©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Dual Specificity Phosphatase VHR Activates Maturation Promotion Factor and Triggers Meiotic Maturation in Xenopus Oocytes (*)

Pilar Aroca , Donald P. Bottaro , Toshio Ishibashi (§) , Stuart A. Aaronson (¶) , Eugenio Santos (**)

From the (1) Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Bacterially expressed, dual specificity phosphatase VHR protein induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes, albeit with slower kinetics than that observed in progesterone- or insulin-induced maturation. A mutant VHR protein missing an essential cysteine residue for its in vitro phosphatase activity completely lacked activity in injected oocytes. VHR injection done in conjunction with progesterone or insulin treatment resulted in highly synergized GVBD responses showing much faster kinetics than that produced by VHR or either hormone alone. The delayed kinetics of VHR-induced GVBD and the synergistic responses obtained in the presence of hormones suggested that this protein may be promoting G/M transition by weakly mimicking the action of cdc25, the dual specificity phosphatase that physiologically activates the maturation promotion factor.

Various experimental observations are consistent with such a role for the injected VHR in oocytes: 1) as opposed to hormone-treated oocytes, histone H1 kinase activation is not preceded by MAPK activation in the process of GVBD in VHR-injected oocytes; 2) incubation of purified VHR with highly concentrated cell-free extracts of untreated oocytes resulted in activation of histone H1 kinase activity in the lysates; 3) coinjection of VHR with activated Ras proteins resulted in synergized responses, faster than those produced by either protein alone; 4) coinjection of VHR with the purified amino-terminal SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (which blocks insulin-induced GVBD) does not affect VHR-induced maturation.

The biological actions of VHR in oocytes clearly distinguish it from other dual specificity phosphatases, which have shown inhibitory effects when tested in oocytes. We speculate that VHR may represent a dual specificity phosphatase responsible for activation of cdk-cyclin complex(es) at a still undetermined stage of the cell cycle.


INTRODUCTION

The process of signal transduction from surface tyrosine kinase receptors to the nucleus is mainly regulated by mechanisms of reversible protein phosphorylation and dephosphorylation. Increasing attention has been focused recently on the family of protein tyrosine phosphatases involved in signaling pathways and regulation of cell proliferation (1, 2) . Dual specificity phosphatases constitute a novel subfamily of protein tyrosine phosphatases characterized by their ability to dephosphorylate not only Tyr but also Ser/Thr residues in substrates in vitro. The first described member in this subfamily, cdc25, is known to activate the p34cdc2-cyclin kinase complex that regulates entry into the M phase, through dephosphorylation of specific Tyr and Thr residues (3) . A number of other dual specificity phosphatases have been recently described (4-10). While some of these have been shown to dephosphorylate MAP() kinase in vivo or in vitro(10, 11, 12) , others appear to form complexes with cdk molecules involved in regulating the cell cycle (reviewed in Ref. 13).

VHR is a dual specificity phosphatase recently isolated in our laboratory by means of expression cloning (14) . Searches of available data bases have failed to identify significant similarity between VHR and other protein tyrosine phosphatases, except for a motif, HCXXXXGR, which seems to constitute the catalytic core common to all protein tyrosine phosphatases (9, 14, 15) . In fact, cdc25 is more similar in sequence to VH1 and VHR than to the other phosphatases (16) . Also, in contrast to most other members of the dual specificity protein tyrosine phosphatase subfamily, VHR exhibits broad substrate specificity in vitro(14) . No physiological role is yet known for this newly identified protein tyrosine phosphatase.

