Article |
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Abstract |
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During oocyte maturation, eggs acquire the ability to generate specialized Ca2+ signals in response to sperm entry. Such Ca2+ signals are crucial for egg activation and the initiation of embryonic development. We examined the regulation during Xenopus oocyte maturation of store-operated Ca2+ entry (SOCE), an important Ca2+ influx pathway in oocytes and other nonexcitable cells. We have previously shown that SOCE inactivates during Xenopus oocyte meiosis. SOCE inactivation may be important in preventing premature egg activation. In this study, we investigated the correlation between SOCE inactivation and the Mosmitogen-activated protein kinase (MAPK)maturation-promoting factor (MPF) kinase cascade, which drives Xenopus oocyte maturation. SOCE inactivation at germinal vesicle breakdown coincides with an increase in the levels of MAPK and MPF. By differentially inducing Mos, MAPK, and MPF, we demonstrate that the activation of MPF is necessary for SOCE inactivation during oocyte maturation. In contrast, sustained high levels of Mos kinase and the MAPK cascade have no effect on SOCE activation. We further show that preactivated SOCE is not inactivated by MPF, suggesting that MPF does not block Ca2+ influx through SOCE channels, but rather inhibits coupling between store depletion and SOCE activation.
Key Words: calcium signaling; store-operated Ca2+ entry; oocyte maturation; Xenopus laevis; maturation-promoting factor
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Introduction |
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The amphibian oocyte represents one of the best-characterized models for oocyte maturation, at a biochemical as well as morphological level (Bement and Capco, 1990). In Xenopus laevis, fully grown oocytes (stage VI) in the ovary are arrested at prophase of meiosis I. During oocyte maturation, cells enter meiosis, undergo germinal vesicle (nuclear envelope) breakdown (GVBD),* complete meiosis I with the extrusion of a polar body, and arrest at metaphase of the second meiotic division (MII) until fertilization (Masui and Clarke, 1979). We will refer to mature cells arrested at MII as eggs, to differentiate them from immature oocytes arrested at prophase I. Xenopus oocyte maturation is initiated in response to the hormone progesterone, which induces a signal transduction cascade culminating in the activation of maturation-promoting factor (MPF) (for recent review see Nebreda and Ferby, 2000). MPF is a complex, composed of a serine/threonine kinase (p34cdc2) subunit and a regulatory cyclin B subunit. MPF activation is necessary and sufficient for GVBD and entry into meiosis (Smith, 1989). Progesterone-induced maturation requires translation of Mos kinase, which induces the MAP kinase cascade, resulting in MPF activation (Sagata, 1997). In addition to the Mosmitogen-activated protein kinase (MAPK) cascade, other signaling cascades are activated in parallel, resulting in rapid MPF induction. The polo-like kinase cascade is thought to activate Cdc25C phosphatase, which dephosphorylates and activates MPF (Qian et al., 1999, 2001).
An important component of oocyte maturation is differentiation of the Ca2+ signaling machinery, which is essential for egg activation after fertilization. An increase in cytoplasmic Ca2+ at fertilization is the universal signal to initiate development in all species investigated (Homa et al., 1993; Stricker, 2000). Ca2+ signals generated at fertilization in Xenopus have specific spatial and temporal features that are presumably important for successful egg activation. For example, Ca2+ signals are crucial for the block to polyspermy (Cross, 1981; Kline and Nuccitelli, 1985), through the induction of membrane depolarization (Machaca et al., 2001) and the activation of cortical granule fusion (Wolf, 1974; Grey et al., 1976). Increased Ca2+ is also responsible for releasing the MII block by inactivating cytostatic factor (CSF) (Meyerhof and Masui, 1979). Therefore, during oocyte maturation, Ca2+ signaling pathways undergo their own differentiation followed by functionally significant consequences. In this study, we investigate the regulation of store-operated Ca2+ entry (SOCE) during Xenopus oocyte maturation. SOCE is a ubiquitous Ca2+ entry pathway in nonexcitable cells, activated in response to depletion of intracellular Ca2+ stores (Parekh and Penner, 1997). SOCE is important for a variety of physiological functions, including regulation of exocytosis (Koizumi and Inoue, 1998; Fomina and Nowycky, 1999), modulation of Ca2+ waves (Girard and Clapham, 1993; Dolmetsch and Lewis, 1994), and activation of T lymphocytes (Fanger et al., 1995; Serafini et al., 1995). The mechanism by which Ca2+ store depletion activates SOCE is controversial. Three primary models have been proposed with experimental evidence for each: direct physical coupling between SOCE channels and a Ca2+ sensor (Berridge, 1995; Kiselyov et al., 1998); activation of SOCE channels by a diffusible messenger (Randriamampita and Tsien, 1993; Csutora et al., 1999); and exocytotic insertion of SOCE channels in the plasma membrane (Somasundaram et al., 1995; Yao et al., 1999).
