DEVELOPMENTAL
BIOLOGY
139,42’7-431
(1990)
BRIEF NOTES In Viva Progesterone Regulation of Protein Phosphatase Activity in Xenopus Oocytes PATRICK CORMIER, ODILE MULNER-LORILLON, AND ROBERTBELLE Physiologic de .?aReproduction, CNRS UA 555, INRA, 4 place Jussieu, 75256 Paris ceclox 05, France Accepted January A,1990 Exogenous B casein, previously phosphorylated in vitro by protein kinase A and casein kinase II, was microinjected into Xenopus oocytes to monitor in viwo protein phosphatase activities. Phosphatase activities were 1.6 and 3.4 fmol/ min/oocyte, respectively, for p casein phosphorylated by casein kinase II and @casein phosphorylated by protein kinase A. Progesterone induced an early decrease (35% after 10 min) in phosphatase activity restricted to the protein kinase A sites of p casein. 0 1990 Academic Press, Inc. INTRODUCTION
Protein phosphorylation/dephosphorylation is known to play a major role in meiotic maturation of Xenopus oocytes. A decrease in the level of phosphorylation of putative maturation protein(s) termed Mp is believed to be sufficient for the resumption of meiosis (Masui and Clarke, 1979; Maller and Krebs, 1980; Ozon et aZ., 1987). It has been shown that inhibitor-l (Huchon et al, 1981) and inhibitor-2 (Foulkes and Maller, 1982) of protein phosphatase-1 inhibited progesterone induction of maturation, thus suggesting that protein phosphatase-1 is involved in Mp’s dephosphorylation. A decrease in protein phosphorylation has been described to occur 1 or 2 hr after progesterone treatment and has been related to inhibition of CAMP-dependent protein kinase (protein kinase A) mediated by CAMP decrease (Boyer et aZ., 1986; Ozon et al., 1987). In order to investigate protein phosphatase involvement in oocyte induction of maturation, we have microinjected into oocytes fi casein as an exogenous substrate which was previously phosphorylated on different sites by protein kinase A or casein kinase II (Mulner-Lorillon et cd, 1988). In this report we show that progesterone provokes a decrease in protein phosphatase activity in viva, thus indicating that protein phosphatase(s) can be under hormonal control in Xenopus oocytes. MATERIALS
AND METHODS
Materials Xenopus la&s adult females were purchased from CRBM Montpellier (France) and maintained under laboratory conditions. [y-32P]adenosine 5’-triphosphate (ATP g 3000 Ci/mmol) was supplied by Amersham (France). DEAE-Sepharose for chromatography was from Pharmacia (France). 6 casein and the catalytic
subunit of protein kinase A purified from beef heart were purchased from Sigma. Casein kinase II was prepared in the laboratory from Xenopus ovaries (MulnerLorillon et aZ., 1988). Phospho-inhibitor-l of protein phosphatase-1, the ATP, Mg-dependent protein phosphatase, was prepared as in Waelkens et al. (1987). Inhibitor-2 or modulator of ATP, Mg-dependent protein phosphatase was purified as described in Yang et al. (1981). Both inhibitors were a generous gift from Dr. J. Goris (Louvain, Belgium). Activity of both inhibitors toward dephosphorylation of phosphorylase a by-protein phosphatase-1, (or ATP, Mg-dependent phosphatase) was 700,000 U/ml (Waelkens et cd, 1987; Yang et al, 1981). Oocyte Preparation
and Microinjection
Ovaries were surgically removed from anaesthetized (MS 222) animals. Defolliculated full-grown oocytes of stage VI (Dumont, 1972) were isolated by dispase/collagenase treatment in medium A containing 88 mlM NaCl, 0.33 mMCa(N03)2, 1 mMKC1,0.41 mMCaC12, 0.82 mMMgS04, and 10 mMHepes, pH 7.6 (Belle et cd, 1986), and carefully selected under a stereotaxic microscope. Micropipets made from Drummond Microcaps were calibrated to distribute ten times 50 nl (Cormier et cd., 1989). Each oocyte was microinjected at the equator level with the appropriate solution in 50 mM Hepes, 1 mM DTT, pH 7.4. Progesterone treatment was performed by incubating oocytes in 1 pii! progesteronecontaining medium A. p Casein Labeling Labeling of 2 mg p casein was performed in 300-~1 final volume containing 1 p&l [y32JATP (0.2 &pmol), 5 mM MgC12, 5 mM 4-nitrophenyl-phosphate (pNPP), 50
427
0012-1606/90 $3.00 Copyright All rights
0 1990 by Academic Press. Inc. of reproduction in any form reserved.
428
DEVELOPMENTAL
BIOLOGY
mM Hepes, pH 7.4, and either casein kinase II (6 U/300 ~1: 1 U transfers 1 pmol phosphate per min) or the catalytic subunit of protein kinase A (300 Sigma U/300 ~1). The reaction started with the addition of ATP; after 2 hr incubation at room temperature with agitation, the incubations were diluted to 5 ml in buffer TK (50 mM Tris, 1 mM DTT, 2 mM EDTA, pH 7) and subjected to a 2-ml DEAE-Sepharose chromatography to separate the labeled fi casein from the enzyme and the pNPP. The chromatography was performed at 27 ml/hr and 4°C. After washing (15 ml TK, pH 6.5), fi casein was eluted from the column with 0.5 M NaCl, pH 6.5, in TK buffer. Fractions of 2.2 ml were collected, ,f3casein was eluted in fractions 3 to 7. The pooled fractions were dialyzed against TK diluted l:lOOO, then lyophilized. The lyophilisate was dissolved in 100 ~150 mM Hepes, 1 mM DTT, pH 7.5, aliquoted and stored at -20°C. p casein phosphorylation was quantified by trichloroacetic acid (TCA) precipitation of aliquots spotted on filter paper (Whatman 31 ET), as already described (Boyer et aL, 1983). The phosphorylation varied between 140 and 280 pmol of phosphate on p casein with the two enzymes. It was verified that no dephosphorylation occurred in vitro in an aliquot of p casein which remained at room temperature during the microinjection procedure; p casein in microinjection buffer was incubated at 20°C for various times. At 0, 10,30, and 60 min, radioactivity in fi casein was analyzed by electrophoresis and autoradiography. It was also verified that no loss of radioactivity associated with p casein occurred after homogenization; p casein was introduced into homogenates and analyzed by electrophoresis and autoradiography before and after the entire processing described below.