Xenopus oocytes are arrested at the G/M transition of the first meiotic prophase. Progesterone or insulin induces these cells to enter metaphase and undergo meiotic maturation involving germinal vesicle breakdown (GVBD), in a process that involves significant changes in cellular phosphorylation (17, 18) . Most of the phosphorylations associated with GVBD are triggered by activation of maturation promoting factor (MPF), a universal regulator of M phase transition in eukaryotic cells (19, 20) . MPF is is a complex composed of p34cdc2 (a serine-threonine protein kinase) and B cyclin (18, 19, 20) whose kinase activity is up-regulated by cdc25, a dual specificity phosphatase (3) . Recent evidence indicates also that MAP kinase kinase and MAP kinase are essential elements in the cascade of phosphorylations culminating in MPF activation and oocyte maturation (21, 22).

Xenopus oocytes possess at least two independent pathways of MPF activation through phosphorylation. In one, progesterone leads to decreased adenylate cyclase activity with a resulting drop in overall cAMP levels and protein kinase A-dependent phosphorylations. In the other, insulin or insulin-like growth factor-1 triggers a cascade of phosphorylations initiated by tyrosine phosphorylation of their receptors (17, 18) . Ras and other oncogenic proteins also have been shown to induce meiotic maturation when microinjected into Xenopus oocytes (21, 23, 24, 25, 26, 27, 28) . Several lines of evidence indicate that Ras proteins are essential components in insulin-induced maturation (29, 30, 31) . The universality of MPF and the possibility of activating it by microinjection of various oncogene products into Xenopus oocytes makes this a very useful experimental model for the analysis of the role of various cellular gene products in progression through the eukaryotic cell cycle. In the present study we utilized the Xenopus oocyte system in order to investigate possible in vivo function(s) of VHR.


EXPERIMENTAL PROCEDURES

Oocyte Preparation and Microinjection

Adult female Xenopus laevis were obtained from Nasco (Fort Atkinson, WI) and stimulated to ovulate by microinjecting 50 units of pregnant mare serum gonadotropin (Calbiochem) 3 days before oocyte extraction. Ovarian fragments were surgically removed from frogs anesthetized by hypothermia. Fully grown stage VI oocytes were manually dissected into ND-96 medium (5 mM Hepes, 96 mM NaCl, 1 mM MgCl, 2 mM KCl, 1.8 mM CaCl, pH 7.8, and 10 µg/ml each of penicillin and streptomycin sulfate). The oocytes were allowed to recover overnight in the same buffer before further treatment and were always maintained at 20 °C.

For induction of meiotic maturation, groups of 10-30 oocytes were incubated in ND-96 without KCl in the presence of progesterone (15 µM; Sigma) or insulin (7.5 µM; Sigma) or were microinjected into the cytoplasm with 30-60 nl of normal or mutant VHR (1 mg/ml, in 20 mM Tris-HCl, pH 7.5). Controls were microinjected with buffer. Meiotic maturation was assayed by scoring the disappearance of the nucleus (GVBD) in oocytes fixed with 10% trichloroacetic acid. In most cases, the absence of the nucleus correlated with the appearance of a white spot in the animal pole.

VHR Protein Expression and Purification

Plasmid pGEX-VHR, a pGEX-KT-derived expression vector (32) , was used to express wild-type and mutant VHR as a fusion protein with glutathione S-transferase (GST). Expression and purification of GST-VHR were essentially as previously described (14, 33) . GST-VHR and GST-VHR (C124S) were >98% pure as determined by Coomassie Blue staining of SDS-polyacrylamide gels. VHR proteins obtained by thrombin cleavage of purified GST fusion protein as described elsewhere (14) were then dialyzed extensively against 20 mM Tris-HCl, pH 7.5, and stored at high protein concentration at -80 °C. No loss of enzymatic activity occurred during long term storage under those conditions. For microinjection, the proteins were used at a concentration of 1 mg/ml in all assays.

Preparation of Oocyte Lysates

Lysates of VHR-microinjected oocytes were prepared as previously described (21) . Oocytes were homogenized in ice-cold buffer (20 µl/oocyte) containing 20 mM Hepes, pH 7.4, 10 mM EGTA, 15 mM MgCl, 1 mM dithiothreitol, 100 mM -glycerophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin and leupeptin.

