experimental Procedures") were treated with or without 1-MeAde, after which Na ϩ was recovered by 48 mM Na ϩ -containing seawater (10% NaSW). A linear increase in pH i after replacement with 10% NaSW was observed in maturing oocytes, but not in the control immature oocytes (Fig. 6A). The 1-MeAde-stimulated activation of Na ϩ /H ϩ exchange peaked at 5 min after 1-MeAde treatment and its activity remained significantly after 90 min (Fig. 6B).
To ensure that this sustained NHE activity is independent of the Mos-MEK-MAPK-p90Rsk pathway, maturing oocytes were treated with 10 M U0126 and tested for NHE activity. We found that the rate of pH i increase in both U0126-treated and non-treated oocytes was quite similar (Fig. 6, C and D), indicating that the Mos-MEK-MAPK-p90Rsk pathway is not involved. Next, immature oocytes placed in 1% NaSW were treated with 1-MeAde for only 5 min, after which Na ϩ was recovered at various time points. Under these conditions, although no GVBD occurred due to the short exposure with 1-MeAde, oocytes increased pH i significantly by the addition of Na ϩ even after 60 min (Fig. 6E). Finally, MI-arrested spawned oocytes obtained by intercoelomic injection with 1-MeAde were tested for NHE activity (Fig. 6F). Oocytes spawned at different time points were immediately replaced in 1% NaSW containing 10 M U0126. Even when the oocytes were collected 100 min after 1-MeAde injection, MAPK remained inactive (Fig. 6F, WB) and NHE activity was high (Fig. 6F, lanes 3-5). In contrast, treatment with LY294002, a PI3K inhibitor alone or in combination with U0126 lowered NHE activity (Fig. 6F, lanes 6 and 7). These results suggest that 1-MeAde-stimulated, PI3K-dependent initial activation of ApNHE3 is maintained until spawning. Therefore, the increase in pH i upon spawning may result from the G␤␥-PI3K pathway rather than the Mos-MEK-MAPK-p90Rsk pathway.
Both Elevated pH i and ApNHE3 (Ser-606) Phosphorylation Are Maintained during Post-meiotic GI Arrest-How long does an elevated cytoplasmic pH i continue after completion of meiosis? We measured cytoplasmic pH i for an extended time period until the cell begins apoptosis. As shown in Fig. 7A, oocytes established pH i homeostasis at pH ϳ 7.0 after ϳ1 h of 1-MeAde administration, which continued until onset of membrane blebbing (arrowhead), a characteristic of an initial phase of apoptosis. Previously, we had shown that sustained activation of MAPK (ϳ8 h) followed by spontaneous inactivation is a prerequisite for apoptosis (29). Accordingly, in cohort oocytes used for pH i measurements (Fig. 7A), MAPK inactivation was seen between 7 and 8 h after 1-MeAde treatment (Fig. 7B, top). A concomitant decrease in the level of phosphorylated ApNHE3 was observed (Fig. 7B, middle). From these results we suggest that p90Rsk-dependent ApNHE3 phosphorylation may ensure the pH i homeostasis of the egg at a high level.

DISCUSSION
During oocyte maturation, an increase in cytoplasmic pH is observed in amphibians, ascidians, molluscs, and echinoderms (30 -32). However, molecular mechanisms of pH i regulation as well as its physiological role, if any, remain largely unknown. Regarding the physiological role of cytoplasmic alkalization during oocyte maturation and egg activation, in early studies, it has been hypothesized that protein synthesis is controlled by FIGURE 6. PI3K-dependent ApNHE3 activation is maintained until spawning. A, GV oocytes were placed in 4.8 mM Na ϩ -containing seawater (1% NaSW) and loaded with BCECF. One part of oocytes was treated with 1-MeAde to give rise to maturing oocytes. Thereafter, these oocytes were replaced (arrowhead) with 48 mM Na ϩ -containing seawater (10% NaSW). After replacement, the pH i was increased in maturing oocytes (open diamonds), but not in GV oocytes (closed diamonds). To quantify NHE activity, the initial slope at 2 min was determined (inset). B, GV oocytes were treated with 1 M 1-MeAde, and then replaced with 10% NaSW at the time points indicated. C and D, BCECF-loaded, GV oocytes were incubated in 1% NaSW with 1-MeAde for 70 min in the presence of 10 M U0126 (C) or DMSO (D). In both cases, GVBD occurred at ϳ20 min. Thereafter, oocytes were replaced with 10% NaSW containing 10 M U0126 (C) or DMSO (D) at the points indicated by the arrows. The increases of pH i in both U0126-treated and MOCK-treated oocytes were similar. Duplicated representative traces were shown. E, similar to B, oocytes treated with 1-MeAde for 5 min were washed with 1% NaSW, after which they were placed in 10% NaSW. F, females were injected with 1-MeAde. Spawning was triggered after ϳ35-40 min. Oocytes spawned at the time points indicated were pooled and placed in 1% NaSW containing 10 M U0126 and/or 200 M LY294002 for 30 min. Thereafter, NHE activity was measured. Immature (IM) and maturing (M) oocytes were tested as well. Western blot (top) shows active (arrow) and inactive (arrowhead) MAPK in these oocytes. Shown are the mean Ϯ S.E. of three to six independent experiments (B, E, and F).

