A novel p34cdc2-binding and activating protein that is necessary and sufficient to trigger G2/M progression in Xenopus oocytes

A novel p34cdc2-binding and activating protein that is necessary and sufficient to trigger G2/M progression in Xenopus oocytes
of 13
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
  A novel p34 cdc2 -binding and activatingprotein that is necessary and sufficientto trigger G 2 /M progressionin  Xenopus  oocytes Ingvar Ferby, Montserrat Blazquez, Amparo Palmer, Ramon Eritja, and Angel R. Nebreda 1 European Molecular Biology Laboratory, 69117 Heidelberg, Germany The activation of maturation-promoting factor (MPF) is required for G 2 /M progression in eukaryotic cells.  Xenopus  oocytes are arrested in G 2  and are induced to enter M phase of meiosis by progesterone stimulation.This process is known as meiotic maturation and requires the translation of specific maternal mRNAs storedin the oocytes. We have used an expression cloning strategy to functionally identify proteins involved inG 2 /M progression in  Xenopus  oocytes. Here we report the cloning of two novel cDNAs that when expressedin oocytes induce meiotic maturation efficiently. The two cDNAs encode proteins of 33 kD that are 88%identical and have no significant homologies to other sequences in databases. These proteins, which we referto as p33 ringo (rapid inducer of G 2 /M progression in oocytes), induce very rapid MPF activation incycloheximide-treated oocytes. Conversely, ablation of endogenous p33 ringo mRNAs using antisenseoligonucleotides inhibits progesterone-induced maturation, suggesting that synthesis of p33 ringo is required forthis process. We also show that p33 ringo binds to and activates the kinase activity of p34 cdc2 but does notassociate with p34 cdc2 /cyclin B complexes. Our results identify a novel p34 cdc2 binding and activating proteinthat regulates the G 2 /M transition during oocyte maturation. [ Key Words : Cell cycle; meiotic maturation; MPF; M phase; oocyte; Cdc2] Received March 5, 1999; revised version accepted June 29, 1999. Entry of eukaryotic cells into M phase of the cell cycle isregulated by maturation-promoting factor (MPF), an ac-tivity composed of a B-type cyclin and the protein kinasep34 cdc2 . Cyclin B is usually synthesized and associateswith p34 cdc2 throughout late S phase and early G 2 , butthe p34 cdc2 /cyclin B complex is maintained inactive bythe phosphorylation of p34 cdc2 on Thr-14 and Tyr-15.Dephosphorylation of p34 cdc2 leads to activation of theMPF kinase activity, which can phosphorylate manyproteins responsible for both the G 2 /M transition andprogression through M phase (for review, see Nurse1990; Coleman and Dunphy 1994; Morgan 1997).  Xenopus  oocytes are naturally arrested in late G 2  andare induced to enter into M phase of meiosis by proges-terone. This process is known as meiotic maturation andis associated with the activation of MPF (Masui andClarke 1979). In G 2 -arrested  Xenopus  oocytes there is apreformed stock of p34 cdc2 /cyclin B complexes (pre-MPF) that is maintained inactive by the phosphorylationof p34 cdc2 on Thr-14 and Tyr-15 (Cyert and Kirschner1988; Gautier and Maller 1991; Kobayashi et al. 1991b).The inhibitory phosphorylation of p34 cdc2 in immatureoocytes is likely to be due to the membrane-bound pro-tein kinase Myt1 (Atherton-Fessler et al. 1994; Korn-bluth et al. 1994; Mueller et al. 1995; Palmer et al. 1998),whereas the activating dephosphorylation probably in-volves the phosphatase Cdc25 (Dunphy and Kumagai1991; Gautier et al. 1991; Kumagai and Dunphy 1991;Strausfeld et al. 1991). Thus, either an increased activityof Cdc25 or the inhibition of Myt1 may bring about theactivation of pre-MPF during oocyte maturation.An essential requirement for progesterone-inducedmaturation is the translation of maternal mRNAs storedin the oocyte. Although several mRNAs are known to betranslated de novo during oocyte maturation (Sagata etal. 1988; Kobayashi et al. 1991b; Gabrielli et al. 1992;Rempel et al. 1995; Murakami and Vande Woude 1998),only the mRNA encoding the protein kinase p39 mos hasbeen found so far to be necessary for oocyte maturation(Sagata et al. 1988, 1989; Freeman et al. 1990; Sheets etal. 1995). Injection of either p39 mos mRNA or recombi-nant p39 mos protein into  Xenopus  oocytes induces matu-ration in the absence of progesterone (Sagata et al. 1988,1989; Yew et al. 1992). Conversely, injection of p39 mos antisense oligonucleotides blocks progesterone-inducedMPF activation indicating that p39 mos is necessary forthe initiation of maturation (Sagata et al. 1988). 