233
Biol Cell (1990) 69, 233-236
© Elsevier, Paris
PROTEIN PHOSPHATASE ACTIVITIES IN VlVO IN XENOPUS LAEVIS OOCYTE: INHIBITION BY OKADAIC ACID Patrick CORMIER, Odile MULNER-LORILLON, Ren60ZON and Robert BELLI~ Laboratoire de Physiologie de la Reproduction, UA CNRS 555, INRA, UPMC, 4 place Jussieu 75256 Paris cedex 05 - .France-.
SUMMARY
MATERIAL AND METHODS
Protein phosphatase activities were analyzed in vivo in Xenopus oocytes. The dephosphorylation of microinjected 8 casein was inhibited when the tumor promoter okadaic acid was microinjected into oocytes. Inhibition was dose dependent and reversible; 50 % of activity was recovered 15-30 minutes
Xenopus laevis adult females were purchased from CRBM Montpellier (France) and maintained under laboratory condition. [732p] adenosine 5'-triphosphate (ATP ~ 3,000 Ci/mmol) was supplied by Amersham France. casein and the catalytic subunit of protein kinase A, purified from beef heart, was purchased from Sigma. Casein kinase II was prepared in the laboratory from X e n o p u s ovaries [9]. 13 casein was phosphorylated in vitro by protein kinase A or by casein kinase II as already described [4]. Okadaic acid was a gift of Professor Daisuke Uemura (Shizuoka University, Japan). Isolated full-grown oocytes obtained as in [1] were microinjected at the equator level with 50 n l o f 50 mM Hepes, 1 mM DTT, pH 7.4 and with 20 to 30 fmoles of 32p-13 casein, alone or together with okadaic acid. Phosphate released from 32p-1~ casein was determined after homogenization of single oocytes in 50 ILl buffer containing 50 mM Tris, 75 mM KCI, 50 mM NaF, l mM Na 2HPO4, 1 mM EDTA, 10 mM ATP, 0.1 mM orthovanadate, 5 mM 4-nitrophenyl phosphate and antiproteases as described [4].
after microinjection. Key words : Protein phosphatase in vivookadaic acid- Xenopus oocyte-protein phosphatase 2,4.
INTRODUCTION Okadaic acid is a tumor promoter which is a potent inhibitor of protein phosphatases in vitro [2] and described as a new probe for the study of cellular regulation [3]. Okadaic acid triggers M-Phase promoting factor (MPF) in Amphibian [6,13] and in Starfish [11,12] oocytes. Moreover, in oocytes and in oocyte extracts, okadaic acid was shown to activate histone H1 kinase [5,11], which is related to the cell cycle control (cdc) gene cdc2 product [10]. We report here that okadaic acid inhibits protein ph,osphatase activities in vivo measured after microinjection into oocytes of B casein. Our results thus provide strong experimental support to interpret the in vivo effects of okadaic acid as an inhibition of specific protein phosphatases.
RESULTS 1. Okadaic acid inhibits protein phosphatase in vivo Protein phosphatase activities were measured in vivo by the dephosphorylation of microinjected 8 caseih previously phosphorylated in vitro by either casein kinase II or
234
P Cormier et al
(pipet concentrations) for casein kinase II and protein kinase A sites of phosphorylation. .>_ >,
.E •*--'
2. Kinetic of protein phosphatase activities after okadaic acid microinjection into oocyte.
O
0850 (D
O
¢Uo~ "t" e's (D O ca.