Determination of Remaining /3 Casein after Microinjecticrn of [/3-““P] Casein
The dephosphorylation in vivo was analyzed by microinjection of phosphorylated substrate in batches of three oocytes. After incubation for the indicated time, oocytes were homogenized in buffer (5 per 100 ~1; containing 50 mM Tris, 75 mM KCl, 50 mM NaF, 1 mM Na2HP04, 1 mM EDTA, 10 mM ATP, 0.1 mM orthovanadate, 5 mM 4-nitrophenyl phosphate and antiproteases), 0.3 mM L-1-tosylamide-2-phenyl ethyl chloromethyl ketone (TPCK), 0.3 mM Na-benzoyl-1-arginine methyl ester (BAME), 1.14 mM phenyl methyl sulfonyl fluoride (PMSF) 5 PM soybean trypsin inhibitor (STI), 1 mM benzamidine, and 0.04 U/ml aprotinine, pH 7.4. Cytosoluble fractions were then prepared by successive centrifugations: 8OOg for 5 min, 2OOOgfor 15 min (Beckman J6; rotor JA2) and 250,OOOg for 10 min (TLl 100; rotor TLA 100). All manipulations were carried at 4°C.
VOLUME
139,199O
Fractions were analyzed by SDS-polyacrylamide gel electrophoresis according to Laemmli (1970). Gels were stained, dried, and autoradiographed as described (Mulner-Lorillon et al, 1988). Densitometric determination was performed using an Shimadzu densitometerintegrator (Boyer et ah, 1986). Determination of the 32P Released after Microinjection of [p-““PI Casein
Oocytes were microinjected (three at a time) and after appropriate incubation time, independently homogenized (50 ~1 buffer per oocyte). Trichloroacetic acid (TCA) was immediately added (950 ~1) and the 32P-labeled inorganic phosphate, released from the /3 casein was separated from the precipitated protein by centrifugation: 4000g for 30 min. The radioactivities of the supernatant and of the pellet were measured in a liquid scintillation spectrometer (Cerenkov effect). Reproducibility of microinjections into oocytes was in the limit of ?30-50%. Under our experimental conditions the 32P release was directly proportional to the microinjected radioactivity. Therefore, total radioactivity in each homogenate fraction was determined by Cerenkov counting and corrections were made to standardize the counts of the TCA-insoluble and TCA-soluble fractions to the same total value corresponding to 25 fmol of 32P which was the amount of phosphate microinjected into oocytes in all experiments. RESULTS
A. Dephosphwylution
of [p-s2P] Casein in Vivo
When /3 casein, phosphorylated in vitro by protein kinase A or by casein kinase II, was microinjected into oocytes, the radioactivity remaining in the protein after 12 min incubation was, respectively, 6 and 36% (Fig. 1). No other phosphopeptides were detected on the gel indicating that the decrease of radioactivity in fl casein was not due to proteolysis but to dephosphorylation. In order to quantify the in vivo parameters of dephosphorylation, we have developed a procedure to determine the 32P released after b casein microinjection into oocytes (see Materials and Methods). Kinetics of dephosphorylation depending on the microinjected substrate are shown in Fig. 2. All further experiments were performed during a 4-min incubation. In vivo phosphatase activities analyzed in independent experiments showed reproducible (P < 0.001) differences between activity on casein kinase II site for /3 casein and activity on protein kinase A sites for p casein, which were, respectively, 1.6 and 3.4 fmol/min/oocyte (see Table I). Experiments were performed using okadaic acid (Bialojan and Takai, 1988) which is a potent inhibitor of
BRIEF
429
NOTES
TABLE IN VIVO PHOSPHATASE
ACTIVITY
1
AFTER
PROGESTERONE
STIMULATION
Phosphatase activity (fmol/min/oocyte) c p casein
*
cP
casein
'12
‘0
FIG. 1. Autoradiography of SDS-PAGE of cytosol, after microinjection of 32P-@casein into oocytes. (A) p casein phosphorylated by the catalytic subunit of protein kinase A. (B) j3 casein phosphorylated by casein kinase II. Batches of three oocytes were microinjected with 642 cpm fi casein and incubated for 12 min in medium A (T,J. The zero time (T,) phosphorylation was determinated by directly adding 32P-o casein to the cytosol. Remaining radioactivity after 12 min dephosphorylation was 39 cpm in A and 232 cpm in B, determined by densitometric analysis of the autoradiography. The arrow indicates the position of fl casein on the whole gel.