Concentrated, cell-free lysates from untreated oocytes for in vitro assays were prepared essentially as previously described (34), with some minor modifications. Stage VI oocytes were washed 10 times in cytostatic factor extraction buffer (CSF-XB, 100 mM KCl, 0.1 mM CaCl, 2 mM MgCl, 10 mM Hepes-potassium, pH 7.7, 50 mM sucrose, 5 mM EGTA, pH 7.7) and then twice in the same buffer containing protease inhibitors (leupeptin, pepstatin, and chymostatin, 10 µg/ml each). The oocytes were then transferred to SW50.1Ti ultracentrifuge tubes (Beckman) containing CSF-XB buffer plus protease inhibitors and 100 µg/ml cytochalasin B and spun at 1000 rpm at room temperature for 60 s and then 30 s at 2000 rpm. After removing all CSF-XB, a crushing spin was performed at 30,000 rpm for 20 min at 4 °C. The cytoplasmic layer was then collected by using a needle and syringe via side puncture. Protease inhibitors (leupeptin, pepstatin, and chymostatin, 10 µg/ml each), 100 µg/ml cytochalasin B, 0.05 volume of CSF-energy mix (150 mM creatine phosphate, 20 mM ATP, 2 mM EGTA, pH 7.7, 20 mM MgCl) and 0.2 M sucrose were then added to this cytoplasmic fraction. The final protein concentration of these extracts was approximately 80 mg/ml.

Protein Kinase Assays

For measurement of GVBD-associated kinases in VHR-microinjected oocytes, histone H1 kinase was assayed essentially as described elsewhere (21) . 5 µl of clarified extracts were incubated in a final volume of 15 µl with 3 µg of exogenous histone (type III-S calf thymus; Sigma), 100 µM cold ATP, and 4 µCi of [-P]ATP (3000 Ci/mmol, Amersham Corp.) for 15 min at room temperature. Phosphorylated histone H1 was visualized by autoradiography after SDS-PAGE on 10-20% gels, and radioactivity in the histone H1 bands was quantitated on the whole gels with a PhosphorImager (Molecular Dynamics). Myelin basic protein (MBP) kinase assay was performed as described elsewhere (35) , but using 1 mg/ml of MBP peptide substrate (UBI) in the presence of protein kinase A inhibitor peptide (7 µM; Life Technologies, Inc.). 5 µl of oocyte extracts were incubated with 35 µl of reaction buffer at 30 °C for 10 min and then spotted on P81 filters (Whatman), washed, and counted using Biofluor as scintillation liquid.

Protein kinase activation in highly concentrated cell-free extracts incubated in the presence of exogenous VHR was assayed basically as follows. 6 µl of purified VHR protein (1 mg/ml) were incubated with 22 µl of extract prepared as described above and and 1 µl of ATP-regenerating buffer (20 µM creatine phosphate, 2 mM ATP, 5 µg/ml creatine phosphokinase; Sigma). After mixing on ice, extract samples were incubated at 21 °C, and equal aliquots were removed at different times and diluted with an equal volume of extraction buffer buffer B (EB; 80 mM -glycerophosphate, 20 mM EGTA, 15 mM MgCl in 10 mM Hepes-potassium, pH 7.7) before performing kinase assays.

Histone H1 kinase and MBP kinase assays were performed following described procedures (36) after diluting the samples 60 or 100 times, respectively, in buffer B. MAP kinase activation was also determined by assays of mobility shift in Western immunoblots (10% SDS-PAGE gels) done using anti-rat MAP kinase R2 antibodies (Upstate Biotechnology, Inc. catalog no. 06-182).


