Mitogen-activated Protein Kinase Kinase Is Required for the Mos ...

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Jul 5, 1994 - The product of the c-mos proto-oncogene functions ..... Thomas. G. (1992) Cell 68.3-6. ~~. Anderson, N. G., Maller, J: L., Tonks, N. IC, and Sturgill, T. W. (1990) .... Leevers, S. J., and Marshall, C. J. (1992) EMBO J. 11,569-574.

THE JOURNALOF BIOIOXCAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 45, Issue of November 11, pp. 2835628358, 1994 Printed in U.S.A.

Mitogen-activated Protein Kinase KinaseIs Required for the Mos-induced Metaphase Arrest* (Received for publication, July 5, 1994, and in revised form, August 19, 1994)

Hidetaka KosakoSP, Yukiko Gotohf, and Eisuke Nishidaa From the $Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-01 and the §Department of Biophysics and Biochemistry, Faculty of Science, University ofTokyo, Hongo, Tokyo 113, Japan

Theproductof the c-mos proto-oncogene functions The product of the c-mos proto-oncogene, a 39-kDa germ-cellnot onlyas an initiator of oocyte maturation but also as specific serinelthreonine protein kinase, an is active component a componentof cytostatic factor thatcauses the natural of CSF (7). The Mos protein is absent from immature oocytes, is arrest of the unfertilized egg at the second meiotic met- synthesized from maternal mRNA in response to progesterone, aphase. It has been shown thatMos can phosphorylate and then is degraded after fertilization (8, 9). Translation of and activate mitogen-activated protein( M A P ) kinase ki- Mos is necessary for progesterone-induced oocyte maturation nase (MAPKK) in vitro, leading to activation of MAP ki- (lo), and the Mos protein alone can initiate maturation without nase. In this study, by using an anti-MAPKK antibody any hormonal stimulation, as shown by injecting Mos mRNAor that can specifically inhibit Xenopus MAPKK activity, bacterially-expressed Mos protein into oocytes (8, 11).The Mos MAPKK mediates thecytostatic fac- mRNA or the Mos protein injected into one blastomere of a we have shown that tor activity of Mos. Coinjection of this anti-MAPKK an- two-cell embryo mimics CSF and arrests cleavage of the intibody with the bacterially expressedMos protein into a jected half of the embryo at metaphase (7, 11).Moreover, the two-cell embryo prevented the Mos-induced cleavage arrest as well as the Mos-inducedMAP kinase activation. CSF activity in the unfertilized egg cytoplasm is neutralized The analysis of individual embryos indicated that the with anti-Mos antibody (7). Thus, Mos is thoughtt o play crucial roles in both the release from prophase arrest in immature degree of the cleavage arrest was correlated with the oocytes and the induction of meiotic metaphase arrest in the extent of the MAP kinase activation in the Mos- and the unfertilized egg. Moslantibody-iqjected embryos. These observations suggest the involvement of a signal transmission path- Mitogen-activated protein ( M A P ) kinase is also activated way consisting of Mos, MAPKK, and MAP kinase in the during Xenopus oocyte maturation (12-14). Thisserine/ threonine protein kinase is highly conserved throughout evometaphase arrest. lutionandisactivated commonly by variousextracellular stimuli (15-18). Activation of MAP kinase requiresphosphorylFully grown Xenopus laevisoocytes are naturally arrested at ation on both tyrosine and threonine residues (19, 20). A 45the first meiotic prophase. Progesterone