protein phosphatase-1, 2A, and 2B (Haystead et al., 1989). B casein loss of phosphate in vivo was inhibited more than 95% by coinjection of 12.5 PLM (pipet concentration) okadaic acid and 6 casein phosphorylated by protein kinase A or casein kinase II. Under our experimental conditions, oocytes remained morphologicaly healthy after okadaic acid microinjection and did not mature. This result confirms that proteolysis of /3 ca-
Substrate
Control (SD = 0.30) na = 9
@caseinPKA sites
3.36
fl caseinCKn site
1.57 (SD = 0.37) na = 5
2.13 (SD = 0.70)b na = 5 1.34 (SD = 0.25) na = 4
a Number of independent determinations. b Significant difference by Student t test. Note. In viva phosphatase activities (4 min dephosphorylation) on fi casein phosphorylated by protein kinase A (/3 easeinPKA sites) and @ casein phosphorylated by casein kinase II (/3 caseinCKn site) were determined as described under Materials and Methods.
sein was not responsible for the loss of phosphate from /3 casein and indicates that p casein phosphatase activity was okadaic acid-dependent. Specific protein phosphatase-1 (or ATP, Mg-dependent phosphatase) inhibitors were microinjected together with fi casein into oocytes. As shown in Table 2 no significant inhibition of @ casein dephosphorylation was observed after microinjection of phospho-inhibitor-l or inhibitor-2 (modulator) at an activity as high as 350,000 U/ml (pipet concentration). B. Eflect of Progesterone Activity in Vivo
on Phosphatase
Phosphatase activity in vivo was investigated during the course of oocyte maturation. Unexpectedly (see Introduction) a transitory decrease in activity was found at as early as 5 min and lasted at least 30 min after TABLE EFFECT
25-
10 min progesterone
OF INHIBITOR-~
AND
2 INHIBITOR-2
DEPHOSPHORYLATION
ON fi CASEIN
IN VIVO
Phosphatase activity (W of control) Microinjection
, p casein
0
5
10
PKAsites
15
20 Minutes
2. Kinetics of in wiwo p casein dephosphorylation. Oocytes were microinjected with %‘-@ casein (25 fmol szP) phosphorylated by casein kinase II (/3 caseinCKn si* ) or phosphorylated by protein kinase A (0 caseinPKA sites).The =P released at the indicated time was determined as described under Materials and Methods. Each point was performed in duplicate. FIG.
fl casein alone (control) fl easein + phosphoinhibitor-l @casein + inhibitor-2
Experiment
1
100 101 (SD = ‘7; n = 6) 109 (SD = 6; n = 6)
Experiment
2
100 88 (SD = 4; n = 6) 93 (SD = 5; n = 6)
Note. Oocytes were microinjected with [j3-32P] casein (25 fmol =P) phosphorylated by protein kinase A and protein phosphatase inhibitor-l or inhibitor-2 (modulator) at 350,000 U/ml in the pipet. /3 casein dephosphorylation was determined as described under Material and Methods by the release of 32P.Results of two independent experiments are expressed in percentage of control value with the standard deviation from six individual oocytes.
430
DEVELOPMENTAL
BIOLOGY
progesterone stimulation. The decrease was consistently observed for protein kinase A sites of /3 casein whereas no decrease occurred for casein kinase II site of p casein (Fig. 3). The effect of progesterone (1 p&f) on phosphatase activity was further tested in experiments at 10 min after hormonal treatment. A 35% decrease was reproducibly obtained for phosphatase activity on protein kinase A sites of p casein. Phosphatase activity on casein kinase II site of @ casein was stable after progesterone treatment (Table 1). Phosphatase activity was analyzed at 10 min for varying concentrations of progesterone. Figure 4 shows that phosphatase inhibition was dependent on progesterone concentration; ED5,, was 0.5 pJf corresponding to ED% for induction of maturation measured in parallel. DISCUSSION
Our results show measurements of in viva phosphatase activity in full-grown Xenopus oocytes. Phosphatase activity was different for @casein phosphorylated by protein kinase A or casein kinase II. Progesterone provoked a dose-dependent decrease in phosphatase activity in viva for protein kinase A sites of p casein and no change in phosphatase activity for casein kinase II site of /3 casein. Casein kinase II phosphorylates /3 casein in vitro on its single threonine-41 amino acid (Pinna et a& 1979; Tuazon et aL, 1979), whereas protein kinase A phosphorylates serine residues and not the threonine site phosphorylated by casein kinase II (Mulner-Lorillon et al, 1989). Therefore an in wivo protein phosphatase whose specificity is restricted to the protein kinase A sites of /3 casein is regulated by hormonal trigger as early as 5 min after stimulation. This is the first report of an in viva phosphatase change
.
4l
O-
5
15
30
60 Minutes
FIG. 3. Phosphatase activities measured after progesterone stimulation of meiotic maturation. Each point corresponds to single microinjected oocyte. In viva phosphatase activities (4 min dephosphorylation) on 0 casein phosphorylated by casein kinase II (@ casei#‘” siti) and fl casein phosphorylated by protein kinase A (/3 caseinPKA ‘im) were determinated as described under Materials and Methods.
VOLUME
139.1990
10-7
10-8 Progeslerone
FIG. 4. Dose-response curve for in viva phosphatase function of progesterone concentration. Phosphatase dephosphorylation) toward p casein phosphorylated tein kinase A was measured as described under Methods 10 min after progesterone stimulation. In indicated the number of single oocyte determinations. sent SEM.