RESULTS

Microinjected VHR Protein Induces Slow Meiotic Maturation of Xenopus Oocytes

A purified, bacterially expressed 20-kDa polypeptide encoded by the VHR gene (14) was injected into full-grown X. laevis oocytes, and its biological effects were evaluated. The injected protein was highly stable and detectable in the cytosol of the injected oocytes using specific antibodies, even after 30 h of microinjection (not shown). Interestingly, we observed that injection of VHR resulted in GVBD after rather prolonged incubation times (Fig. 1A). Typically, the start of GVBD induced by VHR was significantly delayed (by about 10 h) as compared to progesterone or insulin. In addition, VHR-induced GVBD was variable in intensity, occasionally reaching 100% maturation, but most often remaining in the range 60-70% (Fig. 1A). The induction of GVBD by microinjected VHR was reproducible and dependent on its enzymatic activity since injection of buffer or an inactive mutant (Cys Ser) that lacks phosphatase activity (14) failed to produce maturation under similar experimental conditions (Fig. 1A).


Figure 1: Effect of purified VHR phosphatase on Xenopus oocyte maturation. A, induction of GVBD in Xenopus oocytes by microinjected VHR. Time course of GVBD in oocytes microinjected with purified, wild-type, and mutant (C124S) VHR protein, or treated with concentrations of insulin or progesterone producing fast, optimal induction of meiotic maturation. Symbols: , 50 ng purified wild-type VHR protein; , 50 ng of mutant C124S VHR-purified protein; , 7.5 µM insulin; , 20 µM progesterone. Data represented in this panel are the average of 10 separate experiments where the standard deviations was always lower than 20% of values plotted for each time point. B, synergism between injected VHR and hormones insulin or progesterone in the induction of Xenopus oocyte maturation. Time course of GVBD in oocytes treated with low concentrations of insulin or progesterone producing suboptimal (delayed) induction of meiotic maturation, alone or in the presence of microinjected, purified VHR protein. Symbols: , oocytes treated with 0.75 µM insulin alone; , oocytes treated with 0.2 µM of progesterone alone; , oocytes microinjected with 50 ng of VHR and incubated in the presence of 0.75 µM of insulin; , oocytes microinjected with 50 ng VHR and incubated in the presence of 0.2 µM progesterone. Results presented in this panel correspond to a representative experiment, and similar synergistic results were obtained in three additional, separate experiments.



The process of GVBD induced by VHR was also highly dependent on the concentration of VHR present in the injected oocytes. No GVBD was observed when less than 30 ng of purified VHR were injected (not shown).

Synergism between Hormones and VHR in Induction of GVBD in X. laevis Oocytes

Xenopus oocytes possess at least two distinct pathways that eventually converge in activation of the MPF kinase complex and subsequent GVBD. In one, progesterone down-regulates the oocyte adenylate cyclase activity and subsequent protein kinase A-dependent phosphorylations. In the other, insulin or insulin-like growth factor-1 triggers cascades of phosphorylation initiated by tyrosine phosphorylation of the specific receptor (17, 18) . Several lines of evidence indicate the participation of Ras in insulin-induced maturation (29, 30, 31) .

In order to determine if VHR could interact with components of any of these pathways, we microinjected VHR in oocytes that were also treated with either insulin or progesterone. As shown in Fig. 1B, microinjected VHR produced a marked acceleration in the rate of GVBD induced by either progesterone or insulin at a variety of concentrations of either hormone. The synergistic effect of VHR was most obvious when suboptimal concentrations of either hormone were used (Fig. 1B presents results obtained using 1/10 and 1/100 of the optimal concentrations for insulin and progesterone, respectively). The synergism between hormone-induced maturation and VHR is in contrast to the behavior shown by several other phosphatases, which block hormone-induced GVBD and/or kinase activation (6, 37) . The GVBD-inducing activity of VHR and its cooperation with both progesterone and insulin are consistent with a model whereby VHR might act at a late biochemical step, common to both progesterone- and insulin-initiated pathways of GVBD.