releases this arrest kDa protein that can inducephosphorylation and activationof kinase in vitro was purified from Xenopus unfertilized and induces progression through meiosis, leading to the production of the unfertilized egg, which is arrested at the second eggs (21) andfrom mammalian somatic cells (22-25). This MAP meiotic metaphase (metaphase 11). During this meiotic matu- kinase activating factor (for review, see Ref. 26) can undergo ration process, a key event is the activation of maturation- autophosphorylation on serine, threonine, and tyrosine resikinase-deficient mutant of promoting factor (MPF)’ that causes germinal vesicle break- dues (23,271 and phosphorylate the down and chromosome condensation (1).MPF, a complex of the MAP kinase on the regulatory tyrosine and threonine residues B, is ubiqui- (22,24,28-30). Therefore, this factor is a dual specificity protein serinelthreonine protein kinase~ 3 4 “and ~ ‘ cyclin ~ also tous in eukaryotes and promotes transition from G,to M phase kinase and has been called MAP kinase kinase MAPKK (28,3 1called MEK). cDNA cloning and sequencing of in both meiosis and mitosis (2-5). The activationof MPF precedes germinal vesicle breakdown, 34) revealedthat vertebrate MAPKK shows high similarities to and its activity drops after metaphase I and then rises again several yeast protein kinases functioning in various signal that the “APkinase I1 arrest in the unfer- transduction pathways, suggesting and remains high during the metaphase tilized egg. Fertilization releasesthis meiotic arrest and brings cascade functions universally in eukaryotes(35, 36). MAPM is inactivated by protein phosphatase2A treatment of MPF. about the degradationof cyclin B and the inactivation A cytostatic factor (CSF) present in theunfertilized egg is be- in vitro (21, 37), and activationof MAPKK in cells is accompalieved t o stabilize MPF, resulting in the metaphase I1 arrest (6). nied by its phosphorylation on serine and threonine residues (27,38). Therefore, MAPKK itself is thought tobe activated by * This workwas supported in part by grants-in-aidfrom the Ministry phosphorylationcatalyzed by an upstream serinelthreonine kinase (39, 40). Several MAPKK kiof Education, Science and Culture of Japan, the Asahi Glass Founda- protein kinase, tion, and the Toray Science Foundation. The costsof publication of this nases have been reported, including Raf-l(41-43), MEKK (441, article were defrayed in part by the payment of page charges. This and Mos (45, 46). Bacterially expressed Mos protein can actiarticle must thereforebe hereby marked “advertisement”in accordance vate MAP kinase wheninjected into immatureoocytes or added with 18 U.S.C. Section 1734 solely to indicate this fact. to cell-free extracts ofXenopus oocytes ( 4 6 4 8 ) a n dcan directly 8 Recipient of JSPS Fellowships for Japanese Junior Scientists. ll To whom correspondence should be addressed: Institute for Virus phosphorylate and activateMAPKK in vitro (45, 46). Research, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan. Fax: 81MPF, durMos, MAPKK, and MAP kinase are activated, like 75-751-3992. ing Xenopus oocyte maturation and are inactivated shortlyafTheabbreviationsused are: MPF, maturation-promoting factor; CSF, cytostatic factor; M A P , mitogen-activated protein;M A P K K , MAP ter fertilization (8, 9, 12-14, 21). MPF is reactivated in subsequent mitoticcell cycles, although neitherMos nor MAP kinase kinase kinase.