10.5 (M)
activity as a activity (4 min in vitro by proMaterials and parentheses is The bars repre-
occurring after hormonal treatment. Protein phosphatases of types 1 and 2 have both been characterized in oocytes; it has been reported that oocytes contain two major protein phosphatase activities, namely 1 and 2A (Pondaven and Cohen, 1987). Andres et al. (1987) have demonstrated protein phosphatase activity in viva corresponding to protein phosphatase-1 and 2B; dephosphorylation of phosphorylase A and phosphorylase kinase was inhibited by coinjection into oocytes of inhibitor-2 indicating the presence of active protein phosphatase-1 in viva (Andres et aL, 1987). Using p casein as a substrate, no inhibition of protein phosphatase activity was found in our experiments after microinjection of inhibitor-2 or phospho-inhibitor-l. Therefore, p casein allowed detection of protein phosphatase activities in viva corresponding rather to type 2 phosphatases and it is likely that progesterone-induced decrease in fl casein phosphatase in viva was due to inhibition of a type 2 phosphatase suggesting that the dephosphorylation necessary for induction of maturation (see Introduction) could be mediated by protein phosphatase-2 decrease. We are very pho-inhibitor-l This work was fique (CNRS). (INRA), Minis&e
grateful to Dr. J. Goris for the generous gift of phosand modulator of ATP, Mg-dependent phosphatase. supported by Centre National de la Recherche ScientiInstitut National de la Recherche Agronomique de la Recherche et de 1’Enseignement Sup&ieur. REFERENCES
ANDRES, J. L., JOHANSON, J. W., and MALLER, J. L. (1987). Identification of protein phosphatase 1 and 2B as ribosomal protein S6 phosphatases in witro and in tivo. J. Bid Chem 262,14,389-14,393.
BRIEF
BELLE, R., MULNER-LORILLON, O., MAROT, J., and OZON, R. (1986). A possible role for Mg2+ ions in the induction of meiotic maturation of Xenopus oocyte. Cell. oifl 19,253-261. BIALOJAN, C., and TAKAI, A. (1988). Inhibitory effect of a marinesponge toxin, okadaic acid on protein phosphatases, specificity and kinetics. Biochem. J. 256,283-290. BOYER, J., BELLE, R., CAPONY, J. P., and OZON, R. (1983). Early increase of a 105,000-dalton phosphoprotein during meiotic maturation of Xenopus Levis oocyte. Biochimie 65,15-23. BOYER, J., ASSELIN, J., BELLE, R., and OZON, R. (1986). Progesterone and CAMP-dependent protein kinase regulate in wivo the level of phosphorylation of two proteins (M, 20,000) and (Mr 32,000) in Xenopus oocytes. Dev. BioL 113,420-428. CORMIER, P., MULNER-LORILLON, O., OZON, R., and BELLE, R. (1989). Involvement of protein kinase A and Casein kinase II in the in wivo protein kinase activities in prophase arrested Xenopus oocytes. Bioscience Rep. 9,351-358. DUMONT, J. N. (1972). Oogenesis in Xenopus la~vis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. MorphoL 136,153-180. FOULKES, J. G., and MALLER, J. L. (1982). In tivo actions of protein phosphatase inhibitor-2 in Xenopus oocytes. FEBS Lett. 150, 155-160. HAYSTEAD, T. A. J., SIM, A. T. R., CARLING, D., HONNOR, R. C., TSUKITANI, Y., COHEN, P., and HARDIE, D. G. (1989). Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature (London) 337,78-81. HUCHON, D., OZON, R., and DEMAILLE, J. G. (1981). Protein phosphatase-1 is involved in Xenom oocyte maturation. Nature (London) 294,358-359. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the bead of bacteriophage T4. Nature (London) 227, 680-685. MALLER, J. L., and KREBS, E. G. (1980). Regulation of oocyte maturation. Curr. Topics Cell. Reg. 16, 271-311.
NOTES
MASUI, Y., and CLARKE, H. (1979). Oocyte maturation.
431 Int. Rev. CytoL
57.185-282.
MULNER-LORILLON, O., MAROT, J., CAYLA, X., POUHLE, R., and BELLE, R. (1988). Purification and characterization of a casein-kinase-IItype enzyme from Xenopus laevis ovary. Biological effects on the meiotic cell division of full-grown oocyte. Eur. J. Biochem. 171, 107-117. MULNER-LORILLON, O., CREUZET, C., BELLE, R., and LOEB, J. (1989). The resistance to alkali treatment of the phosphorylation of fi casein versus (Ycasein is specific for casein kinase II. Arch Int. PhysioL B&him. 97,25-28. OZON, R., MULNER, O., BOYER, J., and BELLE, R. (1987). Role of protein phosphorylation in Xenopus oocyte meiotic maturation In “Molecular Regulation of Nuclear Events in Mitosis and Meiosis” (R. A. Schlegel, M. S. Halleck, and P. N. Rao, Eds.), pp. 111-130. Academic Press, New York. PINNA, L. A., DONELLA-DEANA, A., and MEGGIO, F. (1979). Structural features determinating the site specificity of a rat liver CAMP-independent protein kinase. Biochem. Biophys. Res. Commun. 87, 114-120. PONDAVEN, P., and COHEN, P. (1987). Identification of protein phosphatases-1 and 2A and inhibitor-2 in oocytes of the starfish Asterias rubens and Marthasterias glacialis. Eur. J. Biochem. 167, 135-140. TUAZON, T., BINGHAM, W., TRAUGH, A. (1979). Cyclic nucleotide-independent protein kinases from rabbit reticulocytes. Site-specific phosphorylation of casein variants. Eur. J. B&hem. 94,497-504. WAELKENS, E., GORIS, J., and MERLEVEDE, W. (1987). Purification and properties of polycation-stimulated phosphorylase phosphatase from rabbit skeletal muscle. J. BioL Chem. 262, 1049-1059. YANG, S. D., VANDENHEEDE, J. R., and MERLEVEDE, W. (1981). A simplified procedure for the purification of protein phosphatase modulator (inhibitor-2) from rabbit skeletal muscle. FEBS I,&. 132,293-295.