In Vivo Activation of Intracellular Kinases in VHR-microinjected Oocytes

It has been shown previously that cascades of phosphorylation involving activation of MAP kinase and the cdc2 kinase component of MPF are essential for the process of GVBD induced by insulin or progesterone (21, 22, 38) . We showed that microinjected Ras proteins can activate these kinases, even in the absence of protein synthesis, in a process in which the peak of activation of MAP kinase precedes the peak of activation of the p34cdc2 kinase component of MPF (19, 20), as measured as histone H1 kinase activity (21) . In fact, purified Ras proteins have been shown to activate MAP kinase in vitro when added to oocyte cell-free extracts (19, 38) .

We sought to analyze activation of early and late acting kinases during the process of GVBD induced by microinjected VHR. Fig. 2presents results obtained in experiments in which the kinetics of GVBD (Panel A), the kinetics of activation of the MAP kinase (Panels D and E) and the p34cdc2 kinase (assayed as histone H1 kinase activity, Panels B and C) were analyzed in parallel.


Figure 2: Kinetics of in vivo activation of histone H1 and MAP kinase in oocytes microinjected with VHR. Representative experiment determining the kinetics of GVBD (Panel A) and parallel activation of cytoplasmic kinases (histone H1 kinase, Panels B and C; MBP kinase, Panels D and E) in VHR-injected oocytes. Similar results were obtained in two more separate experiments. A, time course of GVBD in oocytes microinjected with 50 ng of VHR. B, histone H1 kinase assay in cytosolic extracts of oocytes microinjected with VHR (15 oocytes used per each time point). Kinase activity is shown in arbitrary units, normalized to the activity (PhosphorImager signal) detected in control untreated oocytes at time 0. The maximal H1 kinase peak is coincident with the beginning of oocyte maturation (see Panel A). For comparison purposes, control oocytes had 4314 PhosphorImager (Molecular Dynamics) units, with a blank of 186 PhosphorImager units for the same sample. C, autoradiogram of the same H1 kinase assays analyzed in Panel B, performed on a 4-20% PAGE-SDS minigel. Mobility position of histone H1 protein band is indicated by the arrow. D, MBP kinase in 5 µl of clarified extract (15 oocytes used per each time point), performed as described under ``Experimental Procedures.'' Results presented as -fold increase, normalizing all values to radioactivity incorporated in sample of control, untreated oocytes at time 0. Blank values substracted from all samples were obtained by performing the kinase assays in the absence of the exogenously added substrate. For comparison purposes, control untreated oocytes gave 5154 cpm, with a blank of 3528 cpm for the same sample. E, Western blot analysis with anti-MAP-2 kinase antibodies of the same cytosolic extracts of analyzed in Panel D.



As described for hormone-treated oocytes (19, 20, 21) , we observed that the rapid increase in H1 kinase activity coincided with the onset of oocyte maturation triggered by microinjected VHR (compare Fig. 2, A, with B and C). Also as described previously for hormone-matured oocytes (39) , H1 kinase activity of oocytes induced to mature by VHR peaked at GVBD and then fell to a minimum before gradually rising again, suggesting that the VHR-injected oocytes underwent a complete meiotic maturation process (39) .

The process of VHR-induced GVBD was also accompanied by the significant activation of MAP kinase activity that could be detected either by direct kinase assays of oocyte lysates (Fig. 2D) or by mobility-shift assays using specific antibodies against the 42-kDa MAP kinase molecule (Fig. 2E). In contrast to hormone-treated- or Ras-injected oocytes, in which the peak of MAP kinase activation clearly precedes histone H1 kinase activation (21) , the peak of MAP kinase activation in VHR-injected oocytes (Fig. 2C) occurred several hours after maximal histone H1 kinase activity was already achieved, coinciding, in fact, with a minimum in the histone H1 kinase activity (compare Fig. 2, B and C).