We have previously demonstrated that SOCE inactivates during Xenopus oocyte maturation (Machaca and Haun, 2000), and were interested in elucidating the mechanisms mediating this inactivation. In this study, we show that the induction of MPF is necessary for SOCE inactivation. In contrast, the activation of kinase cascades upstream of MPF does not affect SOCE. However, MPF does not appear to inhibit Ca2+ influx through SOCE channels, rather it blocks the coupling mechanism between store depletion and SOCE activation.
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Results |
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We also examined ISOCE levels in oocytes incubated in progesterone for prolonged periods of time, without undergoing GVBD (GVBD negative) (Fig. 1 G). In the majority of such cells (34/44), ISOCE was detected at levels similar to those in control oocytes (Fig. 1 G), indicating a correlation between ISOCE inactivation and GVBD. However, there was a trend for decreased levels of ISOCE at time points longer than 0.8 GVBD50 (Fig. 1 G). GVBD is followed by white spot appearance, indicating completion of nuclear envelope breakdown. Nonetheless, GVBD initiates before white spot appearance as the nuclear envelope breaks down gradually, in a polarized fashion beginning at the vegetal face of the nucleus (Colman et al., 1985). Therefore, cells in which ISOCE activity was decreased could have initiated GVBD, consistent with a temporal correlation between GVBD and ISOCE inactivation.
ISOCE inactivation could be a direct downstream consequence of GVBD. To test this possibility, we removed the nucleus from oocytes (enucleated), and matured them in progesterone. We recorded ISOCE from enucleated cells when 100% of the control oocyte group underwent GVBD. Under these conditions in both control and enucleated cells, ISOCE was completely inactivated (unpublished data). Furthermore, enucleated cells incubated for the same time in the absence of progesterone had ISOCE levels similar to those of untreated oocytes, indicating that removal of the nucleus did not lead to ISOCE inactivation. Thus, physical breakdown of the nuclear envelope is not a prerequisite for ISOCE inactivation.
Time course of Mos, MAPK, and MPF activation during oocyte maturation
As discussed above, oocyte maturation is driven by kinase cascades that lead to MPF activation and entry into meiosis. We next examined whether these kinases are involved in ISOCE inactivation. We focused our attention on the MosMAPK signal transduction cascade, which is the primary pathway for MPF activation in Xenopus oocytes (Palmer and Nebreda, 2000). Fig. 2 summarizes the time course of Mos, MAPK, and MPF activation during progesterone-induced oocyte maturation. In this experiment, GVBDi occurred at 5.5 h, and GVBD50 was reached 7 h after progesterone addition (Fig. 2 A). MPF activity increased at GVBDi and then decreased slightly 30 min after GVBD, indicative of the transition between meiosis I and II. This was followed by a plateau phase of high MPF activity for the reminder of maturation (Fig. 2 B). MAPK activation could be followed with a specific antibody that recognized the phosphorylated (activated) form of the kinase (Fig. 2 C). Phospho-MAPK (P-MAPK) was first detected 3 h after progesterone addition, and increased slowly with a jump at GVBD (Fig. 2 C). Mos kinase levels rose to a low level plateau 2 h after progesterone addition (Fig. 2 D), before any increase in P-MAPK was observed. This is consistent with Mos's role as the upstream kinase that activates the MAPK cascade (Sagata, 1997). Mos protein levels remained at the low plateau level until 30 min after GVBD, when a significant increase was observed (Fig. 2 D). This is presumably due to the activation of MPF, which stabilizes the Mos protein (Sagata, 1997). As shown in Fig. 1, SOCE inactivated at the GVBD stage of meiosis, at the same time that MPF and P-MAPK levels increased (Fig. 2, B and C). It is therefore possible that either MPF, MAPK, or both are involved in SOCE inactivation.