BIOLOGY
139,42’7-431
(1990)
BRIEF NOTES In Viva Progesterone Regulation of Protein Phosphatase Activity in Xenopus Oocytes PATRICK CORMIER, ODILE MULNER-LORILLON, AND ROBERTBELLE Physiologic de .?aReproduction, CNRS UA 555, INRA, 4 place Jussieu, 75256 Paris ceclox 05, France Accepted January A,1990 Exogenous B casein, previously phosphorylated in vitro by protein kinase A and casein kinase II, was microinjected into Xenopus oocytes to monitor in viwo protein phosphatase activities. Phosphatase activities were 1.6 and 3.4 fmol/ min/oocyte, respectively, for p casein phosphorylated by casein kinase II and @casein phosphorylated by protein kinase A. Progesterone induced an early decrease (35% after 10 min) in phosphatase activity restricted to the protein kinase A sites of p casein. 0 1990 Academic Press, Inc. INTRODUCTION
Protein phosphorylation/dephosphorylation is known to play a major role in meiotic maturation of Xenopus oocytes. A decrease in the level of phosphorylation of putative maturation protein(s) termed Mp is believed to be sufficient for the resumption of meiosis (Masui and Clarke, 1979; Maller and Krebs, 1980; Ozon et aZ., 1987). It has been shown that inhibitor-l (Huchon et al, 1981) and inhibitor-2 (Foulkes and Maller, 1982) of protein phosphatase-1 inhibited progesterone induction of maturation, thus suggesting that protein phosphatase-1 is involved in Mp’s dephosphorylation. A decrease in protein phosphorylation has been described to occur 1 or 2 hr after progesterone treatment and has been related to inhibition of CAMP-dependent protein kinase (protein kinase A) mediated by CAMP decrease (Boyer et aZ., 1986; Ozon et al., 1987). In order to investigate protein phosphatase involvement in oocyte induction of maturation, we have microinjected into oocytes fi casein as an exogenous substrate which was previously phosphorylated on different sites by protein kinase A or casein kinase II (Mulner-Lorillon et cd, 1988). In this report we show that progesterone provokes a decrease in protein phosphatase activity in viva, thus indicating that protein phosphatase(s) can be under hormonal control in Xenopus oocytes. MATERIALS
AND METHODS
Materials Xenopus la&s adult females were purchased from CRBM Montpellier (France) and maintained under laboratory conditions. [y-32P]adenosine 5’-triphosphate (ATP g 3000 Ci/mmol) was supplied by Amersham (France). DEAE-Sepharose for chromatography was from Pharmacia (France). 6 casein and the catalytic
subunit of protein kinase A purified from beef heart were purchased from Sigma. Casein kinase II was prepared in the laboratory from Xenopus ovaries (MulnerLorillon et aZ., 1988). Phospho-inhibitor-l of protein phosphatase-1, the ATP, Mg-dependent protein phosphatase, was prepared as in Waelkens et al. (1987). Inhibitor-2 or modulator of ATP, Mg-dependent protein phosphatase was purified as described in Yang et al. (1981). Both inhibitors were a generous gift from Dr. J. Goris (Louvain, Belgium). Activity of both inhibitors toward dephosphorylation of phosphorylase a by-protein phosphatase-1, (or ATP, Mg-dependent phosphatase) was 700,000 U/ml (Waelkens et cd, 1987; Yang et al, 1981). Oocyte Preparation
and Microinjection
Ovaries were surgically removed from anaesthetized (MS 222) animals. Defolliculated full-grown oocytes of stage VI (Dumont, 1972) were isolated by dispase/collagenase treatment in medium A containing 88 mlM NaCl, 0.33 mMCa(N03)2, 1 mMKC1,0.41 mMCaC12, 0.82 mMMgS04, and 10 mMHepes, pH 7.6 (Belle et cd, 1986), and carefully selected under a stereotaxic microscope. Micropipets made from Drummond Microcaps were calibrated to distribute ten times 50 nl (Cormier et cd., 1989). Each oocyte was microinjected at the equator level with the appropriate solution in 50 mM Hepes, 1 mM DTT, pH 7.4. Progesterone treatment was performed by incubating oocytes in 1 pii! progesteronecontaining medium A. p Casein Labeling Labeling of 2 mg p casein was performed in 300-~1 final volume containing 1 p&l [y32JATP (0.2 &pmol), 5 mM MgC12, 5 mM 4-nitrophenyl-phosphate (pNPP), 50
427
0012-1606/90 $3.00 Copyright All rights
0 1990 by Academic Press. Inc. of reproduction in any form reserved.
428
DEVELOPMENTAL
BIOLOGY
mM Hepes, pH 7.4, and either casein kinase II (6 U/300 ~1: 1 U transfers 1 pmol phosphate per min) or the catalytic subunit of protein kinase A (300 Sigma U/300 ~1). The reaction started with the addition of ATP; after 2 hr incubation at room temperature with agitation, the incubations were diluted to 5 ml in buffer TK (50 mM Tris, 1 mM DTT, 2 mM EDTA, pH 7) and subjected to a 2-ml DEAE-Sepharose chromatography to separate the labeled fi casein from the enzyme and the pNPP. The chromatography was performed at 27 ml/hr and 4°C. After washing (15 ml TK, pH 6.5), fi casein was eluted from the column with 0.5 M NaCl, pH 6.5, in TK buffer. Fractions of 2.2 ml were collected, ,f3casein was eluted in fractions 3 to 7. The pooled fractions were dialyzed against TK diluted l:lOOO, then lyophilized. The lyophilisate was dissolved in 100 ~150 mM Hepes, 1 mM DTT, pH 7.5, aliquoted and stored at -20°C. p casein phosphorylation was quantified by trichloroacetic acid (TCA) precipitation of aliquots spotted on filter paper (Whatman 31 ET), as already described (Boyer et aL, 1983). The phosphorylation varied between 140 and 280 pmol of phosphate on p casein with the two enzymes. It was verified that no dephosphorylation occurred in vitro in an aliquot of p casein which remained at room temperature during the microinjection procedure; p casein in microinjection buffer was incubated at 20°C for various times. At 0, 10,30, and 60 min, radioactivity in fi casein was analyzed by electrophoresis and autoradiography. It was also verified that no loss of radioactivity associated with p casein occurred after homogenization; p casein was introduced into homogenates and analyzed by electrophoresis and autoradiography before and after the entire processing described below.