p90Rsk-dependent pH i Regulation
the pH i rise. In sea urchins, the increase in pH i that occurs at fertilization is implicated in the increased rate of protein synthesis and initiation of DNA synthesis (33,34). Subsequent studies suggested that the raised pH i is only one part of the factors controlling translation and has some other effects on early developmental processes.
Xenopus has been one of the most studied organisms in pH i regulation during oocyte maturation. Increased phosphorylation of 40 S ribosomal protein S6 was proposed to be responsible for the increased rate of protein synthesis (35,36). Temporal correlation between the pH i increase and S6 phosphorylation led to the hypothesis that increased pH i regulates protein synthesis required for meiotic maturation. However, later investigations found that increased pH i is not necessary for S6 phosphorylation and that increased S6 phosphorylation is not sufficient for GVBD or increased protein synthesis (37).
In this study, to understand the molecular mechanisms of the pH i increase during meiosis, we cloned starfish NHE (ApNHE3) and found that the potential phosphorylation sites for various kinases including p90Rsk and MAPK are within the C-terminal domain of ApNHE3 (Fig. 1). MAPK by itself, however, did not directly phosphorylate ApNHE3. Whereas, ApNHE3 phosphorylation sites for p90Rsk were phosphorylated in the MAPKdependent pathway (Fig. 3). In an in vitro maturation system, p90Rsk is activated shortly after GVBD (Fig. 4B, Ref. 4) concomitantly with increased phosphorylation of ApNHE3 at Ser-606 (Fig. 4B). Moreover, microinjection of constitutively active p90Rsk into immature oocytes caused an increase in pH i even in the absence of 1-MeAde signaling, indicating that the pH i increase is induced by the p90Rsk-dependent phosphorylation of ApNHE3 (Fig. 2).
In mammalian cells, p90Rsk phosphorylates NHE1 at Ser-703 in transfected 293 cells (26) and plays an important role in the regulation of Na ϩ /H ϩ exchanger activity during ischemia and reperfusion of rat myocardium (38) and during ischemic neuronal death (39). In Xenopus oocytes, NHE1 is regulated by c-Mos (40) and independently by Raf-1 (41), however, the downstream effecter that directly up-regulates Na ϩ /H ϩ exchanger activity remains unknown. In mouse oocytes, NHE and HCO 3 Ϫ /Cl Ϫ exchangers are essentially inactive during oocyte growth until they reach the fully grown GV stage (42). In both organisms, the presence of active p90Rsk is evident at the MII stage, but there are no reports suggesting the involvement of p90Rsk in pH i regulation. Interestingly, mouse HCO 3 Ϫ /Cl Ϫ exchanger Ae2 is inactivated during meiotic maturation and becomes quiescent at the MII stage (43). This inactivation correlates with MEK/MAPK-regulated Ae2 loss from the plasma membrane, suggesting that membrane trafficking is involved. In this study we suggested, however, that trafficking is unlikely involved in ApNHE3 regulation (supplemental Fig. S4). More precise investigations are required to clarify this problem.