(MAP=,

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Requirement for MAP Kinase Kinase in CSF Arrest seems to be reactivated markedly during earlyembryogenesis. These suggest that theMAPKWMAP kinase cascade may mediate someof the actions ofMos.Recently,we produced an anti-Xenopus MAPKK antibody that can specifically inhibit Xenopus MAPKK activity in vitro (49). Microinjection of this neutralizing antibody against MAPKK into immature oocytes prevented progesterone- or Mos-induced activation of MAP kinase (49). Moreover,progesterone- or Mos-induced activation of MPF was also blocked, as evidenced by inhibition of both germinal vesicle breakdown and histone H1 kinase activation (49). This suggested that MAPKK plays a significant role in mediating thematuration-initiating function of Mos. In thisreport, we have examined whether MAPKK mediates another function of Mos, a function as a component of CSF. Injection of bacterially expressed Mos protein into a two-cell embryoinduced MAP kinase activation and cleavage arrest, while coinjection of the neutralizing antibody against MAPKK with the Mos protein prevented the cleavage arrest aswell as theMAP kinase activation. These results indicate that MAPKK is required for the Mos-induced metaphase arrest. MATERIALS AND METHODS Reagents-The neutralizing antibody against Xenopus MAPKK was prepared by injecting mice with bacterially expressed glutathione Stransferase-MAPKK as described previously (49). Recombinant MAPKK was prepared by cleaving glutathione S-transferase-MAPKK with Factor Xa (28) and by removing glutathione S-transferase with a glutathione-agarose column. Recombinant mulE-mos protein (Xenopus c-mos fused downstream of the maltose-binding protein of Escherichia coli) was expressed in and purified from E. coli as described previously (11, 47,49). Immunoblotting-After SDS-polyacrylamidegel electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore) in a solution containing 25 mM Tris, 192 mM glycine, and 20% methanol. After blocking with 5% skim milk (Difco)in TBS-T (20 mMTris-CI (pH 7.6), 137 mM NaCl, 0.1% Tween-20),membranes were incubated with mouse anti-MAPKKneutralizing antibody or with mouse monoclonal anti-phosphotyrosine antibody (4G10, UBI), or with rabbit anti-MAP kinase antibody (50) in TBS-T for2 h. Immunoreactive bands were detected by horseradish peroxidase-conjugated second antibodies and the ECL Western blotting detection system (Amersham Corp.). Preparation of Cell-free Extructs-Concentrated cell-free extracts were prepared from Xenopusimmature oocytes essentially as described by Shibuya et ul. (51). Dissected ovaries were treated with 2 mg/ml of collagenase in MBS (10 m HEPES pH 7.5,88 mM NaCl, 1mM KC], 2.4 m NaHCO,, 0.3 mM Ca(NO,),, 0.41 mM CaCl,, 0.82 m MgSO,) for 2 h a t 18 "C and then extensively washed with MBS. Stage VI oocytes were sorted by hand, washed twice in EB (20 mM HEPES pH 7.2, 0.25 M sucrose, 0.1 M NaCI, 2.5 mM MgCI,) and transferred to a 1.5-ml tube containing EB with aprotinin, pepstatin, chymostatin, and leupeptin (each 10 pg/ml) and with cytochalasin B (50 &mU. Excess EB was removed, and oocytes were crushed by centrifugation a t 15,000 x g for 15 min at 2 "C. The supernatant between the lipid cap and packed yolk was collected and centrifuged again. Aliquots were frozen in liquid nitrogen and stored a t -80 "C. Microinjections-Ovulated eggs were fertilized in vitro with fresh minced testis anddejellied with 2% cysteine (pH 7.8). Dejelliedembryos were washed several times with 0.3 x MBS and then cultured in 0.3 x MBS containing Ficoll 400 (Pharmacia Biotech Inc.) at 22 "C. 90-105 min after fertilization, 50 nl of samples were microinjected into one blastomere of the two-cell embryo using an IM-1 microinjection apparatus (Narishige, Tokyo). Embryos were cultured for 3 h under the same conditions as above and scored for the extent of cleavage arrest. For assays of MAP kinase, groups of 10 embryos or individual embryos were homogenized in 200 or 50 pl of XB (20 mM Tris-CI (pH 7.5), 60 m P-glycerophosphate, 10 mM MgCI,, 10 mM EGTA, 2 m dithiothreitol, 1 mM Na,VO,, 1 mM phenylmethylsulfonyl fluoride, 20 pg/ml aprotinin), respectively. Homogenates were clarified by centrifugation at 15,000 x g for 15 min at 2 "C.

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FIG.1.Reactivity of t h e anti-Xenopus MAPKK neutralizing antibody. Extracts prepared from Xenopus blastula embryos (80 pg of protein, lunes 2 and 5 ) , bacterially expressed recombinant MAPKK (0.1 pg of protein, lunes 3 and 6 ) ,and bacterially expressed mal&-mos (0.95 pg of protein, lunes 4 and 7) were electrophoresed in a 10%acrylamide gel. Molecularweight standards were electrophoresed on lune 1.The gel was stained with Coomassie Blue (lunes 1 4 ) or transferred to Immobilon (Millipore) membrane (lunes 5-7). The membrane was immunoblotted with the anti-MAPKK antibody (0.17 pg/ml).

