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Regulation of cyclin proteolysis inPatella vulgata oocytes
Biol. Cell, Vol 76, 1992
217
CYCLIN B TRANSLATION DURING FIRST MEIOTIC CELL CYCLE OF STARFISH OOCYTES GALAS Simon, DOREE Marcel and PICARD Andr6 CRBM, r o u t e de M e n d e , 34033 Ivlontpellier Laboratoire A r a g o , 6 6 6 5 0 Banyuls sur mer.
REGULATION OF CYCLIN PI~,OTEOLYSIS IN PATELLA VULGATA OOCYTES. COLAS Pierre 1,2, LAUNAY Catherine I, and
van LOON Andre2.1, GUERRIER Pierre I. I: Laboratoire de Biologic, ENS, Lyon, France; 2: Experimental Zoology, University of Utrecht, The Netherlands.
D u r i n g first meiotic cell cycle cyclin B is the most p r o m i n e n t l y s y n t h e s i z e d p r o t e i n , a l t h o u g h an i m p o r t a n t stockpile of cyclin was already present in the oocyte and meiosis r e i n i t i a t i o n does not require cyclin synthesis. At the time of first polar body emission the whole amount of cyclin has been destroyed, what does not support the view that cyclin B could be synthesized in advance during first cycle to allow a rapid entry into second cycle. As stockpile cyclin B b e g i n s d e g r a d a t i o n long before IVletaphase, the p r o m i n e n t n e o s y n t h e s i s allows to maintain a mitotic state and gives the cell time enough to bring a b o u t events of d i a c i n e s i s , p r o m e t a p h a s e and m e t a p h a s e entry. W e have shown that a nuclear factor, different from cyclin B mRNA, is r e q u i r e d for e n h a n c e d c y c l i n B n e o s y n t h e s i s after G e r m i n a l Vesicle Breakdown.
The mitotic cyclins are the reguhuory subunits of the M phase kinase which triggers the G2/M transition of the cell cycle. Accumuhlted in interphase, they undergo an abrupt destruction at mitosis which is the key event governing the exit from M phase and thus the completion of cell division. Oocytes of most species offer an excellent model system to study the regulation of cyclin proteolysis because of the physiological block in M phase occuring in the course of their m:nuration. As far as now, the M phase block has been examined in det:dl only in Xenopus. However, Xenopus and other amphibians are the only species whose metaphase block does not depend on protein synthesis. Thus, it seems tluite pertinent to address this issue using a more represent:ttive model system. In oocytes of the mollusc Patella t,ulgata, the use of inhibitors shows that the M phase block depends on protein synthesis :rod phosphoryhuion. In order to understand these requirements tit the molecular level, cyclin A and B cDNAs have been cloned and sequenced, and specific antibodies have been raised. Upon emetine treatment, both cyclins disappear rapidly from the oocyles, which raises the question of their sttlbility during the metaphase block. The microinjection of specific :mtisense oligonttcleotides reveals that cyclin synthesis is reqtlired for a long-term maintenance of the M phase block. An in vitro approach has been designed to examine the short-tern1 stability of cyclins under various conditions. In a lysate prepared from metaphase I arrested oocytes or from 6-DMAP treated oocytes, cyclins are stable whereas in it lysate prepared from emetine treated oocytes, both cyclins are destroyed rapidly. The phosphatnse inhibitor okad:dc acid triggers cyclin B destruction in a metaphase I lysate whereas it has a cytostatic effect when injcctcd into a blastomcre during early development. Interestingly, the :tddition of the p13 sucl ycasl gene product to a metaphase I lysale turns on cyclin B destruction but does not :d'fect the stability of cyclin A. This observation suggests that the regulalion of cyclin A and cyclin B proteolysis follow different pathw:tys and their p13 suet is involved in the completion of cell division.
H E P A T O C Y T E S : A S U I T A B L E C E L L S Y S T E M FOR STUDYING TIlE CONTROL MECHANISMS OF CELL CYCLE PROGRESSION IN MAMMALIANS. LOYER Pascal, GLAISE Denise, CARIOU Sandrine, MEIJER Laurent* et GUGUEN-GUILLOUZO Christiane. INSERM U49, Unitd de Recherches Hdpatologiques, H~pital Pontchaillou, 35033 Rennes Cedex et * CNRS, Station Biologique, 29680 Roscoff, France.
CYCLIN A A N D LIVER CELL P R O L I F E R A T I O N ZINDY Fr6d6rique, LAMAS E u g 6 n i a , WANG J i a n , C H E N I V E S S E Xavier, HENGLEIN 13erthold a n d B R E C H O T C h r i s t i a n . CItU Necker. 156 m e d e Vaugirard 7 5 0 1 5 Parts.
Cdc2 gene encodes a 34 kd protein kinase which is highly conserved in all eukaryotes. In yeast, p34 cdc2 is involved in both GI/S and G2/M transitions. However in mammalian cells, its involvement in earlier step(s) of the cell cycle still remains unclear. Complexity has been enhanced by cloning of a closely ode2 related gene encoding a kinase called p33 cdk2. Several lines of evidence indicate that p33 cdk2 can be implicated in DNA synthesis and can monitor GI/S transition. In order to address the issue of whether p33 cdk2 and/or p34 ode2 control the G1/S transition in mammalian cells, we have investigated expression and activation of these two kinases in proliferating hepatocytes either in vivo during rat liver regeneration or in vitro in EGFstimulated primary cultures. These two synchroneous cell systems characterized by their long-lasting GI phase were particularly well appropriate for studying this phase and G I/S transition. We show that p34 ode2 is involved in control of G2/M transition as previously reported for all eukaryotes. In addition, our results indicate that neither ode2 nor cdk2 share histone H1 kinase activity in GI and G1/S transition, whereas they are both expressed during S phase a4ad that active phosphorylation of histone HI takes place at that time. Taken together these data let to think that edc2 and cdk2 are not directly involved in the start-point that monitors entry into S phase, but play a critical role in DNA synthesis. The possibility that factor(s) distinct from these two cdc2-related kinases could control the essential check point of late GI phase in mammalian cells will be discussed.
We h a v e p r e v i o u s l y r e p o r t e d the i d e n t i f i c a t i o n of h e p a t i t i s B v i r u s (I-IBVI i n t e g r a t i o n in a n i n t r o n of cyclin A g e n e in a n e a r l y h e p a t o c e l l u l a r c a r c i n o m a a n d h e n c e t h e i s o l a t i o n of h u m a n cyclin A eDNA (ll. 1) We h a v e c o n s t r u c t e d a eDNA library of the original t u m o r ( t u m o r HEN) mad isolated several h y b r i d HBV-cyclin A cDNAs. T h e s e cDNAs h a v e t h e c o d i n g c a p a c i t y lor a HBV-eyclin A f u s i o n p r o t e i n . In t h e c h i m e r i c p r o t e i n , t h e N - t e r m i n a l of c y e l i n A, i n c l u d i n g t h e s i g n a l s for s i g n a l s for e y c l i n clegradation, was deleted and replaced by viral PreS2/S s e q u e n c e s w h i l e t h e r e s t of cyclin A r e m i n d i n t a c t . HBV i n t e g r a t i o n in the eyclin A g e n e r e s u l t e d in the o v e r e x p r e s s i o n of h y b r i d l-IBV-cycliu A t r a n s c r i p t s t h a t code for a s t a b i l i z e d cyclin A. (2). 2) We h a v e i n v e s t i g a t e d cyclin A e x p r e s s i o n in a p r i m a r y c u l t u r e of n o r m a l r a t h e p a t o c y t e s a n d d u r i n g r a t l i v e r r e g e n e r a t i o n after partial h e p a t e c l o m y . In b o t h c a s e s , cyclin A m R N A a n d p r o t e i n a c c u m u l a t e a s t h e cells e n t e r S p h a s e . M o r e o v e r we m i c r o i n j e e t e d a n t i - s e n s e DNA c o n s t r u c t s for cyclin A, restdth-lg in effective i n h i b i t i o n of S p h a s e entry. {3) In c o n c l u s i o n : We h a v e s h o w e d a n a n a l y s i s of the e x p r e s s i o n p a t t e r n of cyclin A g e n e in original t u m o r w h i c h s u p p o r t s t h e h y p o U l e s i s of i n s e r t i o n a l m u t a g e n e s i s of HBV, a n d a s t u d y of t h e role of cyclin A in a n o r m a l cell cycle w h i c h i n d i c a t e s its i n v o l v e m e n t in G 1 / S t r a n s i t i o n . T h a t cyclin A is involved in ,S p h a s e m a y p r o v i d e n e w c l u e s a s to its p o t e n t i a l role in carcinogenesis. I) WANG J., CHENIVESSE X., IIENGI.EIN B., BRECIIOT C. (1990). Nuture,
343, 555-557. 2) ZINDY F., I.AMAS F_. CHENIVHSSE X., SOBCZAK J., WANG J., I-'I.2SQUET D., HENGI.EIN 13., BRI';CHOT C. (1992). Biuphys. Binchcnl. Res. Comm. 182, 1144-1154. 3) WANG J., ZINI.)Y F.. CIIENIVESSE X., I.AMAS E.. HENGLEIN B., BRECHOT C. (1992). Ollcogc,c. ia press. ,,,
217
CYCLIN B TRANSLATION DURING FIRST MEIOTIC CELL CYCLE OF STARFISH OOCYTES GALAS Simon, DOREE Marcel and PICARD Andr6 CRBM, r o u t e de M e n d e , 34033 Ivlontpellier Laboratoire A r a g o , 6 6 6 5 0 Banyuls sur mer.
REGULATION OF CYCLIN PI~,OTEOLYSIS IN PATELLA VULGATA OOCYTES. COLAS Pierre 1,2, LAUNAY Catherine I, and
van LOON Andre2.1, GUERRIER Pierre I. I: Laboratoire de Biologic, ENS, Lyon, France; 2: Experimental Zoology, University of Utrecht, The Netherlands.
D u r i n g first meiotic cell cycle cyclin B is the most p r o m i n e n t l y s y n t h e s i z e d p r o t e i n , a l t h o u g h an i m p o r t a n t stockpile of cyclin was already present in the oocyte and meiosis r e i n i t i a t i o n does not require cyclin synthesis. At the time of first polar body emission the whole amount of cyclin has been destroyed, what does not support the view that cyclin B could be synthesized in advance during first cycle to allow a rapid entry into second cycle. As stockpile cyclin B b e g i n s d e g r a d a t i o n long before IVletaphase, the p r o m i n e n t n e o s y n t h e s i s allows to maintain a mitotic state and gives the cell time enough to bring a b o u t events of d i a c i n e s i s , p r o m e t a p h a s e and m e t a p h a s e entry. W e have shown that a nuclear factor, different from cyclin B mRNA, is r e q u i r e d for e n h a n c e d c y c l i n B n e o s y n t h e s i s after G e r m i n a l Vesicle Breakdown.
The mitotic cyclins are the reguhuory subunits of the M phase kinase which triggers the G2/M transition of the cell cycle. Accumuhlted in interphase, they undergo an abrupt destruction at mitosis which is the key event governing the exit from M phase and thus the completion of cell division. Oocytes of most species offer an excellent model system to study the regulation of cyclin proteolysis because of the physiological block in M phase occuring in the course of their m:nuration. As far as now, the M phase block has been examined in det:dl only in Xenopus. However, Xenopus and other amphibians are the only species whose metaphase block does not depend on protein synthesis. Thus, it seems tluite pertinent to address this issue using a more represent:ttive model system. In oocytes of the mollusc Patella t,ulgata, the use of inhibitors shows that the M phase block depends on protein synthesis :rod phosphoryhuion. In order to understand these requirements tit the molecular level, cyclin A and B cDNAs have been cloned and sequenced, and specific antibodies have been raised. Upon emetine treatment, both cyclins disappear rapidly from the oocyles, which raises the question of their sttlbility during the metaphase block. The microinjection of specific :mtisense oligonttcleotides reveals that cyclin synthesis is reqtlired for a long-term maintenance of the M phase block. An in vitro approach has been designed to examine the short-tern1 stability of cyclins under various conditions. In a lysate prepared from metaphase I arrested oocytes or from 6-DMAP treated oocytes, cyclins are stable whereas in it lysate prepared from emetine treated oocytes, both cyclins are destroyed rapidly. The phosphatnse inhibitor okad:dc acid triggers cyclin B destruction in a metaphase I lysate whereas it has a cytostatic effect when injcctcd into a blastomcre during early development. Interestingly, the :tddition of the p13 sucl ycasl gene product to a metaphase I lysale turns on cyclin B destruction but does not :d'fect the stability of cyclin A. This observation suggests that the regulalion of cyclin A and cyclin B proteolysis follow different pathw:tys and their p13 suet is involved in the completion of cell division.
H E P A T O C Y T E S : A S U I T A B L E C E L L S Y S T E M FOR STUDYING TIlE CONTROL MECHANISMS OF CELL CYCLE PROGRESSION IN MAMMALIANS. LOYER Pascal, GLAISE Denise, CARIOU Sandrine, MEIJER Laurent* et GUGUEN-GUILLOUZO Christiane. INSERM U49, Unitd de Recherches Hdpatologiques, H~pital Pontchaillou, 35033 Rennes Cedex et * CNRS, Station Biologique, 29680 Roscoff, France.
CYCLIN A A N D LIVER CELL P R O L I F E R A T I O N ZINDY Fr6d6rique, LAMAS E u g 6 n i a , WANG J i a n , C H E N I V E S S E Xavier, HENGLEIN 13erthold a n d B R E C H O T C h r i s t i a n . CItU Necker. 156 m e d e Vaugirard 7 5 0 1 5 Parts.
Cdc2 gene encodes a 34 kd protein kinase which is highly conserved in all eukaryotes. In yeast, p34 cdc2 is involved in both GI/S and G2/M transitions. However in mammalian cells, its involvement in earlier step(s) of the cell cycle still remains unclear. Complexity has been enhanced by cloning of a closely ode2 related gene encoding a kinase called p33 cdk2. Several lines of evidence indicate that p33 cdk2 can be implicated in DNA synthesis and can monitor GI/S transition. In order to address the issue of whether p33 cdk2 and/or p34 ode2 control the G1/S transition in mammalian cells, we have investigated expression and activation of these two kinases in proliferating hepatocytes either in vivo during rat liver regeneration or in vitro in EGFstimulated primary cultures. These two synchroneous cell systems characterized by their long-lasting GI phase were particularly well appropriate for studying this phase and G I/S transition. We show that p34 ode2 is involved in control of G2/M transition as previously reported for all eukaryotes. In addition, our results indicate that neither ode2 nor cdk2 share histone H1 kinase activity in GI and G1/S transition, whereas they are both expressed during S phase a4ad that active phosphorylation of histone HI takes place at that time. Taken together these data let to think that edc2 and cdk2 are not directly involved in the start-point that monitors entry into S phase, but play a critical role in DNA synthesis. The possibility that factor(s) distinct from these two cdc2-related kinases could control the essential check point of late GI phase in mammalian cells will be discussed.
We h a v e p r e v i o u s l y r e p o r t e d the i d e n t i f i c a t i o n of h e p a t i t i s B v i r u s (I-IBVI i n t e g r a t i o n in a n i n t r o n of cyclin A g e n e in a n e a r l y h e p a t o c e l l u l a r c a r c i n o m a a n d h e n c e t h e i s o l a t i o n of h u m a n cyclin A eDNA (ll. 1) We h a v e c o n s t r u c t e d a eDNA library of the original t u m o r ( t u m o r HEN) mad isolated several h y b r i d HBV-cyclin A cDNAs. T h e s e cDNAs h a v e t h e c o d i n g c a p a c i t y lor a HBV-eyclin A f u s i o n p r o t e i n . In t h e c h i m e r i c p r o t e i n , t h e N - t e r m i n a l of c y e l i n A, i n c l u d i n g t h e s i g n a l s for s i g n a l s for e y c l i n clegradation, was deleted and replaced by viral PreS2/S s e q u e n c e s w h i l e t h e r e s t of cyclin A r e m i n d i n t a c t . HBV i n t e g r a t i o n in the eyclin A g e n e r e s u l t e d in the o v e r e x p r e s s i o n of h y b r i d l-IBV-cycliu A t r a n s c r i p t s t h a t code for a s t a b i l i z e d cyclin A. (2). 2) We h a v e i n v e s t i g a t e d cyclin A e x p r e s s i o n in a p r i m a r y c u l t u r e of n o r m a l r a t h e p a t o c y t e s a n d d u r i n g r a t l i v e r r e g e n e r a t i o n after partial h e p a t e c l o m y . In b o t h c a s e s , cyclin A m R N A a n d p r o t e i n a c c u m u l a t e a s t h e cells e n t e r S p h a s e . M o r e o v e r we m i c r o i n j e e t e d a n t i - s e n s e DNA c o n s t r u c t s for cyclin A, restdth-lg in effective i n h i b i t i o n of S p h a s e entry. {3) In c o n c l u s i o n : We h a v e s h o w e d a n a n a l y s i s of the e x p r e s s i o n p a t t e r n of cyclin A g e n e in original t u m o r w h i c h s u p p o r t s t h e h y p o U l e s i s of i n s e r t i o n a l m u t a g e n e s i s of HBV, a n d a s t u d y of t h e role of cyclin A in a n o r m a l cell cycle w h i c h i n d i c a t e s its i n v o l v e m e n t in G 1 / S t r a n s i t i o n . T h a t cyclin A is involved in ,S p h a s e m a y p r o v i d e n e w c l u e s a s to its p o t e n t i a l role in carcinogenesis. I) WANG J., CHENIVESSE X., IIENGI.EIN B., BRECIIOT C. (1990). Nuture,
343, 555-557. 2) ZINDY F., I.AMAS F_. CHENIVHSSE X., SOBCZAK J., WANG J., I-'I.2SQUET D., HENGI.EIN 13., BRI';CHOT C. (1992). Biuphys. Binchcnl. Res. Comm. 182, 1144-1154. 3) WANG J., ZINI.)Y F.. CIIENIVESSE X., I.AMAS E.. HENGLEIN B., BRECHOT C. (1992). Ollcogc,c. ia press. ,,,