When assayed with an in vitro maturation system, 1-MeAde stimulated the increase in pH i before GVBD was triggered. This increase in pH i by 1-MeAde is not mediated by the Mos-MEK-MAPK-p90Rsk pathway, because MAPK is inactive before GVBD; instead, it is regulated by the G protein ␤␥ subunit-PI3K pathway (6). NHE activity reached its maximum after 5-10 min of 1-MeAde treatment and continues for ϳ90 min (Fig. 6B). Accordingly, ApNHE3 shifted its mobility upward by 10 min after hormone treatment and this band shift was continued for at least 1 h (Fig. 4B). This 1-MeAde-induced rapid increase in pH i was blocked by LY294002 (6), a PI3K inhibitor, but not by roscovitine, a cdc2 inhibitor (data not shown). Furthermore, Akt inhibitors such as a phosphatidylinositol analogue (SH-6) (44) or an Akt peptide (45) had no effect on the 1-MeAdeinduced pH i increase (data not shown). From these data, we concluded that downstream G␤␥-PI3K signaling promotes Na ϩ /H ϩ exchanger activity. In mammalian cells, the PI3K-SGK1 pathway (46) activates NHE3 (47)(48)(49). Interestingly, the putative phosphorylation site by SGK1 is present in the C-terminal domain of ApNHE3 ( Fig. 2A). Furthermore, ApNHE3 was phosphorylated by the active form of the recombinant human SGK1 (supplemental Fig. S7), suggesting that the early phase of ApNHE3 activation may be driven by SGK1 signaling. However, it is still unknown why such a large increase in pH i observed in an in vitro system is suppressed in the ovarian oocytes.
Given the observations for a spawning-associated pH i increase and emergence of MAPK activation after spawning, we first hypothesized that p90Rsk-dependent ApNHE3 activation leads to MI exit. However, U0126-treated oocytes exited MI and extruded the first polar body (10). This observation suggests that p90Rsk activation is not the primary event necessary for MI exit. Because 1-MeAde-evoked Na ϩ /H ϩ exchanger p90Rsk-dependent pH i Regulation activity was kept for ϳ40 min after replacement with fresh 1% NaSW in vitro (Fig. 6E), the pH i increase observed at spawning may be mediated primarily, if not all, by this active ApNHE3.
It should be noted that the pH i increase is still observed when the extracellular pH is raised from 6.4 (estimated pH of ovarian fluid) to 8.0 (pH of normal seawater) in the absence of external Na ϩ (data not shown). In this context, it is conceivable that the concerted action of active (NHE involved) and passive (spontaneous) mechanisms in pH i regulation governs the transition from metaphase I to anaphase I. The spawning-stimulated pH i increase may also regulate the timing of MI exit because the accelerated pH i increase caused by NH 4 Cl treatment, or insemination, shortened the MI period (10,50).
After completion of meiosis, sustained MAPK activity for as long as ϳ8 h is required to acquire apoptosis competence, and this is referred to as the MAPK-dependent period (29). After the MAPK-dependent period, spontaneous MAPK inactivation initiates caspase-3 activation followed by membrane blebbing (51,52). In this study, we found that sustained high pH i continues in GI-arrested mature eggs until the onset of membrane blebbing, a characteristic of the early phase of apoptosis (Fig.  7A). Increased NHE activity by PI3K signaling is expected to return at a basal level by the end of meiosis (Fig. 6E), therefore the sustained high pH i thereafter could be reasoned for a mechanism other than the PI3K pathway.
It could be possible that constitutive MAPK-p90Rsk signaling maintains NHE activity, and following spontaneous MAPK inactivation, results in NHE inactivation (Fig. 8). The other possibility involves suppression of NHE-deactivating enzymes such as tyrosine phosphatase SHP-1 or p38 MAPK. SHP-1 is necessary for the execution of acidification-dependent apoptosis (53,54). SHP-1 may trigger caspase-8 activation, by which NHE becomes inactivated through direct action or via inhibition of p90Rsk (55). Low density lipoproteins down-regulate NHE via activation of p38 MAPK in human platelets (56). In starfish, MAPK inactivation is prerequisite for the p38 MAPK activation necessary for apoptotic body formation (29).
The physiological role of sustained high pH i in eggs after completion of meiosis remains unknown. However, one can easily assume that such change in cytoplasmic pH by ϳ0.4 units will impact a vast number of cellular metabolisms and signaling, by which eggs may increase the duration of post-meiotic fertilizing capacity (avoidance from apoptosis execution), fertilization success rate, or post-fertilization developmental competence. Further studies are required to address these possibilities and determine the exact role of post-meiotic pH i homeostasis.