In Vitro Activation of Kinases by VHR Protein Exogenously Added to Cell-free Oocyte Lysates

The linkage of the GVBD-inducing ability of VHR to its phosphatase activity (Fig. 1) and the unusual kinetics of kinase activation accompanying this process (Fig. 2) suggested that VHR might be acting directly at the level of activation of MPF, mimicking in a sense the action of cdc25, another dual specificity phosphatase known to be the physiological activator of the p34cdc2 kinase activity in the MPF complex during this G/M transition (3, 19, 20) . In order to test this hypothesis, we to sought determine whether purified VHR could activate MPF kinase in vitro, when added to highly concentrated cell-free oocyte extracts.

Under experimental conditions similar to those used with Ras and Mos (11, 22, 38, 40), we observed that addition of purified VHR to highly concentrated oocyte lysates resulted in the rapid in vitro activation of the soluble cytosolic kinase activities (Fig. 3). In particular, histone H1 kinase showed significant activation very shortly after the start of incubation in the presence of VHR, reaching a maximum at 6-9 h (Fig. 3, A and B). The pattern of activation of MAP kinase activity paralleled the activation of histone H1 activity, with a maximum at 6-9 h, although it did not decline to base-line values afterward (Fig. 3C). Since it has been shown that MAPK can be a substrate for MPF (41, 42, 43) , we interpret these results as an indication that the MAP kinase activation observed may be due to the histone H1 kinase activity.


Figure 3: Assays of in vitro histone H1 and MAP kinase activities in oocyte cytosolic extracts incubated in the presence of VHR. Kinase assay activity of highly concentrated oocyte cytosolic extracts after incubation with VHR phosphatase at room temperature. Equal aliquots were removed at the indicated times and assayed for the kinases as described under ``Experimental Procedures.'' A, autoradiogram of histone H1 kinase activity analyzed using SDS-PAGE as described under ``Experimental Procedures.'' B, profile of histone H1 kinase activity detected using a PhosphorImager on the gel shown in Panel A. Kinase activity is shown in arbitrary units (as -fold increase), normalized to the activity (PhosphorImager signal) detected in control, untreated extracts at time 0. For comparison purposes, control extracts at time 0 of treatment yielded 12,108 PhosphorImager units, with a blank of 73 PhosphorImager units. C, MBP kinase activity of samples removed at the indicated times and assayed as described under ``Experimental Procedures.'' Control untreated extract: 7880 cpm, with a blank of 2834 cpm for the same sample.



Effect of GVBD Inducers and Blockers on Meiotic Maturation Produced by Microinjected VHR Protein

The above results implied a slow, direct activation of the MPF kinase by VHR, acting at a late biochemical step of the GVBD process, common to both progesterone- and insulin-initiated pathways. If that were the case, it would be expected that GVBD-promoting agents acting upstream of the point of convergence of progesterone and insulin pathways should also synergize with VHR-induced GVBD. However, GVBD-blocking agents acting upstream of such a convergence point would not be expected to inhibit VHR-induced GVBD.

We tested those predictions by coinjecting VHR with activated Ras (Lys-12) protein (21) (Fig. 4A), which induces GVBD by acting in the insulin pathway, upstream of MPF activation (29, 30, 31) . We also coinjected VHR with a peptide corresponding to the amino-terminal SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (Fig. 4B), which blocks insulin-induced GVBD (44) by acting at a step near the activated receptor tyrosine kinase and upstream of Ras activation by insulin. Fig. 4A shows that coinjection of activated Ras (Lys-12) and VHR proteins resulted in a significant acceleration of the onset of GVBD, as compared to injection with either VHR or Ras (Lys-12) alone. In contrast, the amino-terminal SH2 p85 peptide completely blocked insulin-induced GVBD (Fig. 4B) without affecting progesterone-induced GVBD (not shown). Of note, the kinetics of GVBD observed in oocytes coinjected with both VHR and the p85 SH2 peptide were no different from that of oocytes injected with VHR alone (Fig. 4B), indicating that the peptide could not inhibit maturation induced by VHR. All of our findings support a model (Fig. 5) whereby microinjected VHR weakly mimics the physiological phosphatase activity of cdc25, activating the MPF kinase complex and triggering the process of meiotic maturation in Xenopus oocytes.