Determination of Remaining /3 Casein after Microinjecticrn of [/3-““P] Casein
The dephosphorylation in vivo was analyzed by microinjection of phosphorylated substrate in batches of three oocytes. After incubation for the indicated time, oocytes were homogenized in buffer (5 per 100 ~1; containing 50 mM Tris, 75 mM KCl, 50 mM NaF, 1 mM Na2HP04, 1 mM EDTA, 10 mM ATP, 0.1 mM orthovanadate, 5 mM 4-nitrophenyl phosphate and antiproteases), 0.3 mM L-1-tosylamide-2-phenyl ethyl chloromethyl ketone (TPCK), 0.3 mM Na-benzoyl-1-arginine methyl ester (BAME), 1.14 mM phenyl methyl sulfonyl fluoride (PMSF) 5 PM soybean trypsin inhibitor (STI), 1 mM benzamidine, and 0.04 U/ml aprotinine, pH 7.4. Cytosoluble fractions were then prepared by successive centrifugations: 8OOg for 5 min, 2OOOgfor 15 min (Beckman J6; rotor JA2) and 250,OOOg for 10 min (TLl 100; rotor TLA 100). All manipulations were carried at 4°C.
VOLUME
139,199O
Fractions were analyzed by SDS-polyacrylamide gel electrophoresis according to Laemmli (1970). Gels were stained, dried, and autoradiographed as described (Mulner-Lorillon et al, 1988). Densitometric determination was performed using an Shimadzu densitometerintegrator (Boyer et ah, 1986). Determination of the 32P Released after Microinjection of [p-““PI Casein
Oocytes were microinjected (three at a time) and after appropriate incubation time, independently homogenized (50 ~1 buffer per oocyte). Trichloroacetic acid (TCA) was immediately added (950 ~1) and the 32P-labeled inorganic phosphate, released from the /3 casein was separated from the precipitated protein by centrifugation: 4000g for 30 min. The radioactivities of the supernatant and of the pellet were measured in a liquid scintillation spectrometer (Cerenkov effect). Reproducibility of microinjections into oocytes was in the limit of ?30-50%. Under our experimental conditions the 32P release was directly proportional to the microinjected radioactivity. Therefore, total radioactivity in each homogenate fraction was determined by Cerenkov counting and corrections were made to standardize the counts of the TCA-insoluble and TCA-soluble fractions to the same total value corresponding to 25 fmol of 32P which was the amount of phosphate microinjected into oocytes in all experiments. RESULTS
A. Dephosphwylution
of [p-s2P] Casein in Vivo
When /3 casein, phosphorylated in vitro by protein kinase A or by casein kinase II, was microinjected into oocytes, the radioactivity remaining in the protein after 12 min incubation was, respectively, 6 and 36% (Fig. 1). No other phosphopeptides were detected on the gel indicating that the decrease of radioactivity in fl casein was not due to proteolysis but to dephosphorylation. In order to quantify the in vivo parameters of dephosphorylation, we have developed a procedure to determine the 32P released after b casein microinjection into oocytes (see Materials and Methods). Kinetics of dephosphorylation depending on the microinjected substrate are shown in Fig. 2. All further experiments were performed during a 4-min incubation. In vivo phosphatase activities analyzed in independent experiments showed reproducible (P < 0.001) differences between activity on casein kinase II site for /3 casein and activity on protein kinase A sites for p casein, which were, respectively, 1.6 and 3.4 fmol/min/oocyte (see Table I). Experiments were performed using okadaic acid (Bialojan and Takai, 1988) which is a potent inhibitor of
BRIEF
429
NOTES
TABLE IN VIVO PHOSPHATASE
ACTIVITY
1
AFTER
PROGESTERONE
STIMULATION
Phosphatase activity (fmol/min/oocyte) c p casein
*
cP
casein
'12
‘0
FIG. 1. Autoradiography of SDS-PAGE of cytosol, after microinjection of 32P-@casein into oocytes. (A) p casein phosphorylated by the catalytic subunit of protein kinase A. (B) j3 casein phosphorylated by casein kinase II. Batches of three oocytes were microinjected with 642 cpm fi casein and incubated for 12 min in medium A (T,J. The zero time (T,) phosphorylation was determinated by directly adding 32P-o casein to the cytosol. Remaining radioactivity after 12 min dephosphorylation was 39 cpm in A and 232 cpm in B, determined by densitometric analysis of the autoradiography. The arrow indicates the position of fl casein on the whole gel.