1 Corresponding author.E-MAIL; FAX 49 6221 387166.GENES & DEVELOPMENT 13:2177–2189 © 1999 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/99 $5.00; 2177  The function of p39 mos in oocyte maturation is mostlikely related to its ability to activate the p42 mpk1 MAPkinase pathway (Nebreda et al. 1993; Nebreda and Hunt1993; Posada et al. 1993; Shibuya and Ruderman 1993).This is supported by the injection of neutralizing anti-MAP kinase kinase antibodies that inhibit p39 mos -in-duced oocyte maturation (Kosako et al. 1994, 1996) andis also consistent with the observation that upon ectopicp39 mos expression, activation of p42 mpk1 always pre-cedes the activation of pre-MPF (Nebreda and Hunt1993; Posada et al. 1993; Shibuya and Ruderman 1993).The idea that activation of p42 mpk1 plays an importantrole in meiotic maturation is further supported by thedelay in progesterone-induced maturation observed uponinjection of either anti-MAP kinase kinase antibodies(Kosako et al. 1994, 1996) or a specific MAP kinase phos-phatase (Gotoh et al. 1995). Conversely, microinjectionof constitutively active MAP kinase kinase (Huang et al.1995) or active thiophosphorylated p42 mpk1 (Haccard etal. 1995) can induce oocyte maturation in the absence ofprogesterone. Taken together, the results suggest thatactivation of the p42 mpk1 cascade may be both necessaryand sufficient to release the oocyte from the G 2 -phasearrest. A possible connection between the activation ofp42 mpk1 and MPF during oocyte maturation may be pro-vided by the p42 mpk1 -activated protein kinase p90 rsk ,which can phosphorylate and down-regulate Myt1(Palmer et al. 1998).In spite of the central role of p39 mos and the p42 mpk1 pathway in oocyte maturation, there is evidence thatsynthesis of other proteins in addition to p39 mos is alsorequired for progesterone to initiate oocyte maturation(Nebreda et al. 1995; Barkoff et al. 1998). Thus, in manybatches of oocytes, the ability of p39 mos or a constitu-tively active MAP kinase kinase mutant to activate pre-MPF is significantly reduced when protein synthesis isinhibited, whereas p42 mpk1 is normally activated underthese conditions (Yew et al. 1992; Daar et al. 1993; Ne-breda and Hunt 1993; Shibuya and Ruderman 1993;Huang et al. 1995; Murakami and Vande Woude 1997).These data indicate that p42 mpk1 activation alone is notalways sufficient to activate pre-MPF. In contrast, theability of recombinant cyclin A to activate pre-MPF isthe same when injected into either untreated or cyclo-heximide-treated oocytes (Nebreda et al. 1995). Further-more, overexpression of a dominant-negative p34 cdc2 mutant or injection of a neutralizing anti-p34 cdc2 anti-body blocks progesterone-induced p39 mos accumulationand the activation of both p42 mpk1 and MPF (Nebreda etal. 1995). These results were taken to suggest that acti-vation of the free p34 cdc2 present in the oocyte (which isin a notable excess over the p34 cdc2 complexed with cy-clin B) is normally required for progesterone-inducedmaturation. However, there is no evidence that newlysynthesized A- and B-type cyclins, which would be theobvious candidates to activate free p34 cdc2 , are requiredfor progesterone-induced MPF activation (Minshull et al.1991).In this paper we have used an expression cloning strat-egy to identify novel proteins that can trigger oocytematuration. We have cloned two cDNAs that upon ex-pression in oocytes (either as synthetic mRNAs or re-combinant proteins), can potently induce MPF activa-tion and germinal vesicle breakdown (GVBD) in the ab-sence of progesterone. Moreover, antisense-directedablation of the endogenous mRNAs in the oocyte inhib-its progesterone-induced maturation. These cDNAs codefor closely related proteins that can bind to and activatethe protein kinase p34 cdc2 . Our results indicate that thisnovel p34 cdc2 binding and activating protein plays an im-portant role in oocyte maturation. Results Cloning of two novel cDNAs that potently induceG 2 /M progression in  Xenopus  oocytes To identify novel proteins involved in G 2 /M progressionduring the meiotic maturation of  Xenopus  oocytes, weused an expression cloning strategy in which we