specificallyinhibit the activity ofXenopus MAPKK as described previously (49). Immunoblot analysis revealed that this antiMAPKK antibody (mouse polyclonal antibody) reacted specifically with the 45-kDa MAPKK band in total extracts of embryos at the blastula stage(Fig. 1,lanes 2 and 5).This antibody reacted strongly with bacterially expressed recombinant MAPKK (Fig. 1,lanes 3 and 6)but not at all with bacterially expressed Mos (malE-mosprotein, a fusion protein between the E. coli maltose-binding protein and theXenopus c-mos protein kinase; Fig. 1, lanes 4 and 7). The recombinant MAPKK contains 11extra amino acids (GIPGNSALTPN) on the authentic N-terminus of MAPKK (28). To examine whether this anti-MAPKK antibody prevents Mos-induced activation of MAP kinase in a cell-free system, we prepared concentrated cell-free extracts from immature oocytes (51). ARer incubation with control mouse IgG or the same amount of the anti-MAPKK antibody at 0 "C, malE-nos was added to the extracts, and the incubation was performed at 22 "C. The malE-mos induced full activation of MAP kinase within 1h in the control IgG-incubated extracts, as judged by the shift in electrophoretic mobility of the 42-kDa polypeptide recognized by anti-MAPkinase antibody (Fig. 2, lanes 5-8).In contrast, the activation of MAP kinase was markedly diminished in the anti-MAPKK antibody-incubated extracts (Fig. 2, lanes 9-12).When this anti-MAPKK antibody had been preincubated with the recombinant MAPKK, MAP kinase activation induced by malE-mos was restored (Fig. 2, lanes 13-16). Thus, the neutralizing antibody against MAPKK blocked the malEmos-induced activation of MAP kinase by inhibiting the MAPKK activity in a cell-free system. Mos-induced Metaphase Arrest Is Inhibited in the Embryos Injected with the Neutralizing Antibody against MAPKK-It has been shown that microinjection of malE-mos into one blastomere of a two-cell embryoresults incleavage arrest atmitotic metaphase (11)and inrapid activation of MAP kinase (46). To examine the effect of the neutralizing antibody against MAPKK on the Mos-induced metaphase arrest, wemicroinRESULTS jected malE-mos with the control IgG orwith the anti-MAPKK The Neutralizing Antibody against MAPKK Prevents Mos- antibody into one of the blastomeres of two-cell embryos. Miinduced Activation of MAP Kinase in Cell-free Extracts of Xe- croinjection of mulE-nos with the control IgG resulted in cleavnopus Oocytes-We prepared a neutralizing antibody that can age arrest at the injected half of embryos and caused hemiblas-

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IgG IgG (aMAPKK+MAPKK) aMAPKK FIG. 2. The anti-MAPKK neutralizing antibody prevents the maZE-me-induced activation of MAP kinase in a cell-free system. Phosphate-buffered saline (0.5 p1) containing control mouse IgG or anti"APKK antibody (each 2.8 pg) was mixed with 0.5 p1 of buffer or recombinantMAF'KK (0.8pg) for 30 min at 0 "C. Then, the mixtures (1 pl) were incubatedwithxenopus cell-free extracts (8 p1) for 30 min at 0 "C. After the addition of 0.5 pl of buffer or maZE-mos(0.19pg) plus 0.5 pl of a solution consisting of 10 mMATP, 10 mM MgCl,, 0.1 M creatine phosphate, and 0.5 mg/ml creatine kinase, the incubation was performed at 22 "C for the indicated times. Samples (each 0.8 p1) were analyzed by immunoblotting with anti-MAPkinase antibody.