protein phosphatase-1, 2A, and 2B (Haystead et al., 1989). B casein loss of phosphate in vivo was inhibited more than 95% by coinjection of 12.5 PLM (pipet concentration) okadaic acid and 6 casein phosphorylated by protein kinase A or casein kinase II. Under our experimental conditions, oocytes remained morphologicaly healthy after okadaic acid microinjection and did not mature. This result confirms that proteolysis of /3 ca-
Substrate
Control (SD = 0.30) na = 9
@caseinPKA sites
3.36
fl caseinCKn site
1.57 (SD = 0.37) na = 5
2.13 (SD = 0.70)b na = 5 1.34 (SD = 0.25) na = 4
a Number of independent determinations. b Significant difference by Student t test. Note. In viva phosphatase activities (4 min dephosphorylation) on fi casein phosphorylated by protein kinase A (/3 easeinPKA sites) and @ casein phosphorylated by casein kinase II (/3 caseinCKn site) were determined as described under Materials and Methods.
sein was not responsible for the loss of phosphate from /3 casein and indicates that p casein phosphatase activity was okadaic acid-dependent. Specific protein phosphatase-1 (or ATP, Mg-dependent phosphatase) inhibitors were microinjected together with fi casein into oocytes. As shown in Table 2 no significant inhibition of @ casein dephosphorylation was observed after microinjection of phospho-inhibitor-l or inhibitor-2 (modulator) at an activity as high as 350,000 U/ml (pipet concentration). B. Eflect of Progesterone Activity in Vivo
on Phosphatase
Phosphatase activity in vivo was investigated during the course of oocyte maturation. Unexpectedly (see Introduction) a transitory decrease in activity was found at as early as 5 min and lasted at least 30 min after TABLE EFFECT
25-
10 min progesterone
OF INHIBITOR-~
AND
2 INHIBITOR-2
DEPHOSPHORYLATION
ON fi CASEIN
IN VIVO
Phosphatase activity (W of control) Microinjection
, p casein
0
5
10
PKAsites
15
20 Minutes
2. Kinetics of in wiwo p casein dephosphorylation. Oocytes were microinjected with %‘-@ casein (25 fmol szP) phosphorylated by casein kinase II (/3 caseinCKn si* ) or phosphorylated by protein kinase A (0 caseinPKA sites).The =P released at the indicated time was determined as described under Materials and Methods. Each point was performed in duplicate. FIG.
fl casein alone (control) fl easein + phosphoinhibitor-l @casein + inhibitor-2
Experiment
1
100 101 (SD = ‘7; n = 6) 109 (SD = 6; n = 6)
Experiment
2
100 88 (SD = 4; n = 6) 93 (SD = 5; n = 6)
Note. Oocytes were microinjected with [j3-32P] casein (25 fmol =P) phosphorylated by protein kinase A and protein phosphatase inhibitor-l or inhibitor-2 (modulator) at 350,000 U/ml in the pipet. /3 casein dephosphorylation was determined as described under Material and Methods by the release of 32P.Results of two independent experiments are expressed in percentage of control value with the standard deviation from six individual oocytes.
430
DEVELOPMENTAL
BIOLOGY
progesterone stimulation. The decrease was consistently observed for protein kinase A sites of /3 casein whereas no decrease occurred for casein kinase II site of p casein (Fig. 3). The effect of progesterone (1 p&f) on phosphatase activity was further tested in experiments at 10 min after hormonal treatment. A 35% decrease was reproducibly obtained for phosphatase activity on protein kinase A sites of p casein. Phosphatase activity on casein kinase II site of @ casein was stable after progesterone treatment (Table 1). Phosphatase activity was analyzed at 10 min for varying concentrations of progesterone. Figure 4 shows that phosphatase inhibition was dependent on progesterone concentration; ED5,, was 0.5 pJf corresponding to ED% for induction of maturation measured in parallel. DISCUSSION
Our results show measurements of in viva phosphatase activity in full-grown Xenopus oocytes. Phosphatase activity was different for @casein phosphorylated by protein kinase A or casein kinase II. Progesterone provoked a dose-dependent decrease in phosphatase activity in viva for protein kinase A sites of p casein and no change in phosphatase activity for casein kinase II site of /3 casein. Casein kinase II phosphorylates /3 casein in vitro on its single threonine-41 amino acid (Pinna et a& 1979; Tuazon et aL, 1979), whereas protein kinase A phosphorylates serine residues and not the threonine site phosphorylated by casein kinase II (Mulner-Lorillon et al, 1989). Therefore an in wivo protein phosphatase whose specificity is restricted to the protein kinase A sites of /3 casein is regulated by hormonal trigger as early as 5 min after stimulation. This is the first report of an in viva phosphatase change
.
4l
O-
5
15
30
60 Minutes
FIG. 3. Phosphatase activities measured after progesterone stimulation of meiotic maturation. Each point corresponds to single microinjected oocyte. In viva phosphatase activities (4 min dephosphorylation) on 0 casein phosphorylated by casein kinase II (@ casei#‘” siti) and fl casein phosphorylated by protein kinase A (/3 caseinPKA ‘im) were determinated as described under Materials and Methods.
VOLUME
139.1990
10-7
10-8 Progeslerone
FIG. 4. Dose-response curve for in viva phosphatase function of progesterone concentration. Phosphatase dephosphorylation) toward p casein phosphorylated tein kinase A was measured as described under Methods 10 min after progesterone stimulation. In indicated the number of single oocyte determinations. sent SEM.