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SOCE inactivates independently of the MAPK cascade
The time course of ISOCE inhibition and kinase activation during progesterone-induced maturation (Figs. 1 and 2), suggest that the MAPK cascade and/or MPF could be involved in SOCE inactivation. To directly test this possibility we manipulated the levels of MAPK and MPF, and determined the effect on SOCE. This was accomplished by injecting cyclin B1 protein in the presence or absence of a mitogen-activated protein kinase kinase (MEK) inhibitor (Fig. 3). MEK is the upstream kinase that phosphorylates and activates MAPK (Palmer and Nebreda, 2000). Cyclin B1 binds and activates p34cdc2, the catalytic subunit of MPF, thus rapidly increasing MPF activity. Cyclin B1 injections have been shown to induce GVBD in the absence of progesterone (Freeman et al., 1991).
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The time delay between MPF activation and ISOCE inactivation was also evident for GVBD, which is a known target of MPF (Bement and Capco, 1990). Therefore, ectopic activation of MPF by cyclin B1 injection did not faithfully reproduce the downstream effects of progesterone-induced MPF activation. In the case of progesterone, an increase in MPF levels coincided with GVBD (Fig. 2 B). The reason for the lag period between MPF activation by cyclin B1 and GVBD is not clear to us. We suggest that it could be due to mislocalization of MPF after cyclin B1 injection, thus delaying MPF action on downstream targets.
To directly test whether MAPK activation is required for ISOCE inactivation we treated cells with the MEK inhibitor PD98059, which blocks MAPK activation. This allowed us to obtain a population of cells containing high MPF activity, without activating the MAPK cascade (Fig. 3, C and D). Injection of cyclin B1 into PD98059-treated oocytes induced the activation of MPF (Fig. 3 C) with a similar time course and to comparable levels as control oocytes (Fig. 3 A). In this case there also was a strong temporal correlation between GVBD and ISOCE inactivation. Pretreating oocytes with PD98059 delayed but did not completely block MAPK activation, as some cells at later time points showed high levels of P-MAPK (Fig. 3 D). However, we could easily define a population of cells, between 0.9 and 1.6 GVBDi, in which MAPK activity was completely blocked by PD98059 (Fig. 3 D, box). All of these cells had high levels of MPF activity (one- to fourfold that in eggs) (Fig. 3 C, box). Such cells with high MPF activity and no detectable P-MAPK had completely inactivated ISOCE (Fig. 3, C and D). This indicates that activation of the MAPK cascade is not required for ISOCE inactivation.
Fig. 4 shows examples of single cells illustrating the need for MPF, but not MAPK, activation for ISOCE inactivation. In a control untreated oocyte MPF, MAPK, and Mos activities were undetectable, and ISOCE was activated in response to store depletion (Fig. 4 A). In contrast, no ISOCE could be detected after store depletion in a cyclin B1injected cell (Fig. 4 B). In this case, cyclin B1 injection induced MPF and P-MAPK activation (1.6- and 0.9-fold egg levels, respectively), but little Mos protein was detected (Fig. 4 B). This argues that Mos kinase is not required for SOCE inactivation.
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MPF activation is necessary for ISOCE inactivation
We next examined whether MPF activation is required for SOCE inactivation. We initiated oocyte maturation by injecting Mos RNA into oocytes in the presence or absence of a specific MPF inhibitor, the Wee1 kinase (Mueller et al., 1995) (Fig. 5). These experiments also allowed us to test whether prolonged activation of Mos and the MAPK cascade can induce ISOCE inactivation. Mos kinase is necessary and sufficient for oocyte maturation (Sagata, 1997).
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If MPF is directly responsible for ISOCE inhibition, then specifically blocking MPF activation should prevent ISOCE inactivation. We accomplished this by injecting a constitutively active Wee1 kinase (GST107Wee1), which phosphorylates MPF and blocks its activation (Mueller et al., 1995; Howard et al., 1999). Injection of Wee1 did not significantly alter the activation time course of either Mos or MAPK in response to Mos RNA injection (Fig. 5, D and E). However, Wee1 inhibited MPF activation (Fig. 5 F) and prevented GVBD. In these cells with high Mos and MAPK levels and no MPF activity, store depletion activated ISOCE (Fig. 5 F). This shows that MPF activation is necessary for ISOCE inactivation. Note that in Wee1-injected cells, it was possible to record from oocytes for a significantly longer period of time (up to 1.6 GVBDi or 10 h) because they did not undergo GVBD. Even at these latter time points (1.31.6 GVBDi), ISOCE was clearly detectable in response to store depletion at levels only slightly lower than those in oocytes. Oocytes injected with Wee1 alone (without Mos injection), had ISOCE levels similar to those of control oocytes (1 ± 0.09, n = 4), and no detectable MPF or MAPK activation, indicating that Wee1 does not affect ISOCE. Together, these data demonstrate that MPF activation is necessary for ISOCE inactivation.
Examples of single-cell data from Mos- and Wee1/Mosinjected cells are shown in Fig. 6. The oocyte had a robust ISOCE signal and no detectable Mos, MAPK, or MPF (Fig. 6 A). In contrast, a Mos-injected cell showed complete ISOCE inactivation, and high MPF and P-MAPK levels (1.3- and 1.6-fold egg levels, respectively) (Fig. 6 B). Ectopic GSTMos (Fig. 6 B, arrowhead) was 13.1-fold higher than endogenous Mos levels found in eggs (Fig. 6 B, arrow). No endogenous Mos protein was detected in this cell (Fig. 6 B, arrow). A representative Wee1-preinjected cell (Fig. 6 C) showed levels of ectopic Mos and P-MAPK (8.7- and 0.76-fold egg levels, respectively) similar to a Mos-injected cell. However MPF kinase was not significantly activated (0.2-fold egg level), and ISOCE was readily induced in response to store depletion (Fig. 6 C). This shows that despite the activation of both Mos and P-MAPK for extensive time periods, ISOCE does not inactivate in the absence of MPF activity.
MPF blocks the coupling between store depletion and SOCE activation
We next investigated the mechanism of MPF action in mediating SOCE inactivation. MPF could be either blocking Ca2+ influx through SOCE channels, or it could be inhibiting the coupling mechanism between store depletion and SOCE. The molecular identity of the SOCE channel remains unclear, therefore we could not directly test whether MPF phosphorylates SOCE channels. However, by altering the timing of SOCE versus MPF activation, we could determine the target of MPF action. Our rational was to activate ISOCE before MPF levels increased. If MPF phosphorylates SOCE channels and blocks Ca2+ influx, then preactivated ISOCE will be inhibited concurrently with MPF activation. Alternatively if MPF blocks the coupling mechanism, then preactivated ISOCE will not be affected by MPF activation.
We activated ISOCE shortly before GVBD in oocytes treated with progesterone, and continuously recorded ISOCE until, and past, GVBD occurrence (Fig. 7 B). GVBD in this cell was apparent 35 min after store depletion (Fig. 7 B). However, ISOCE remained active with no significant inhibition, even
20 min after GVBD (Fig. 7 B). We confirmed that GVBD appearance was coupled to the characteristic increase in MPF activity (Fig. 7 B), indicating that 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) injection, which was necessary to measure ISOCE, did not interfere with MPF activation. Note that although ISOCE remained activated past GVBD in Fig. 7 B, there was a gradual inhibition in the current over time. Recording ISOCE from a control oocyte for the same prolonged time, shows a similar gradual ISOCE inhibition (Fig. 7 A), indicating that the gradual decline in ISOCE was not due to MPF activation. This shows that once SOCE was activated, increasing MPF levels were not able to inactivate ISOCE. This indicates that MPF does not block Ca2+ influx through SOCE channels. However, if MPF is activated before store depletion, SOCE cannot be activated (Fig. 1 F, Fig. 4, B and C, and Fig. 6 B). Therefore, MPF inhibits the coupling mechanism between store depletion and SOCE activation.
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Interestingly, thapsigargin-induced ISOCE levels were consistently smaller after GVBD as compared with control oocytes (Fig. 8, compare A and B). ISOCE levels measured 1015 min after GVBD in cells treated with thapsigargin, were at levels 40 ± 3% of control oocyte levels (Fig. 8 C). If ISOCE was recorded 1530 min after GVBD occurred, ISOCE levels declined further, to 17 ± 3% of oocyte levels (Fig. 8 C). Therefore, preactivated ISOCE decreases gradually after GVBD.
What is the reason for this gradual downregulation of ISOCE after GVBD? In oocytes and other cell types, Ca2+ store depletion results in the activation of SOCE. Therefore, a positive signal (i.e., the coupling species) is induced resulting in SOCE activation. Once the Ca2+ store is refilled, SOCE is downregulated. This argues for the presence of a constitutive mechanism that downregulates ISOCE in the absence of the coupling species. As shown above, MPF activation blocks coupling between store depletion and SOCE activation. This would eliminate the positive signal to maintain active SOCE and would lead to SOCE downregulation. Therefore, the gradual inhibition of preactivated ISOCE after GVBD is likely due to the block of Ca2+ storeSOCE coupling by MPF and constitutive downregulation of SOCE.
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Discussion |
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Inactivation of ionic currents during M phase is not a peculiar property of ISOCE in Xenopus oocytes, and has been described for other membrane proteins and cell types. For example, in Xenopus oocytes, amino acid and phosphate transporters are downregulated, but not completely inactivated, during oocyte maturation (Richter et al., 1984). In the same respect, surface levels of the Na+K+ATPase (Schmalzing et al., 1990) and of ß1 integrin (Muller et al., 1993) decrease during Xenopus oocyte maturation. Recently, several exogenously expressed channels have been shown to be downregulated during oocyte maturation (Shcherbatko et al., 2001). We have obtained similar results with exogenously expressed glutamate receptor (GluR3; unpublished data). In contrast, the levels of Ca2+-activated Cl- current (ICl,Ca) do not decrease during maturation (Machaca and Haun, 2000). Therefore, there is extensive, specific regulation of membrane proteins during Xenopus oocyte maturation. Regulation of membrane proteins during M phase is not limited to Xenopus oocytes. SOCE has been shown to inactivate during mitosis of HeLa cells (Preston et al., 1991), and membrane trafficking in general is inhibited during mammalian cell mitosis (Warren, 1993). Therefore, determining the molecular mechanisms controlling SOCE inactivation during Xenopus oocyte maturation provides valuable insights into the regulation of plasma membrane protein levels during M phase.
Our results argue that activation of MPF leads to uncoupling of store depletion from SOCE. Several lines of evidence support this conclusion. During progesterone-induced maturation, ISOCE inactivation at GVBD coincides with MPF activation (Figs. 1 and 2). Furthermore, regardless of the method by which we induced oocyte maturation (progesterone, cyclin B1, or Mos injection), every cell in which ISOCE was inactivated had high MPF activity. In addition, cells with low MPF activity always activated ISOCE in response to store depletion (Figs. 36). Out of 155 individual cells from which we recorded ISOCE and measured kinase activity, not a single cell deviated from these criteria. Finally, Mos injection did not lead to SOCE inactivation when MPF was inhibited (Fig. 5 F). Mos injection activates the MAPK cascade (Fig. 5 B), and leads to the activation of other signal transduction cascades important for oocyte maturation. Specifically blocking MPF activation under these conditions with Wee1, reversed ISOCE inactivation. This demonstrates that MPF activation during oocyte maturation is necessary for ISOCE inactivation.
Is MPF activation sufficient for ISOCE inactivation? Progesterone (Figs. 1 and 2) and Mos RNA injection data (Fig. 5 C) argue that it is. Under these conditions, the time course of MPF activation paralleled that of ISOCE inhibition. In contrast, rapid activation of MPF with cyclin B1 injection revealed a delay before GVBD and SOCE inactivation (Fig. 3 A). As discussed above this time lag could be an artifact of ectopic MPF activation, and not a true reflection of endogenous MPF activity. Two pools of p34cdc2 kinase exist in the immature Xenopus oocyte, an inactive preMPF pool and a 10-fold excess-free p34cdc2 protein pool (Kobayashi et al., 1991). Progesterone and cyclin B1 likely activate the two p34cdc2 pools differentially, which would explain the delay in GVBD after cyclin B1 injection. Nonetheless, after progesterone, Mos, or cyclin B1 injection, ISOCE inactivation correlates temporally with GVBD. As GVBD is a target for MPF, we argue that MPF activation is sufficient for ISOCE inactivation.
In contrast to the MPF requirement for SOCE inactivation, we did not observe any correlation between ISOCE levels and P-MAPK and Mos levels (Figs. 36). This indicates that neither Mos kinase nor the MAPK cascade plays a direct role in ISOCE inactivation.
MPF blocks coupling of store depletion to SOCE activation
Activation of MPF before Ca2+ store depletion blocks SOCE activation. This was true whether MPF was activated by progesterone treatment (Figs. 1 and 2), Mos RNA injection (Figs. 5 and 6), or cyclin B1 protein injection (Figs. 3 and 4). In contrast, when MPF was induced after SOCE activation, it was unable to block ISOCE (Figs. 7 and 8). This indicates that MPF blocks SOCE activation and not the current through SOCE channels. MPF could uncouple store depletion from SOCE activation by inhibiting the coupling species. Alternatively, MPF could target SOCE channels and prevent them from responding to the coupling species, without inhibiting the coupling species. In either case, our results demonstrate that MPF blocks coupling of Ca2+ store depletion to SOCE activation. Further investigations of the downstream targets of MPF will provide critical insights into the identity of the elusive coupling mechanism between store depletion and SOCE activation.
MPF is a member of the cyclin-dependent kinase family or cell cycle regulators. Cyclin-dependent kinases are master regulators of entry and progression through both mitosis and meiosis (Morgan, 1997). In that capacity, MPF controls various cellular processes crucial for meiotic progression. For example, MPF induces GVBD and stabilizes Mos kinase, which is a component of CSF (Palmer and Nebreda, 2000). CSF is required to maintain metaphase II arrest until fertilization (Masui, 1991). Our results indicate that SOCE inactivation can now be added to the list of cellular processes driven by MPF. Interestingly, MPF has recently been shown to regulate Ca2+ oscillations at fertilization in ascidian eggs (Levasseur and McDougall, 2000). The differentiation of Ca2+ signaling pathways is an important element of oocyte maturation. The fact that MPF regulates both Ca2+ release and Ca2+ influx in oocytes from different species strongly supports the idea that Ca2+ signaling is tightly controlled and functionally relevant during meiosis.