tulation (Fig. a). There often existed embryos in which part or whole of the uninjected half also ceased cleavage probably because of the leak of the injected samples (Fig. 3, B and C).In contrast, microinjection of malE-mos with the anti-MAPKK antibody induced no cleavage arrest (Fig. 30) or the arrest in the limited area at the injected half (Fig. 3, E andF). The result is represented by the histogram (Fig. 41,in which results obtained from four independent experiments are summarized. The result indicated that the Mos-induced metaphase arrest could be canceled by the neutralizing antibody against MAPKK (Figs. 3 and 4). Microinjection of malE-mos with the control IgG induced marked activation of MAP kinase, as evidenced by the appearance of the electrophoretically retarded MAP kinase band on SDS-polyacrylamide gel electrophoresis (Fig. 5, lane 3,upper) and the corresponding anti-phosphotyrosine-positiveband (Fig. 5, lane 3, lower). In contrast, microinjection of malE-mos with the anti-MAPKK antibody induced almost no appearance of the electrophoretically retarded, tyrosine-phosphorylated form of MAP kinase that represents the activated form of the kinase (Fig. 5, lane 4, upper and lower).Thus, the neutralizing antibody against MAPKK inhibited the Mos-induced metaphase arrest and the Mos-induced MAP kinase activation. To confirm that theeffect of the anti-MAPKK antibody was specifically due to the inhibition of MAPKK activity, we microinjected malE-mos and the anti-MAPKK antibody together with or without the recombinant MAPKK into one of the blastomeres of two-cell embryos.The embryos injected withmalEmos, the anti-MAPKK antibody,and the recombinant MAPKK showed cleavagearrest (Fig. 6 A , lower) and MAP kinase activation (Fig.6B,lane 4 ) to greater extent than did the embryos injected with malE-mos and the anti-MAPKK antibody (Fig. 6 A , upper and Fig. 6B,lane 3). Thus, the effect of the neutralizing antibody against MAPKK was antagonized by the addition of the recombinant MAPKK. The result that theextent of cleavage arrest inFig. 6A was less than thatin Fig. 4 is due to a lesser amount of injected malE-nos in theexperiment shown in Fig. 6. We then examined in more detail the correlation betweenthe cleavage arrest and the MAP kinase activation by analyzing embryos individually.We arbitrarily selected 16 embryos that had been injected with malE-mos and the control IgG or with malE-mos and the anti-MAPKK antibody. After observingthe extent of cleavage arrest of each embryo, that embryo was lysed, and the lysate was analyzed by immunoblotting with anti-MAP kinase antibody. Nearly full activation of MAP kinase had occurred in theembryos of which whole area showed cleavage arrest (Fig. 7, lanes 1 and 2).In theembryos of which

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FIG.3. Morphology of embryos injected with m Z E - m e plus control IgG or with maZE-mos plus the anti-MAPKK antibody. A-C, animal-pole view of distinct embryos injected with malE-mos (3.4 ng) plus controlIgG (0.34 pg). D-F, animal-pole viewof distinct embryos injected with malE-mos (3.4 ng) plus anti-MAF'KK antibody (0.34 pg). Embryos in which oneof the blastomeres (the right sideof the embryos) was injected at the two-cell stage were cultured for 3 h at 22 "C before being fixed in 1%glutaraldehyde.

over the half area showed cleavagearrest, 2 0 4 0 % of total MAP kinase molecules were activated even in the embryos injected with malE-mos and the anti-MAPKK antibody (Fig. 7, lanes

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Frc. 4. Effect of the anti-MAPKK neutralizingantibody on the maZE-mos-induced cleavage arrest. One blastomere of a two-cell embryo was injected with 50 nl of either malE-mos(3.4ng) plus control IgG (0.34pg) or malE-mos (3.4ng) plus anti-MAPKK antibody (0.34 pg). Injected embryos were culturedfor 3 h a t 22 "C and scored forthe extent of cleavage arrest, which was classified into four types. Type I includes embryos showing no cleavage arrest (typically Fig. 30).Type I1 includes embryos in whichbelow a quarter area shows cleavage arrest (typically Fig.3,E and F ) . Type I11 includes embryos in which over the half but not the whole area shows cleavage arrest (typically Fig. 3,A and B ) . Type IV includes embryos in which whole the area showscleavage arrest (typically Fig. 3C).Results obtained from four independent experiments are summarized as the histogram shown here. In each experiment, injected embryos were derivedfrom eggs laid by one frog.

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FIG.6.Effect of the anti-MAPKK neutralizingantibody with or without r e c o m b i n a n t MAPKK on the malE-mos-inducedcleavage arrest (A) and the malE-mos-induced activationof MAP kinase ( B ) .One blastomereof a two-cell embryo was injected with 50 nl of either mal&-mos (1.4 ng) plus anti-MAPKK antibody (0.22 pg) or aP-Tyr f MAPK malE-mos (1.4ng) plus anti-MAPKK antibody(0.22 pg) plus recombinant MAPKK (50 ng). A, injected embryos were cultured for 3 h a t 22 "C, scored for the extentof cleavage arrest, and classifiedas in Fig. 4. B, after being scored for the extent of cleavage arrest, 10 embryos FIG.5. Effect of the anti"APKK neutralizing antibody on the injected with malE-mos and anti-MAPKK antibody (lane 3)or 10 emmdE-mos-induced activation of MAP kinase. In one of the four bryos injected with malE-mos, anti-MAPKK antibody, and recombinant experiments (thesecond one) in Fig.4,after being scoredfor the extent MAPKK (lane 4) were lysed and analyzed by immunoblotting with of cleavage arrest,10 embryos injected with malE-mos plus control IgGanti-" kinase antibody (upper) and anti-phosphotyrosine antibody (lane 3) or with maZE-mos plus anti-MAPKK antibody (lane 4 ) were (lower).The unfertilized eggs(lane 1 ) and uninjected embryos(lane 2) lysed and analyzedby immunoblotting with anti-MAP kinase antibody were also analyzed a s above. (upper) and anti-phosphotyrosine antibody (lower). The unfertilized eggs (lane 1) and uninjected embryos (lane 2) were also analyzed as function as a MAPKK kinase (45-48).MAP kinase is originally above.