10.5 (M)
activity as a activity (4 min in vitro by proMaterials and parentheses is The bars repre-
occurring after hormonal treatment. Protein phosphatases of types 1 and 2 have both been characterized in oocytes; it has been reported that oocytes contain two major protein phosphatase activities, namely 1 and 2A (Pondaven and Cohen, 1987). Andres et al. (1987) have demonstrated protein phosphatase activity in viva corresponding to protein phosphatase-1 and 2B; dephosphorylation of phosphorylase A and phosphorylase kinase was inhibited by coinjection into oocytes of inhibitor-2 indicating the presence of active protein phosphatase-1 in viva (Andres et aL, 1987). Using p casein as a substrate, no inhibition of protein phosphatase activity was found in our experiments after microinjection of inhibitor-2 or phospho-inhibitor-l. Therefore, p casein allowed detection of protein phosphatase activities in viva corresponding rather to type 2 phosphatases and it is likely that progesterone-induced decrease in fl casein phosphatase in viva was due to inhibition of a type 2 phosphatase suggesting that the dephosphorylation necessary for induction of maturation (see Introduction) could be mediated by protein phosphatase-2 decrease. We are very pho-inhibitor-l This work was fique (CNRS). (INRA), Minis&e
grateful to Dr. J. Goris for the generous gift of phosand modulator of ATP, Mg-dependent phosphatase. supported by Centre National de la Recherche ScientiInstitut National de la Recherche Agronomique de la Recherche et de 1’Enseignement Sup&ieur. REFERENCES
ANDRES, J. L., JOHANSON, J. W., and MALLER, J. L. (1987). Identification of protein phosphatase 1 and 2B as ribosomal protein S6 phosphatases in witro and in tivo. J. Bid Chem 262,14,389-14,393.
BRIEF
BELLE, R., MULNER-LORILLON, O., MAROT, J., and OZON, R. (1986). A possible role for Mg2+ ions in the induction of meiotic maturation of Xenopus oocyte. Cell. oifl 19,253-261. BIALOJAN, C., and TAKAI, A. (1988). Inhibitory effect of a marinesponge toxin, okadaic acid on protein phosphatases, specificity and kinetics. Biochem. J. 256,283-290. BOYER, J., BELLE, R., CAPONY, J. P., and OZON, R. (1983). Early increase of a 105,000-dalton phosphoprotein during meiotic maturation of Xenopus Levis oocyte. Biochimie 65,15-23. BOYER, J., ASSELIN, J., BELLE, R., and OZON, R. (1986). Progesterone and CAMP-dependent protein kinase regulate in wivo the level of phosphorylation of two proteins (M, 20,000) and (Mr 32,000) in Xenopus oocytes. Dev. BioL 113,420-428. CORMIER, P., MULNER-LORILLON, O., OZON, R., and BELLE, R. (1989). Involvement of protein kinase A and Casein kinase II in the in wivo protein kinase activities in prophase arrested Xenopus oocytes. Bioscience Rep. 9,351-358. DUMONT, J. N. (1972). Oogenesis in Xenopus la~vis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. MorphoL 136,153-180. FOULKES, J. G., and MALLER, J. L. (1982). In tivo actions of protein phosphatase inhibitor-2 in Xenopus oocytes. FEBS Lett. 150, 155-160. HAYSTEAD, T. A. J., SIM, A. T. R., CARLING, D., HONNOR, R. C., TSUKITANI, Y., COHEN, P., and HARDIE, D. G. (1989). Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature (London) 337,78-81. HUCHON, D., OZON, R., and DEMAILLE, J. G. (1981). Protein phosphatase-1 is involved in Xenom oocyte maturation. Nature (London) 294,358-359. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the bead of bacteriophage T4. Nature (London) 227, 680-685. MALLER, J. L., and KREBS, E. G. (1980). Regulation of oocyte maturation. Curr. Topics Cell. Reg. 16, 271-311.
NOTES
MASUI, Y., and CLARKE, H. (1979). Oocyte maturation.
431 Int. Rev. CytoL
57.185-282.
MULNER-LORILLON, O., MAROT, J., CAYLA, X., POUHLE, R., and BELLE, R. (1988). Purification and characterization of a casein-kinase-IItype enzyme from Xenopus laevis ovary. Biological effects on the meiotic cell division of full-grown oocyte. Eur. J. Biochem. 171, 107-117. MULNER-LORILLON, O., CREUZET, C., BELLE, R., and LOEB, J. (1989). The resistance to alkali treatment of the phosphorylation of fi casein versus (Ycasein is specific for casein kinase II. Arch Int. PhysioL B&him. 97,25-28. OZON, R., MULNER, O., BOYER, J., and BELLE, R. (1987). Role of protein phosphorylation in Xenopus oocyte meiotic maturation In “Molecular Regulation of Nuclear Events in Mitosis and Meiosis” (R. A. Schlegel, M. S. Halleck, and P. N. Rao, Eds.), pp. 111-130. Academic Press, New York. PINNA, L. A., DONELLA-DEANA, A., and MEGGIO, F. (1979). Structural features determinating the site specificity of a rat liver CAMP-independent protein kinase. Biochem. Biophys. Res. Commun. 87, 114-120. PONDAVEN, P., and COHEN, P. (1987). Identification of protein phosphatases-1 and 2A and inhibitor-2 in oocytes of the starfish Asterias rubens and Marthasterias glacialis. Eur. J. Biochem. 167, 135-140. TUAZON, T., BINGHAM, W., TRAUGH, A. (1979). Cyclic nucleotide-independent protein kinases from rabbit reticulocytes. Site-specific phosphorylation of casein variants. Eur. J. B&hem. 94,497-504. WAELKENS, E., GORIS, J., and MERLEVEDE, W. (1987). Purification and properties of polycation-stimulated phosphorylase phosphatase from rabbit skeletal muscle. J. BioL Chem. 262, 1049-1059. YANG, S. D., VANDENHEEDE, J. R., and MERLEVEDE, W. (1981). A simplified procedure for the purification of protein phosphatase modulator (inhibitor-2) from rabbit skeletal muscle. FEBS I,&. 132,293-295.