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Materials and methods |
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Electrophysiological methods
The ISOCE was measured as previously described (Machaca and Haun, 2000). In brief, SOCE was activated by depletion of intracellular Ca2+ stores with either ionomycin (10 µM) or thapsigargin (1 µM for 3 h). Oocytes were injected with 7 nmoles BAPTA before store depletion, to buffer intracellular Ca2+ rise and block the endogenous Ca2+-activated Cl- current (ICl,Ca), which would otherwise mask the ISOCE. Assuming an oocyte volume of 1 µl, this would result in a final concentration of
7 mM BAPTA, which completely blocks ICl,Ca. ISOCE was typically measured at the hyperpolarizing potential of -140 mV. This is because we wanted to maximize ISOCE, to most accurately determine the level of inactivation. Xenopus oocytes or eggs were voltage clamped with two microelectrodes by the use of a GeneClamp 500 (Axon Instruments, Inc.). Electrodes were filled with 3 M KCl and had resistances of 0.52 M
. Voltage stimulation and data acquisition were controlled using Curcap30 (Bill Goolsby, Emory University, Atlanta, GA). Current data was filtered at 10 kHz, digitized, and analyzed using Origin® software (Microcal Software, Inc.). Some experiments used 30Ca or 70Mg solutions (30Ca: 55 mM NaCl, 30 mM CaCl2, 10 mM Hepes, pH 7.4; 70Mg: 70 mM MgCl2, 10 mM Hepes, pH 7.4).
MPF kinase assay
MPF kinase activity was measured by lysing an oocyte in 50 µl extraction buffer (80 mM ß-glycerophosphate, 20 mM Hepes, pH 7.5, 20 mM EGTA, 15 mM MgCl2, 1 mM sodium vanadate, 50 mM NaF, 1 mM DTT, 10 µg/ml aprotinin, 50 µg/ml leupeptin, 1 mM PMSF). Lysates were centrifuged at 16,000 g for 5 min, and the supernatant was stored at -70°C. MPF activity was measured using the SignaTECTTM cdc2 kinase assay system (Promega), according to manufacturer's recommendations. This assay measures MPF activity in terms of the phosphorylation of a specific substrate, i.e., a biotinylated peptide derived from histone H1 (PKTPKKAKKL).
Western blotting
P-MAPK, total MAPK, and Mos protein levels were measured by Western blotting. Lysates were prepared as for the MPF assay, and protein concentration was measured using the Bradford assay (Bio-Rad Laboratories). SDS-PAGE gels were loaded with equal amounts of protein, transferred onto Immobilon P-PVDF membranes (Millipore), and probed with the appropriate primary antibody: antiphospho-MAPK and antitotal MAPK (Cell Signaling Technology) or anti-Mos antibodies (Santa Cruz Biotechnology, Inc.). This was followed by the appropriate HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) and detected using the ECL Plus Western blot detection system (Amersham Pharmacia Biotech). Westerns were visualized using a STORMTM system (Molecular Dynamics), which allowed a linear response to be measured from the band intensity on the Western blot. This was crucial for accurate quantification of P-MAPK and Mos protein levels. To control for variability between different trials, a large batch of lysate from mature eggs was prepared for both P-MAPK and Mos. This control lysate was loaded on each gel used for quantifying P-MAPK and Mos levels. The same lysate was used for all P-MAPK and Mos gels, respectively, which provided a consistent internal control for all the experiments. Using this method we could faithfully reproduce (with <15% error margin) P-MAPK and Mos protein levels from the same sample in different experiments.
Reagents
The following reagents were used: ionomycin and PD98059 (Calbiochem); thapsigargin (Molecular Probes Inc.); progesterone, salts, and other reagents (Sigma-Aldrich). The following constructs were provided by A.M. MacNicol (University of Arkansas Medical Science): GSTMos; GST107Wee1, which encodes a constitutively active Wee1 kinase (Mueller et al., 1995; Howard et al., 1999); and a His6-tagged 87cyclin B1 protein (Kumagai and Dunphy, 1995; Howard et al., 1999). Oocytes were injected with 10 ng of the GSTMos and GST107Wee1 RNAs and
200 pg
87cyclin B1 protein.
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Footnotes |
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Acknowledgments |
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This work was supported by a grant from the National Institutes of Health (GM-61829).
Submitted: 11 October 2001
Revised: 15 November 2001
Accepted: 19 November 2001
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References |
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