3-9). In contrast, the activation of MAP kinase was undetectable in the embryos of which noneor below a quarterof the area showed cleavagearrest (Fig. 7, lanes 10-16).Thus, the extent of the cleavage arrest was correlated with that of the MAP kinase activation. DISCUSSION

Recent studies from several laboratories demonstrated that Mos lies upstream of the M A P K K / " kinase cascade and can

identified as a serinelthreonine protein kinase that is activated rapidly by a variety of mitogens. As MAP kinase may have a critical role in anetwork of protein kinases in mitogenic signal transductions, it is thought that Mos, normally expressed at significant levels onlyin germ cells, can work as a transforming oncogene, when expressedin somatic cells,through activating the MAP kinase pathway. MAP kinase is now recognized as an important element in several signaling pathways in eukaryotes (35, 36). In Xenopus oocytes, MAP kinase and MAPKK are activated during meiotic maturation (12-14, 21). Recently, we

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4. Murray, A. W., and Kirschner, M. W. (1989) Science 246,614-621 5. Nurse, P. (1990.)Nature 344, 503-508 6. Masui, Y., and Markert, C. L. (1971) J. Exp. Zool. 177, 129-146 7. Sagata, N., Watanabe, N., Vande Woude, G. F., and Ikawa, Y. (1989) Nature 342,512-518 8. Sagata, N., Daar, I., Oskarsson, M., Showalter, S. D., and Vande Woude, G. F. (1989) Science 245,643-646 9. Watanabe, N., Vande Woude, G. F., Ikawa, Y., and Sagata, N. (1989) Nature 342,505-511 10. Sagata, N., Oskarsson, M., Copeland, T., Brumbaugh, J., and Vande Woude, G. L F. (1988) Nature 335,519-525 1 v 1 v I l l 111 111 111 111 111 111 11 11 1' I1 I I I 11. Yew, N., Mellini, M. L., and Vande Woude, G. F. (1992)Nature 355,649-652 12. Ferrell, J. E., Wu, M., Gerhart, J. C., and Martin, G. S. (1991)Mol. Cell. Biol. mal€-mos mal€-mos 11,1965-1971 13. Gotoh. Y.. Nishida. E.. Matsuda. S.. Shiina. N.. Kosako, H., Shiokawa, K., aMAPKK IgG Akiyama, T., Ohta, k, and Sakai; H. (1991) Nature 349,251-254 FIG.7. Analysis of both the extent of cleavage arrest and the 14. Posada, J., Sanghera, J., Pelech, S..Aebersold, R., and Cooper,J. A. (1991)Mol. Cell. Bid. li,2517-2528 extent of MAP kinase activation in individual embryos injected with malE-mos and control IgG or with malE-mos and anti- 15. Cobb, M. H., Boulton, T. G., and Robbins, D.J. (1991) Cell Regul. 2,965-978 Pelech, S. L.,and Sanghera, J. S. (1992) %rids Biochem. Sci. 17,233-238 MAPKK antibody. In one of the four experiments (thefourth one) in 16. 17. Ruderman, J. V. (1993) Curr. Opin. Cell B i d . 5, 207-213 Fig. 4, each embryo injected withmaZE-mos and control IgG (lanes 1-6) 18. Thomas., G. (1992) .~ ~.Cell 68.3-6 or with malE-mos and anti-MAPKK antibody (lanes 7-16) was scored 19. Anderson, N. G., Maller, J: L., Tonks, N. IC, and Sturgill, T.W. (1990)Nature for the extentof cleavage arrest andclassified as in Fig. 4 (Z-ZV). Then, 343,651-653 20. Payne, D.M., Rossomando, A. J., Martino, P., Erickson, A.K., Her, J.-H., each embryo was lysed individually and analyzed by immunoblotting Shabanowitz, J., Hunt,D. F., Weber, M. J., and Sturgill,T. W. (1991)EMBO with anti-" kinase antibody. J. 10,885-892 21. Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11,973-982 reported that inhibition of activation of MAP kinase by the C.M., and Erikson, R. L. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, neutralizing antibody against MAPKK blocked Mos-induced 22. Crews, 8205-8209 oocyte maturation (49). In thispaper, we have shown that this 23. Nakielny, S., Campbell, D. G., and Cohen, P. (1992)FEBS Lett. 308, 183-189 R., Ahn, N. G., Posada, J., Munar, E. S., Jensen, A. M., Cooper, J. A., anti-MAPKK antibody, when introduced into one blastomereof 24. Seger, Cobb, M. H., and Krebs, E. G. (1992) J. Biol. Chem. 267, 14373-14381 a two-cell embryo, antagonizes the ability ofMos to induce 25. Shirakabe, K., Gotoh, Y., and Nishida, E. (1992) J. Biol. Chem. 267, 1668516690 metaphase arrest in cleavingembryos. Thus, MAPKK is 26. Ahn, N. G.,Seger, R., and Krebs, E. G. (1992)Curr: Opin. Cell Bid. 4,992-999 thought to play a crucial rolein both initiating oocyte matura- 27. Kosako. H.. Gotoh, Y., Matsuda, S., Ishikawa, M., and Nishida, E. (1992) EMBO J. 11,2903-2908 tion and inducing metaphase arrest downstream of Mos. AlKosako, H., Nishida, E., and Gotoh, Y. (1993) EMBO J. 12,787-794 though MAPKK may have other substrates than MAP kinase 28. 29. Nakielny, S.,Cohen, P., Wu, J., and Sturgill,T.(1992)EMBOJ. 11,2123-2129 and one of these might be the crucial target or functionally 30. Posada, J., and Cooper, J. A. (1992) Science 255,212-215 redundant with MAP kinase, it is hypothesized that MAP ki- 31. Ashworth, A,, Nakielny, S., Cohen, P., and Marshall, C. (1992) Oncogene 7, 2555-2556 nase, the only identified substrate for MAPKK, is necessary for 32. Crews, C. M., Alessandrini, A,, and Erikson,R. L. (1992)Science 258,478480 inducing metaphase arrest. This, however, remains to be tested 33. Seger, R., Seger, D., Lozeman, F. J., Ahn,N. G., Graves, L. M., Campbell, J. S., Ericsson, L., Harrylock, M., Jensen, A. M., and Krebs, E. G. (1992)J. B i d . directly inthe futurework. The mechanismby which the same Chem. 267,25628-25631 protein kinase participates in these apparently different cellu- 34. Wu, J., Harrison, J. IC, Vincent, L. A,, Haystead, C., Haystead, T., Michel, H., Hunt, D., Lynch, IC R., and Sturgill, T.W. (1993) Proc. Natl. Acad. Sci. lar events is unclear, but MAP kinase may directlyor indirectly U.S. A. 90,173-177 regulate a factor(s)involved inactivation and/or stabilization of 35. Errede, B., and Levin, D. E. (1993) Cum Opin. Cell B i d . 5,254-260 36. Nishida, E., and Gotoh, Y. (1993) ?Fends Biochem. Sci. 18, 128-131 MPF. Gomez, N., and Cohen, P. (1991)Nature 353,170-173 Recently, Maller and co-workers (52) reported that microin- 37. 38. Ahn, N. G., Campbell, J. S., Seger, R., Jensen, A. L., Graves, L. M., and Krebs, jection of thiophosphorylated MAP kinase into one blastomere E. G. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,5143-5147 of a two-cell embryo induced cleavage arrest similar to that 39. Gotoh, Y., Matsuda, S., Takenaka, IC, Hattori, S., Iwamatsu.A., Ishikawa, M., Kosako, H., and Nishida, E. (1994) Oncogene 9, 1891-1898 induced by Mos (52), suggesting that active MAP kinase in the 40. Matsuda, S., Gotoh, Y., and Nishida, E.