con-structed a  Xenopus  oocyte cDNA library in the FTX5expression vector. The primary library was subdividedinto pools of 150–200 colonies and plasmid DNA waspurified from the pools and in vitro transcribed to obtainmRNAs. The mRNA pools that upon microinjectioninto oocytes were able either to induce oocyte matura-tion on their own or to accelerate progesterone-inducedmaturation were subdivided into smaller pools and rein-jected until single positive clones were isolated. Usingthis approach, we isolated two clones named ls26 andls27, which by DNA hybridization experiments did notcorrespond to proteins that are known to induce oocytematuration including p39 mos , Cdc25, and several A- andB-type cyclins (data not shown). The mRNAs preparedfrom the ls26 and ls27 clones were able to induce oocytematuration in the absence of progesterone stimulation,even at concentrations as low as 250 pg per oocyte (Fig.1A, left), as well as to significantly accelerate progester-one-induced maturation at a 10-fold lower concentration(Fig. 1A, right). Oocyte maturation induced by expres-sion of ls26 or ls27 was accompanied by the appearanceof a white spot at the animal pole of the oocyte as inprogesterone-matured oocytes (Fig. 2), although a fewhours later ls26/ls27-injected oocytes usually appeared‘overmatured’. This overmaturation is sometimes simi-larly observed upon injection of high concentrations ofcyclin A or p39 mos (not shown). As in progesterone-treated oocytes, the maturation induced by ls26 or ls27correlated with the activation of both p42 mpk1 MAP ki-nase (p42 mpk1 is phosphorylated resulting in an upwardshift in immunoblots) and p34 cdc2 /cyclin B (pre-MPF,p34 cdc2 is dephosphorylated resulting in a downwardshift in immunoblots) as well as with the appearance ofp39 mos (Fig. 1B). We also found that ls26 and ls27 wereable to induce oocyte maturation faster than p39 mos ,based on experiments where all mRNAs were preparedfrom the same expression vector and injected into oo-cytes at equivalent concentrations (Fig. 1C).DNA sequencing of the clones isolated from the ex-pression library showed that the ls26 and ls27 open read- Ferby et al.2178 GENES & DEVELOPMENT  ing frames (ORFs) encoded related proteins that werefused in-frame to the carboxyl terminus of the myc tag inthe FTX5 vector. Using the partial cDNAs as probes, wecloned full-length cDNAs from a    ZAP  Xenopus  oocytecDNA library. The ls26 cDNA was 1574 bp long andencoded a protein of 300 amino acids, whereas ls27 was1357 bp in length and encoded a protein of 298 aminoacids. Both clones contained stop codons upstream of thefirst ATG and in the same frame. The predicted ls26 andls27 proteins were 88% identical (Fig. 3). The cDNAsisolated from the oocyte expression screening encodedMyc-tagged proteins that either lacked the first 11 aminoacids (ls26) or had an amino-terminal extension of 24amino acids corresponding to the 5  -untranslated region(ls27). In a search of the ls26 and ls27 sequences usingBLAST against DNA and protein sequence databases, wecould detect no significant homologies, suggesting thatls26/ls27 belonged to a novel protein family. We werealso unable to identify conserved protein motifs usingPROSITE and GENEQUIZ programs. The only poten-tially relevant homologies were obtained from expressedsequence-tag (EST) databases. The best score in thesesearches was for the human EST clone 757814, whichwas 53% identical over a 49-amino-acid stretch. In con-trast, no homologs were detected in the budding yeastgenome. The close similarity between the sequences ofls26 and ls27 suggests that they might correspond topseudoalloploid alleles; because they induced oocytematuration with the same efficiency, we concentratedon ls26 for further characterization. We named this pro-tein p33 ringo for rapid inducer of G 2 /M progression inoocytes. Recombinant p33  ringo can trigger GVBD and MPF activation in cycloheximide-treated oocytes To further characterize the ability of p33 ringo to induceoocyte maturation, we prepared a maltose-binding pro-tein (malE)–p33 ringo fusion protein. We found that theinjection of 40 ng of bacterially produced malE–p33 ringo was able to induce oocyte maturation considerably fasterthan progesterone treatment; 50% GVBD usually took ∼ 2.5 hr with malE–p33 ringo versus 5–11 hr with proges-terone. Moreover, injection of only 10 ng of malE–p33 ringo per oocyte was still able to induce maturation in100% of the injected oocytes (see Fig. 6a, below). We also Figure 2.  