0
I
I
I
-7 -6 -5 Okadaic acid (log M) Figure 1. Dose-response curve for protein phosphatase activities in vivo as a function of okadaic acid concentration. Oocytes were microinjected with okadaic acid and 32p-13 casein (25 fmoles 3 2 p) previously phosphorylated by protein kinase A (PKA sites; 0-0) or casein kinase II (CK II site; x-x ). Protein phosphatase activities (4 min dephosphorylation ) towards 8 casein was measured as described in material and methods. Each point is expressed in % of control and is the mean of 6 single oocyte determinations with SD. protein kinase A. Protein phosphatase activities were respectively 1.6 fmoles/min/oocyte (SD--0.3; 7 experiments) and 3.4 fmoles/min/oocyte (SD=0.7; 11 experiments). When microinjected at a concerffration of 251.tM (pipet concentration), okadaic acid provoked the activation of MPF and germinal vesicle breakdown. At a concentration of 12.5 I.tM (pipet concentration), okadaic acid totally inhibited dephosphorylation of both casein kinase II site and p'rotein kinase A sites of 8 casein to respectively 0.03 and 0.14 fmoles/oocyte/min. The dose-response curve (figure 1) of phosphatase activity inhibition by okadaic acid shows ID 50 of respectively 1 and 2 p.M
When oocytes were first microinjected with okadaic acid (12.5 I.tM; a dose which was totally inhibitor) and then 15 or 120 minutes later microinjected with 13 casein, it was found that protein phosphatase activity was no more inhibited (figure 2). The time for half recovery of the initial value was around 15-30 minutes for both casein kinase II and protein kinase A phosphorylated sites of 13 casein. 100
•~
PI C
80 6o 4o ao
0. 0 control
0 min
15
min
120
min
time after okadaic acid Figure 2. Protein phosphatase activity in vivo after microinjection of okadaic acid into oocytes. 0 min, 15 min and 120 min after first okadaic microinjection, protein phosphatase activity was determined from the microinjection of 8 casein phosphorylated in vitro by casein kinase II (CK II sites) or by protein kinase A (PKA sites) during a 4 min incubation of oocytes at 20°C. Results represent the mean of 6 single oocyte determinations. After the first okadaic acid microinjection, protein phosphatases were allowed to recover for 15 and 120 minutes and then, 13 casein was microinjected together with .okadaic acid; protein phosphatase activities were again
Protein phosphatase activities in vivo in Xenopus laevis oocyte inhibition by okadaic acid
inhibited both for the casein kinase II and protein kinase A phosphorylated sites.
DISCUSSION Our results show that protein phosphatase activities towards 8 casein are inhibited by microinjection of okadaic acid into oocytes. Okadaic acid is a potent inhibitor [2,7] of protein phosphatases 1 and 2A (AMD and PCS phosphatases) below the p.molar range concentration. 8 casein is an exogenous substrate which is not dephosphorylated in vivo by protein phosphatase 1 in prophase arrested oocytes [4]. Since estimations from the data of Hermann [8] indicate a concentration of protein phosphatase 2A in the micromolar range, protein phosphatase 2A corresponds to the phosphatase okadaicsensitive responsible for 8 casein dephosphorylation in vivo. Following the first microinjection of okadaic acid, protein phosphatase activity was partially restored after 15 minutes. Since a second microinjection of okadaic acid totally inhibited the recovered activity, we conclude that okadaic acid did not induce activation of a new resistant protein phosphatase activity. Interestingly, it was reported that okadaic acid could induce M-phase promoting factor (MPF) in Xenopus oocytes [6,13] and in Starfish oocytes [11,12]. MPF induction was interpreated as a result of protein phosphatase inhibition. Our results give strong experimental support to this hypothesis. In our experiments, MPF induction occurred at a concentration of 25 llM okadaic acid (pipet concentration) which is consistent with previous results [11,12,13] We have consistently observed that in no instance maturation occurred for an injected dose below 12.5 liM (pipet concentration). Therefore total inhibition of protein phosphatases could be achieved by doses of
235
okadaic acid lower than those which provoke MPF induction; in this case, inhibition was rapidly reversible. This indicates that protein phosphatase in vivo must be inhibited for a longer period in order to induce MPF. ACKNOWLEGMENTS We are very grateful to Dr. Josef Goris (Katholieke Universiteit te Leuven, Belgium) for providing Okadaic acid and for helpfull comments and discussion on the manuscript. This work was supported by Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, and Minist~re de la Recherche et de I'Enseignement Sup6rieur.
PEFEPENCES 1. 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 Diff., 19, 253-261. 2. BIALOJAN C. and TAKAI A.,1988. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem. J., 256, 283-290. 3. COHEN P., HOLMES C.F.B. and TSUKITANI Y., 1990. Okadaic acid: a new probe for the study of cellular regulation. Trends Bio. ScL, 15, 98-102. 4. CORMIER P., MULNER-LORILLON O. and BELLE~ R. ,1990. In vivo progesterone regulation of protein phosphatase activity in X e n o p u s oocytes. Develop. BioL, 139, 427-431. 5. FELIX M.A., COHEN P. and KARSANTI E., 1990. Cdc2 kinase is negatively regulated by a type 2A phophatase in the Xenopus early embryonic cell cycle: evidence from the effect of okadaic acid. EMBO J., 9, 675-683. 6. GORIS J., HERMANN J., HENDRIX P., OZON R. and MERLEVEDE W., 1989. Okadaic acid, a specific protein phosphatase inhibitor, -induces maturation and MPF formation in Xenopus laevis oocytes. FEBS Lett., 245, 9194.
236
P Cormier el al
7. HAYSTEAD T.A.J., SlM A.T.R., CARLING D., HONNOR R.C., TSUKITANI Y. and COHEN P, 1989. Effect of the tumor promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature, 337, 78-81.
11. PICARD A., CAPONY J.P., BRAUTIGAN D.L. and DORg:E M. , 1989. Involvement of protein phosphatase 1 and 2A in the control of M Phase-promoting factor activity in Starfish. J. Cell BioL, 109, 3347-3354.
8. HERMANN J., CAYLA X., DUMORTIER K., GORIS J., OZON R. and MERLEVEDE W. , 1988. Modulation of the substrate specificity of the polycation-stimulated protein phosphatase from Xenopus laevis oocytes. Eur. J. Biochem., 173, 17-25.
12. PONDAVEN P., MEIJER L. and BIALOJAN C.,1989. La micro-injection d'acide okada'='que, un inhibiteur de phosphoprot~ine phosphatases, induit la maturation de rovocyte d'6toile de mer. C.R. Acad. ScL, 309, 563-569.
9. MULNER-LORILLON O., MAROT J., CAYLA X., POULHE R. and BELLI~ R., 1988. Purification and characterization of a casein kinase II-type enzyme from Xenopus laevis ovary. Biological effects on the meiotic cell division of fullgrown oocyte. Eur. J. Biochem., 171, 107-117.
13. RIME H., HUCHON D., MERLEVE~DE W. and Characterization of MPF acid in Xenopus oocyte. 29,47-58.
10. NURSE P., 1990. Universal control mechanism regulating onset of M-phase. Nature, 344, 503-508.
JESSUS C., GORIS J., OZON R. 1990. activation by okadaic Cell Diff. Develop., P
Received 2 October 1990 Accepted for publication 9 November 1990
Biol Cell (1990) 69, 233-236
© Elsevier, Paris
PROTEIN PHOSPHATASE ACTIVITIES IN VlVO IN XENOPUS LAEVIS OOCYTE: INHIBITION BY OKADAIC ACID Patrick CORMIER, Odile MULNER-LORILLON, Ren60ZON and Robert BELLI~ Laboratoire de Physiologie de la Reproduction, UA CNRS 555, INRA, UPMC, 4 place Jussieu 75256 Paris cedex 05 - .France-.
SUMMARY
MATERIAL AND METHODS
Protein phosphatase activities were analyzed in vivo in Xenopus oocytes. The dephosphorylation of microinjected 8 casein was inhibited when the tumor promoter okadaic acid was microinjected into oocytes. Inhibition was dose dependent and reversible; 50 % of activity was recovered 15-30 minutes
Xenopus laevis adult females were purchased from CRBM Montpellier (France) and maintained under laboratory condition. [732p] adenosine 5'-triphosphate (ATP ~ 3,000 Ci/mmol) was supplied by Amersham France. casein and the catalytic subunit of protein kinase A, purified from beef heart, was purchased from Sigma. Casein kinase II was prepared in the laboratory from X e n o p u s ovaries [9]. 13 casein was phosphorylated in vitro by protein kinase A or by casein kinase II as already described [4]. Okadaic acid was a gift of Professor Daisuke Uemura (Shizuoka University, Japan). Isolated full-grown oocytes obtained as in [1] were microinjected at the equator level with 50 n l o f 50 mM Hepes, 1 mM DTT, pH 7.4 and with 20 to 30 fmoles of 32p-13 casein, alone or together with okadaic acid. Phosphate released from 32p-1~ casein was determined after homogenization of single oocytes in 50 ILl buffer containing 50 mM Tris, 75 mM KCI, 50 mM NaF, l mM Na 2HPO4, 1 mM EDTA, 10 mM ATP, 0.1 mM orthovanadate, 5 mM 4-nitrophenyl phosphate and antiproteases as described [4].
after microinjection. Key words : Protein phosphatase in vivookadaic acid- Xenopus oocyte-protein phosphatase 2,4.
INTRODUCTION Okadaic acid is a tumor promoter which is a potent inhibitor of protein phosphatases in vitro [2] and described as a new probe for the study of cellular regulation [3]. Okadaic acid triggers M-Phase promoting factor (MPF) in Amphibian [6,13] and in Starfish [11,12] oocytes. Moreover, in oocytes and in oocyte extracts, okadaic acid was shown to activate histone H1 kinase [5,11], which is related to the cell cycle control (cdc) gene cdc2 product [10]. We report here that okadaic acid inhibits protein ph,osphatase activities in vivo measured after microinjection into oocytes of B casein. Our results thus provide strong experimental support to interpret the in vivo effects of okadaic acid as an inhibition of specific protein phosphatases.
RESULTS 1. Okadaic acid inhibits protein phosphatase in vivo Protein phosphatase activities were measured in vivo by the dephosphorylation of microinjected 8 caseih previously phosphorylated in vitro by either casein kinase II or
234
P Cormier et al
(pipet concentrations) for casein kinase II and protein kinase A sites of phosphorylation. .>_ >,
.E •*--'
2. Kinetic of protein phosphatase activities after okadaic acid microinjection into oocyte.
O
0850 (D
O
¢Uo~ "t" e's (D O ca.
0
I
I
I
-7 -6 -5 Okadaic acid (log M) Figure 1. Dose-response curve for protein phosphatase activities in vivo as a function of okadaic acid concentration. Oocytes were microinjected with okadaic acid and 32p-13 casein (25 fmoles 3 2 p) previously phosphorylated by protein kinase A (PKA sites; 0-0) or casein kinase II (CK II site; x-x ). Protein phosphatase activities (4 min dephosphorylation ) towards 8 casein was measured as described in material and methods. Each point is expressed in % of control and is the mean of 6 single oocyte determinations with SD. protein kinase A. Protein phosphatase activities were respectively 1.6 fmoles/min/oocyte (SD--0.3; 7 experiments) and 3.4 fmoles/min/oocyte (SD=0.7; 11 experiments). When microinjected at a concerffration of 251.tM (pipet concentration), okadaic acid provoked the activation of MPF and germinal vesicle breakdown. At a concentration of 12.5 I.tM (pipet concentration), okadaic acid totally inhibited dephosphorylation of both casein kinase II site and p'rotein kinase A sites of 8 casein to respectively 0.03 and 0.14 fmoles/oocyte/min. The dose-response curve (figure 1) of phosphatase activity inhibition by okadaic acid shows ID 50 of respectively 1 and 2 p.M
When oocytes were first microinjected with okadaic acid (12.5 I.tM; a dose which was totally inhibitor) and then 15 or 120 minutes later microinjected with 13 casein, it was found that protein phosphatase activity was no more inhibited (figure 2). The time for half recovery of the initial value was around 15-30 minutes for both casein kinase II and protein kinase A phosphorylated sites of 13 casein. 100
•~
PI C
80 6o 4o ao
0. 0 control
0 min
15
min
120
min
time after okadaic acid Figure 2. Protein phosphatase activity in vivo after microinjection of okadaic acid into oocytes. 0 min, 15 min and 120 min after first okadaic microinjection, protein phosphatase activity was determined from the microinjection of 8 casein phosphorylated in vitro by casein kinase II (CK II sites) or by protein kinase A (PKA sites) during a 4 min incubation of oocytes at 20°C. Results represent the mean of 6 single oocyte determinations. After the first okadaic acid microinjection, protein phosphatases were allowed to recover for 15 and 120 minutes and then, 13 casein was microinjected together with .okadaic acid; protein phosphatase activities were again
Protein phosphatase activities in vivo in Xenopus laevis oocyte inhibition by okadaic acid
inhibited both for the casein kinase II and protein kinase A phosphorylated sites.
DISCUSSION Our results show that protein phosphatase activities towards 8 casein are inhibited by microinjection of okadaic acid into oocytes. Okadaic acid is a potent inhibitor [2,7] of protein phosphatases 1 and 2A (AMD and PCS phosphatases) below the p.molar range concentration. 8 casein is an exogenous substrate which is not dephosphorylated in vivo by protein phosphatase 1 in prophase arrested oocytes [4]. Since estimations from the data of Hermann [8] indicate a concentration of protein phosphatase 2A in the micromolar range, protein phosphatase 2A corresponds to the phosphatase okadaicsensitive responsible for 8 casein dephosphorylation in vivo. Following the first microinjection of okadaic acid, protein phosphatase activity was partially restored after 15 minutes. Since a second microinjection of okadaic acid totally inhibited the recovered activity, we conclude that okadaic acid did not induce activation of a new resistant protein phosphatase activity. Interestingly, it was reported that okadaic acid could induce M-phase promoting factor (MPF) in Xenopus oocytes [6,13] and in Starfish oocytes [11,12]. MPF induction was interpreated as a result of protein phosphatase inhibition. Our results give strong experimental support to this hypothesis. In our experiments, MPF induction occurred at a concentration of 25 llM okadaic acid (pipet concentration) which is consistent with previous results [11,12,13] We have consistently observed that in no instance maturation occurred for an injected dose below 12.5 liM (pipet concentration). Therefore total inhibition of protein phosphatases could be achieved by doses of
235
okadaic acid lower than those which provoke MPF induction; in this case, inhibition was rapidly reversible. This indicates that protein phosphatase in vivo must be inhibited for a longer period in order to induce MPF. ACKNOWLEGMENTS We are very grateful to Dr. Josef Goris (Katholieke Universiteit te Leuven, Belgium) for providing Okadaic acid and for helpfull comments and discussion on the manuscript. This work was supported by Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, and Minist~re de la Recherche et de I'Enseignement Sup6rieur.
PEFEPENCES 1. 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 Diff., 19, 253-261. 2. BIALOJAN C. and TAKAI A.,1988. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem. J., 256, 283-290. 3. COHEN P., HOLMES C.F.B. and TSUKITANI Y., 1990. Okadaic acid: a new probe for the study of cellular regulation. Trends Bio. ScL, 15, 98-102. 4. CORMIER P., MULNER-LORILLON O. and BELLE~ R. ,1990. In vivo progesterone regulation of protein phosphatase activity in X e n o p u s oocytes. Develop. BioL, 139, 427-431. 5. FELIX M.A., COHEN P. and KARSANTI E., 1990. Cdc2 kinase is negatively regulated by a type 2A phophatase in the Xenopus early embryonic cell cycle: evidence from the effect of okadaic acid. EMBO J., 9, 675-683. 6. GORIS J., HERMANN J., HENDRIX P., OZON R. and MERLEVEDE W., 1989. Okadaic acid, a specific protein phosphatase inhibitor, -induces maturation and MPF formation in Xenopus laevis oocytes. FEBS Lett., 245, 9194.
236
P Cormier el al
7. HAYSTEAD T.A.J., SlM A.T.R., CARLING D., HONNOR R.C., TSUKITANI Y. and COHEN P, 1989. Effect of the tumor promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature, 337, 78-81.
11. PICARD A., CAPONY J.P., BRAUTIGAN D.L. and DORg:E M. , 1989. Involvement of protein phosphatase 1 and 2A in the control of M Phase-promoting factor activity in Starfish. J. Cell BioL, 109, 3347-3354.
8. HERMANN J., CAYLA X., DUMORTIER K., GORIS J., OZON R. and MERLEVEDE W. , 1988. Modulation of the substrate specificity of the polycation-stimulated protein phosphatase from Xenopus laevis oocytes. Eur. J. Biochem., 173, 17-25.
12. PONDAVEN P., MEIJER L. and BIALOJAN C.,1989. La micro-injection d'acide okada'='que, un inhibiteur de phosphoprot~ine phosphatases, induit la maturation de rovocyte d'6toile de mer. C.R. Acad. ScL, 309, 563-569.
9. MULNER-LORILLON O., MAROT J., CAYLA X., POULHE R. and BELLI~ R., 1988. Purification and characterization of a casein kinase II-type enzyme from Xenopus laevis ovary. Biological effects on the meiotic cell division of fullgrown oocyte. Eur. J. Biochem., 171, 107-117.
13. RIME H., HUCHON D., MERLEVE~DE W. and Characterization of MPF acid in Xenopus oocyte. 29,47-58.
10. NURSE P., 1990. Universal control mechanism regulating onset of M-phase. Nature, 344, 503-508.
JESSUS C., GORIS J., OZON R. 1990. activation by okadaic Cell Diff. Develop., P
Received 2 October 1990 Accepted for publication 9 November 1990