(1993)J. Bid. Chem. 268,32774281 41. Dent, P., Haser, W., Haystead, T.A. J., Vincent, L.A., Roberts, T.M., and unfertilized egg is sufficient for inducing metaphase I1 arrest. Sturgill, T.W. (1992) Science 257,1404-1407 Thus, the MAPKWMAP kinase cascade maymediate the CSF 42. Howe, L. R., Leevers, S. J., Gomez, N., Nakielny, S., Cohen, P., and Marshall, activity of Mos. The disappearance of CSF activity upon fertiliC. J. (1992) Cell 71,335-342 zation maybe due to the degradation of Mos and inactivation of 43. Kyriakis, J. M.,App, H., Zhang, 2.-F., Banerjee, P., Brautigan, D. L., Rapp, U., and Avruch, J. (1992) Nature 358,417A21 W K K and MAP kinase. Interestingly, in clam oocytes that 44. Lange-Carter, C. A,, Pleiman, C. M., Gardner, A. M., Blumer, K. J., and Johnson, G. L. (1993) Science 260,315-319 are not arrested at metaphase 11, activation of MAP kinase is A. R., Hill, C., Gomez, N., Cohen, P., and Hunt,T. (1993)FEBS Lett. not sustained and inactivated prior to germinal vesicle break- 45. Nebreda, 333, 183-187 down (53). 46. Posada, J., Yew, N., Ahn, N. G., Vande Woude, G. F., and Cooper, J. A. (1993) Mol. Cell. Biol. 13, 2546-2553 The previousreport that Ras has the CSF activity like Mos 47. Nebreda, A. R.,and Hunt, T.(1993)EMBO J. 12,1979-1986 K K / (54)can be explained by Ras-inducedactivation of the " 48. Shibuya, E. IC, and Ruderman, J. V. (1993) Mol. Biol. Cell 4,781-790 MAP kinase cascade (51, 55-57). Ras is supposed to induce 49. Kosako, H., Gotoh, Y., and Nishida, E. (1994)EMBO J. 13,2131-2138 Y., Moriyama, K., Matsuda, S., Okumura, E., Kishimoto, T., Kawasaki, metaphase arrest independently of Mos probably through 50. Gotoh, H., Suzuki, K., Yahara, I., Sakai, H., and Nishida, E. (1991)EMBO J. 10, Raf-1, another MAPKK kinase (41-43).The MAPKWMAP ki2661-2668 nase cascade may function at a convergent point in various 51. Shibuya, E. IC, Polverino, A. J., Chang, E., Wigler, M., and Ruderman, J. V. (1992) Proc. Natl. Acad. Sci. U.S.A. 89,9831-9835 signal transduction pathways resulting in metaphase arrest. 52. Haccard, O.,Sarcevic, B., Lewellyn, A., Hartley, R., Roy, L., Izumi, T., Erikson, E., and Maller J. L. (1993) Science 262, 1262-1265 Acknowledgments-We thank Tim Hunt for providing the plasmid 53. Shibuya, E. K., Boulton, T. G., Cobb, M. H., and Ruderman,J. V. (1992)EMBO J. 11,3963-3975 expressing maZE-mos and Hiroshi Y. Kubota for kind help in thepho54 Daar, I., Nebreda, A. R., Yew, N., Sass, P., Paules, R., Santos, E., Wigler, M., tography of Xenopus embryos. and Vande Woude, G. F. (1991) Science 253,74-76 55. Hattori, S., Fukuda, M., Yamashita, T., Nakamura, S., Gotoh, Y., and Nishida, REFERENCES E. (1992) J. Bid. Chem. 267,20346-20351 56 Itoh, T., Kaibuchi, IC, Masuda, T., Yamamoto, T., Matsuura, Y., Maeda, A., 1. Masui, Y., and Clarke, H. J. (1979) Int. Reu. Cytol. 57, 185-282 Shimizu, K., and Takai,Y. (1993)Proc. Natl. Acad. Sci. U.S. A. 90,975-979 2. Hunt, T. (1989) Curr. Opin. Cell Bid. 1, 268-274 57 Leevers, S. J., and Marshall, C. J. (1992) EMBO J. 11,569-574 3. Maller, J. L.(1990) Biochemistry 29,3157-3166 1

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