Morphological appearance of p33 ringo mRNA-in-jected oocytes (50 ng/oocyte) after 2.5 and 5 hr of incubation.Oocytes treated with progesterone (5 µg/ml) for 10 hr and con-trol (water-injected) oocytes are shown. Figure 1.  Induction of  Xenopus  oocyte maturationby a novel protein. (  A ) The indicated concentrations(total amount per oocyte) of mRNA transcribed invitro from the ls26 cDNA were injected into oocytesand GVBD was scored (  left ). Progesterone was addedto the groups of injected oocytes that showed nowhite spot after 18 hr of incubation (  right ). Similarresults were observed with mRNA prepared fromthe ls27 cDNA (not shown). ( B ) Immunoblot analysisusing anti-p42 mpk1 , anti-p34 cdc2 , and anti-p39 mos antibodies of lysates prepared from oocytes (fouroocytes per time point) either untreated (control),treated with progesterone (5 µg/ml), injected within vitro-transcribed mRNA encoding for p33 ringo (ls26, 50 ng/oocyte) or both injected with mRNAencoding for p33 ringo and treated with progester-one. In this experiment 50% GVBD was observedat 10, 3.5, and 1.5 hr with progesterone, p33 ringo ,and p33 ringo + progesterone, respectively. ( C ) Oo-cytes were either injected with in vitro-transcribedmRNAs (500 pg/oocyte) encoding Myc-tagged formsof the two isoforms of p33 ringo (ls26 and ls27) andp39 mos or treated with progesterone (5 µg/ml) andthen GVBD was scored. Novel protein involved in oocyte maturationGENES & DEVELOPMENT 2179  tested whether malE–p33 ringo was able to induce oocytematuration in the presence of protein synthesis inhibi-tors. Preincubation of the oocytes with cycloheximide(10 µg/ml) is known to block progesterone-inducedmaturation, consistent with the known essential re-quirement for de novo translation of maternal mRNAs.However, we found that cycloheximide had no effect onthe ability of malE–p33 ringo to induce MPF activationand GVBD (Fig. 4).We investigated the kinetics of activation of p42 mpk1 and p34 cdc2 /cyclin B in oocytes induced to mature bymalE–p33 ringo . Consistent with previous work, proges-terone treatment activated both p42 mpk1 and pre-MPF atthe same time, whereas malE–p39 mos injection activatedp42 mpk1 before pre-MPF (Fig. 4). In contrast, injection ofmalE–p33 ringo rapidly activated histone H1 kinase(which correlated with Tyr dephosphorylation ofp34 cdc2 ) somewhat before myelin basic protein (MBP) ki-nase activity and p42 mpk1 phosphorylation, indicatingthat p34 cdc2 /cyclin B preceded p42 mpk1 activation. Inter-estingly, the ability of malE–p33 ringo to activate p34 cdc2 /cyclin B, as determined by both increased histone H1kinase activity and disappearance of the band with re-duced electrophoretic mobility in p34 cdc2 immunoblots,was unaffected in the presence of cycloheximide (Fig. 4).In these oocytes, however, the kinase activity on histoneH1 was not stable and decreased after GVBD (Fig. 4) sug-gesting that p34 cdc2 /cyclin B might be transiently acti-vated by p33 ringo in cycloheximide-treated oocytes. Onthe other hand, oocytes injected with malE–p33 ringo inthe presence of cycloheximide had no detectable MBPkinase activity and most of their p42 mpk1 was unphos-phorylated, suggesting that activation of p42 mpk1 wasvery much reduced (Fig. 4). This observation suggeststhat the activation of p42 mpk1 by p33 ringo may be theconsequence of positive feedback loops that are blockedby cycloheximide. Thus, the function of p33 ringo is morelikely to be related to the activation of p34 cdc2 ratherthan of p42 mpk1 . We also found that as in the case ofprogesterone and p39 mos , oocyte maturation induced byp33 ringo involves de novo synthesis of cyclins B1 and B4(Fig. 4).  p33  ringo  is required for progesterone-induced oocytematuration To evaluate the importance of p33 ringo for oocyte matu-ration we used antisense oligonucleotides. We firsttested the oligonucleotides in an in vitro system andfound that some antisense but not the control oligo-nucleotides could efficiently target the two isolatedp33 ringo clones (Fig. 5A, ls26 and ls27). The p33 ringo an-tisense oligonucleotides, however, did not trigger degra-dation of the synthetic p39 mos mRNA under the same Figure 3.  Amino acid sequence comparison of thels26 and ls27 p33 ringo clones. Identity is indicated bya vertical bar. Figure 4.  