Figure 4: Effects of inducers and blockers of GVBD on meiotic maturation promoted by VHR. A, effect of transforming Ras (Lys-12). Representative experiment showing time course of GVBD in oocytes microinjected with: , 50 ng of purified VHR; , 10 ng of transforming Ras (Lys-12) protein; [, 50 ng of VHR and 10 ng of transforming Ras. Similar results were obtained in two separate experiments. B, effect of the amino-terminal SH2 domain of the p85 subunit of of phosphatidylinositol 3-kinase (N-SH2 p85). Kinetics of GVBD in oocytes treated as follows: , incubated with 7.5 µM insulin alone; , microinjected with 30 ng of N-SH2-p85 of phosphatidylinositol 3-kinase and incubated in the presence of 7.5 µM insulin; , microinjected with 50 ng of VHR; , coinjected with both 50 ng of VHR and 30 ng of N-SH2-p85. Data presented for each time point correspond to the average of four separate experiments where the S.D. was always lower than 15% of values plotted here.




Figure 5: Tentative model for VHR action in Xenopus oocytes. GVBD in Xenopus oocytes can be driven through separate cascades of phosphorylation initiated by either progesterone (protein kinase A-dependent) or insulin (receptor tyrosine kinase-dependent) that eventually converge in activation of MPF kinase, the universal regulator of M phase transition in eukaryotic cells (19, 20). Activation of the MAP kinase kinase/MAP kinase cascade is a requirement for MPF activation in vivo (21, 22, 38), although MAP kinase can also be a substrate for retrophosphorylation by activated MPF (41-43). Ras proteins are essential components in insulin-induced maturation (29-31) and can also induce activation of MAP kinase, but not MPF, in oocyte lysates in vitro (38). Microinjected Ras proteins, even in the absence of protein synthesis, lead to GVBD through a process where MAP kinase activation precedes MPF kinase activation (21). A model explaining the results observed here postulates that VHR acts by mimicking the function of cdc25, the physiological phosphatase activator of MPF (3). This model predicts that agents acting upstream of cdc25 in the pathway of GVBD induction will synergize with microinjected VHR if they exert a positive regulatory effect (progesterone, insulin, microinjected Ras) but will not inhibit the VHR effect if they have a negative regulatory role (N-SH2-PI3K). Another prediction is that the microinjected VHR must localize to a compartment where its phosphatase activity exerts a positive regulatory role instead of the inhibitory effect that would be expected if its high phosphatase activity was exerted randomly throughout the oocyte.




DISCUSSION

In Xenopus oocytes, factors that control cell proliferation and/or regulate the cell cycle (including hormones, growth factors and their receptors, and oncogenes) can be readily analyzed functionally. Oocytes offer models for adenylate cyclase and protein kinase A-dependent (progesterone-induced GVBD) and receptor tyrosine kinase-dependent (insulin or insulin-like growth factor-1-induced GVBD) signaling pathways leading to meiotic maturation (17-20).

The distinct cascades of phosphorylation initiated by either progesterone (protein kinase A-dependent) or insulin receptor tyrosine kinase-dependent) eventually converge in the activation of MPF kinase activity, the universal regulator of M phase transition in eukaryotic cells (19, 20) (Fig. 5). Because the receptor tyrosine kinase-dependent and protein kinase A-dependent signaling pathways have been at least partially characterized, injection of suspected intermediates in some of these pathways may help pinpoint their sites of action in those signaling pathways (see Fig. 5 ).

Our results showed that the purified VHR dual specificity phosphatase protein induced GVBD in the microinjected oocytes. These results were not expected in view of recent reports showing that other tyrosine phosphatases or dual specificity phosphatases block the process of meiotic maturation or of activation of kinase cascades in Xenopus oocytes (6, 37) . GVBD induction by VHR was slow compared to maturation in the presence of physiological concentrations of progesterone or pharmacological doses of insulin. In addition, its ability to induce GVBD was dependent on the presence of large amounts of VHR, with no observable effects when less than 30 ng were injected. Nevertheless, the VHR effects were reproducible and specific since a phosphatase defective VHR mutant did not produce the effect. The slow kinetics and low rate of GVBD, as well as the need for high amounts of exogenous VHR, suggest that VHR-induced GVBD is mediated via its potential functional similarity with cdc25, the dual specificity phosphatase that physiologically promotes transition from G to M phase in Xenopus oocytes (19, 20) and can also induce quick GVBD when injected into the oocytes (45) .

The GVBD-inducing activity of VHR was also unexpected in view of the very high tyrosine phosphatase activity shown by this protein in vitro on a variety of substrates including, for example, the insulin receptor (14) . It is clear that after microinjection of VHR into the cytoplasm of oocytes, multiple potential targets for dephosphorylation exist between the point of hormone contact at the surface and the activation of MPF. In fact, dephosphorylation of one or more of such targets must account for the inhibitory effects on GVBD exhibited by other phosphatases such as 1B or CL100 (6, 37) . For example, microinjected phosphatase 1B (37) has been reported to dephosphorylate at least the insulin receptor and possibly other targets, while CL100 (6) dephoshorylated and inactivated MAP kinase, which is absolutely required for insulin induced GVBD (22) .

The fact that VHR did not block hormone-induced GVBD but actually potentiated it and that the pattern of overall phosphorylation was similar in injected and uninjected oocytes (not shown) indicate that the injected VHR is not indiscriminately exerting its phosphatase activity. These findings suggest that after injection, VHR is directed to a compartment where substrates whose dephosphorylation results in inhibition of GVBD are not accessible. The nucleus is one such candidate compartment, a possibility that can be tested when specific antibodies become available.

All of our findings are compatible with a model (see Fig. 5 ) in which the injected VHR protein, through a low degree of functional similarity to cdc25 phosphatase, activates the cdc2 complex of MPF, by faintly mimicking the effect of the physiological activator, cdc25. The slow kinetics and low rate of the GVBD induced by VHR would be compatible with such a hypothesis. The observation that VHR consistently activates the histone H1 kinase in vitro in oocyte extracts further supports this hypothesis. It is of note that exogenous Ras and Mos can only consistently activate MAP kinase, but not MPF kinase, in similar in vitro experiments with oocyte extracts (11, 38, 40) .

Even if VHR were distinct from cdc25 and most likely not normally involved in the process of G/M transition in oocytes, this mammalian dual specificity phosphatase may be a physiological component of the machinery involved in modulation of the kinase activity of cdk-cyclin complexes involved in different steps of the cell cycle. The existence of multiple forms of these complexes acting at different steps of the cell cycle has been recently reported (13, 46, 47) . For example, a dual specificity phosphatase (KAP) with significant sequence similarity to VHR has been recently shown to form complexes with cdk in mammalian cells (9) . Characterization of the effects of VHR exogenously expressed in transfected mammalian cells may help in determining whether such a hypothesis is correct.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Otolaryngology, University of Tokyo, Japan.

Current address: Mount Sinai Medical Center, New York, NY 10029.

**
To whom correspondence should be addressed: LCMB, NCI, NIH, Bldg. 37, Rm. 1D28, Bethesda, MD 20892. Tel.: 301-496-1070; Fax: 301-496-8479.

The abbreviations used are: MAP, microtubule-associated protein; GVBD, germinal vesicle breakdown; MPF, maturation promotion factor; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MBP, myelin basic protein.


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