MPF activation by recombinant malE–p33 ringo in cycloheximide-treated oocytes. Oo-cytes were injected with malE–p33 ringo (40 ng) ormalE–p39 mos (40 ng), or treated with progesterone(5 µg/ml) in the presence or absence of 10 µg/mlcycloheximide, as indicated. Cycloheximide wasadded 30 min prior to the injection or progester-one treatment and maintained during the subse-quent incubation. At the indicated times, groupsof five oocytes were collected, lysed, and analyzedboth by in vitro kinase assay using as a substrateeither histone H1 or MBP and by immunoblottingwith anti-p42 mpk1 , anti-p34 cdc2 , and anti-cyclinsB1 and B4 antibodies. Ferby et al.2180 GENES & DEVELOPMENT  conditions (Fig. 5A, p39 mos ). Next, we injected thep33 ringo antisense oligonucleotides into oocytes andfound that they were able to strongly delay progesterone-induced oocyte maturation, whereas injection of controloligonucleotides had no inhibitory effect (Fig. 5B). ByNorthern blot we found that the p33 ringo antisense oli-gonucleotides were able to ablate the endogenousp33 ringo mRNA(s) but not the endogenous p39 mos mRNA(Fig. 5C).To test the specificity of the antisense oligonucleo-tides, we tried to rescue the inhibition of progesterone-induced GVBD by adding back small amounts of purifiedp33 ringo protein. For this experiment, oocytes that hadbeen injected with control or antisense oligonucleotideswere injected a second time with different amounts ofpurified malE–p33 ringo prior to progesterone stimulation.As shown in Figure 6A, the inhibitory effect of thep33 ringo antisense oligonucleotide on progesterone-in-duced maturation, could be readily reversed by coinjec-tion of only 2.5 ng of malE–p33 ringo , an amount that wasnot enough to trigger oocyte maturation in the absenceof progesterone. These results indicate that the p33 ringo Figure 5.  Inhibition of progesterone-induced oocyte matura-tion by p33 ringo antisense oligonucleotides. (  A ) In vitro-tran-scribed mRNAs encoding the two isoforms of p33 ringo (ls26 andls27) and p39 mos were translated in rabbit reticulocyte lysateswith [ 35 S]methionine either alone (lane  1 ) or in the presence ofp33 ringo antisense oligonucleotides (lanes  2–8 ) or with controloligonucleotides (lane  9 ) and analyzed by SDS-PAGE and auto-radiography. ( B ) Oocytes were injected with 100 ng of the indi-cated oligonucleotides and incubated for 6 hr prior to progester-one stimulation. GVBD was scored at the indicated times. Weobserved essentially the same results in four experiments usingtwo different oligonucleotide preparations. ( C ) RNA was ex-tracted from groups of 20 oocytes taken after 16 hr of incubationas in  B  and analyzed by Northern blot using as a probe thep33 ringo cDNA ( top ). The same RNA blot was reprobed with thep39 mos cDNA ( bottom ). Figure 6.  Requirement for endogenous p33 ringo during proges-terone-induced oocyte maturation. (  A ) Oocytes were injectedwith 100 ng of either antisense-6 or control-9 oligonucleotides(indicated by + and −, respectively) and incubated for 6 hr priorto the injection of the indicated amounts of malE–p33 ringo and/or progesterone stimulation. After 8 hr, GVBD was scored andgroups of five oocytes were taken, lysed, and analyzed by im-munoblotting with anti-p34 cdc2 and anti-p42 mpk1 antibodies. ( B )Oocytes were either noninjected or injected with 100 ng of theindicated oligonucleotides and incubated for 6 hr prior to pro-gesterone stimulation. After 12 hr, lysates were prepared fromgroups of five oocytes and analyzed by immunoblotting usingaffinity-purified anti-p33 ringo antibodies. In lanes  5  and  6 , theantibodies were preincubated with malE–p33 ringo protein beforeimmunoblotting. The arrow indicates one band that cross-re-acts with anti-p33 ringo antibodies in mature oocytes. ( C ) Groupsof 25 oocytes were injected with the indicated oligonucleotidesand incubated with [ 35 S]methionine in the presence or absenceof progesterone. After 14 hr, oocytes were lysed and immuno-precipitated with either preimmune (lanes  1 , 2 ) or anti-p33 ringo antiserum (lanes  3–6 ). The arrows indicate two proteins that areimmunoprecipitated by anti-p33 ringo antibodies in mature oo-cytes. Novel protein involved in oocyte maturationGENES & DEVELOPMENT 2181


Dec 31, 2018
Similar documents
View more...
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks