Control
of M-phase
by maturation-promoting
factor
M. Do&e Centre
de Recherches
sur la Biochimie Current
des Macromolecules, Opinion
in Cell Biology
Introduction The alternation between interphase and M-phaseis controlled by periodic changesin the activity of maturationpromoting factor (MPF). Recently,MPF was purikd from Xenopuseggs(khka et al, Proc NatlAcudSci USA1988, 85:3009-3013) and was shown to contain a p34 protein homologue of the yeast cell-cycle-controlgene c&2 (Gamier et al, Cell 1988, 54:433-439;Dunphy et al, Cell 1988,54:423-431>.Moreover, evidencewas obtained that cyclins, a family of proteins whose levels follow a sawtooth oscillation during the cell cycle, may control MPF activation in both marine invertebrates (Swenson et al., Cell 1986, 47867-870; Pines and Hunt, EMBO J 1987, 6:2987-2327;Hagan et al, J Cell Sci 1988,91:587-595).In this review, I discuss recent progress which clariIies the subunit structure of MPF and sheds some light on the mechanisms responsible for its periodic changes in activity during meiotic maturation and early embryogenesis in animal cells. The accompanyingreview by Glover (this issue, pp 258-261) will specikally focus on Drasophila.
MPF: identification Mitotic
and subunit structure
cdc 2 kinases
Arion et al. [l] substantially puriiied an H, histone kinase (HrK) from starfish oocytes, that had been shown to peak at M-phaseduring meiosis and early cleavagein echoderms (Picard et al, De0 Biol 1985, 109:311-320, and Dev Growth Dfl&. 1987, 291:93-103) and amphibians (Cicirelli et al, J Biol them 1988, 263:2009-2019; Labbe et al, Dev Biol 1988, 127:157-169). The iinal preparation contained CdQ, shown by immunoblotting and binding to p13=‘, a yeast protein which interacts physically with cdc2. Demonstration that the M-phase-specilic HrK corresponds to MPF came from Iabbe et al. [2] who also purified H,K from stariish oocytes (Labbk et al, Nature 1988, 335:251-254). The iinal preparation (specik activity, 0.5 l.rmol of phosphate per min/mg of enzyme to Hr histone) contained Cdc2 as the single major protein. MPF activity was found to co-migrate with H,K through-
CNRS and INSERM, Montpellier,
France
1990, 2:269-273
out purilication (no ATP-Crswas used at any time to ‘stabilize’MPF, see below). The mammalian growth-associated H1K, an enzyme whose activity is sharply elevatedat mitosis, was purified from Novikoff rat hepatoma cells [3]. The final preparation (spectic activity, 0.5 urn01of phosphate per min/mg of enzyme) also contained Cdc2. In addition, it was found to catalyzephosphorylation of the same sites in Hr as did Xen0pu.sMPF.An H,K containing Cdc2 was also purikd from human cervical carcinoma (HeIa) cells arrested at M-phase by nocodazole treatment [4]. The kinase was probably substantially purikl, although its ~%ralspeciiic activity was lower than star&h MPF by severalorders of magnitude. MpF is a heterodimer
containing
cyclin
and cdc2
Although starfish p34a retaining both H, kinase and MPF activities could be separated from other proteins following extensive purikation by conventional column chromatography,various lines of evidencesuggestedthat it formed part of a multiprotein complex in the living cell or when directly assayedin crude extracts. A 62 kD protein, subsequently identiiied as cyclin B [ 51, was found to coimmunoprecipitate with p34a from G2 but not Gl HeIa cells (Draetta and Beach, Cell 1988, 54:17-26X In the clam, Spisuh, anticyclin B immunoprecipitates were found to have H,K activity [ 61. Moreover, cyclin B coeluted with the main peak of H,K activity after gel iiltration of crude extracts, although a second Hr kinase peak of lower apparent M, was detected, which did not contain any cyclin. Cyclin B and cdc2 were also found to coelute with H,K activity at the last step, after its partial purification from sea-urchin mitotic extracts [7]. A cyclin ACdc2 complex was also reported to be a minor component of the M-phase-specificHrK in early clam embryos [6]. However, at variancewith anticyclin B reactivity, anticyclin A reactivity ran ahead of H,K activity on a sizing column, suggestingthat most cyclin A associates with proteins other than Cdc2. Using an improved procedure based on pl3aQephar ose a&inky chromatography, Iabbe et al [8] purilkd MPF to apparent homogeneity and with high recovery
Abbreviations CSF-cytostatic factor; H,K-H, histone kinase; MPF-maturation-promoting factor; mRNA-messenger RNA; PSTAIR+onserved ECVPSTAIREISLLKE region of cdc2.
Current
Science Ltd ISSN 0955*74
269
270
Cell multidication
(50%). At variancewith their previous results, they found the m itotic kinase (specific activity, 8 urn01of phosphate per m in/mg of protein to HI histone) to consist of a complex formed by a 1: 1 stoichiometric associationof Cdc2 with cyclin B, identified by direct m icrosequencing.Their previous Ending was explained if one assumedthat, during the alternative procedure which comprised six steps of conventional chromatography extended over 2 days, either the stoichiometric complex became disrupted or the cyclin component underwent proteol@s. In any case, the previous result indicates that an intact cyclin subunit is not required for Cdc2 kinase activity. Probably, cyclin B is required in vim for Cdc2 kinase activation. Whether MPF also exists as a cyclin A-CdQ complex during later development remains to be investigated since MPF was purified to homogeneity from oocytes at first meiotic metaphaseonly, which do not contain cyclin A
[61. Cyclin B and MPF activation p34a is stable during meiosis and early embryogenesis (Labti et al, 1988) [6,7]. However, protein synthesisis required at each cell cycle for MPF activation (Wagenaar, Exp Gdf Res 1983, 144:39-l; Gerhart et al, Cytobias 1985, 43~335-347;Picard et al, 1985 and 1987; Karsenti et al, Dev Bioll987, 119442453) making cyclin an attractive candidate for an MPF-activatorprotein. This was demonstratedby showing that speciEcdestruction of endogenous cyclin B messengerRNA (mRNA) arrests frogegg extracts in interphase [9] and that addition of exogenous cyclin B mRNA to similar extracts is sufficient to produce multiple cell cycles [lo]. There is a delay in viva between the time after which protein synthesis is no longer required and the time when kinase activation occurs. This suggeststhat, although cydin B accumulation is required for MPF activation, it is not the only factor involved in the activation process. Post-translationalmodification of either cyclin or Cdc2 may be required for complex formation. Human cells in early Gl synthesizecyclin B, but it is not associatedwith Cdc2 151.In the clam, it was proposed [ 111that maternal cyclin B is masked and the interaction with Cdc2 prevented until fertilization. Alternatively, part of the Cdc2 pool may be associated,through its conserved EGVPSTAIREISLLKE region (called PSTAIR),with an inhibitory molecule whose normal function is to prevent formation of the active cyclin B-Cdc2 complex. Indeed, m icroinjection of the PSTAIRpeptide was sufhcient to activate MPF in star&h oocytes [2], and it was found to accelerate its action in [email protected] (Gauthier et al, 1988). Further evident that cyclin may interact with Cdc2 through its PSTAIRregion came from Pines and Hunter [ 51, who showed that cyclin B does not coprecipitate with antibodies to the PSTAIRsequence.Formation of the cyclin B-Cdc2 complex is also not sufficient to activate MPF. Indeed the complex can be isolated from G2-arrested oocytes, yet it does not contain active MPF (IabM and I.e Bouffant, personal communication). Thus, other steps
in the pathway that leads to MPF activation may be subject to regulation.They may involve the product of homologues of the c&25+ gene,which acts in yeast as a dosedependent activator of the m itosis-inducing function of c&+ [ 12,131(Russeland Nurse, cell 1986,451:145-153).
Control of MPF activity by phosphorylation and dephosphorylation reactions Despite some early reports which emphasized the correlation between increased phosphoryfation of p34a and activation of its catalytic activity during the cell cycle (Draetta and Beach, 1988; Draetta et aL, Nature 1988, 336:73%743) there are good evidencesthat p34d dephosphoryfation controls MPF activation. In starfish, it was shown that while p34a is phosphorylated, its protein kinase activity is inhibited, preventing entry into Mphase, but once p34C&Qis dephosphorylated,its protein kinase activity increasesand M-phaseis initiated [ 21. Similar correlations were observed in Xenopus eggs [ 14,151, mammalian cells in culture [ 161 and yeast [ 181. Moreover, Cdc2 kinasecould be activatedin vitm simply by removing ATP from crude extracts [2] (Labb+ et al, 1988). Dephosphorylation of tyrosine accompaniescdc2 kinase activation during entry into m itosis [ 15-171. In yeast, the site of tyrosine phosphotylation has been mapped to tyrosine 15, a residue within the presumptive ATP-binding site. Substitution of this tyrosine by pheny&nine advanced cells prematurely into m itosis [ 171.In mouse 3T3 fibroblasts, inhibition of tyrosine dephosphoryfation by exposure of cells to vanadatewas found to arrest them reversiblyat G2. Nonetheless,specific dephosphoryfation of tyrosine residuesby phosphotyrosine phosphatase1B failed to activateCdc2 kinase in interphasic extracts on tyrosine, but also on threonine and serine residues,which undergo dephosphoryfation at the time of Cdc2 activation. The corresponding phosphataseis not known, but it is neither type 1 nor type 2A phosphatasebecauseit is not sensitive to okadaic acid [18] (Felix et al, EMBO J 1990, in press). Besides p34dCa dephosphoryfation, cyclin B phosphoryfation may be involved in Cdc2 kinase activation under physiological conditions. Indeed, both events occur almost simultaneouslyduring the first m itotic cell cycle in the sea urchin [7]. However, cyclin B in an excellent substrate for Cdc2 kinase, at least in vitro [8]. Thus, it is diKicult to conclude from temporal correlation alone that cyclin phosphorytation is causal for kinase activation. Nonetheless, other reports indicate that a phosphoryfation event is required for activation of the cdc2-cyclic B complex. Entry of sea-urchin eggs into m itotic metaphasewas shown to be reversibly prevented by 6dimethyfadenine, an inhibitor of protein phosphorylation [ 191.Although other interpretations are possible (see below), Cdc2 kinase activation in oocytes m icroinjected with okadaic acid [18] (Chris et al, FEBS Lett 1989,245:91-94) or a-naphthyiphosphate,a broad-spectrum phosphataseinhibitor (Pondaven and Meijer, Ezap
Control
Cd Res1986,163:447-; Iabbe et aA, Dev GrcrwLbD$ fZ 1988,30:197-207) may also reflect cyclin phosphorylation resulting from inhibition of the reverse reaction. According to this view, MPF would be subject to both negative control (at the level of its Cdc2 subunit) and positive control (at the level of its cyclin B subunit) by phosphotylation. The report by Felix et al [20] that cellfree extracts, prepared from activatedXen~us eggs,display one cycle of Cdc2 kinase activation even in the absence of ATP-Mg*+ and in the presence of 6dimethyladenenine shows that cyclin B phosphotylation, if required, is not the linal event triggering Cdc2 kinase activation. The initial activation of MPF following hormonal stimulation, at least in amphibians, requires translation of the c-mos protooncogene [ 20-231. Nonetheless,serial transfer of MPF remains effective even in the absenceof protein synthesis(Wassermanand Masui, E&0 Cell Res1975, 91:381-388; Dome, Exp Cell Res 1982, 139:127-133). Thus, expression of c-mosdoes not appear to be a compulsory event for MPF activation.
Evidence for active factors monitored and distinct from cdc2 kinase
as MPF
Active factors monitored as MPF in biological assaysmay not invariably have been Cdc2 kinase itself. In concordance with this view, two p&s of MPF activity could be separated from Xfznopuseggs after treating a O-33% ammonium sulfate fraction with ATP-US.The first peak correspond to the Cdc2 kinase. The second one neither contained significant H,K nor any material which cross reacted with antibodies to Cdc2 [24]. Both peaks were active in the absence of protein synthesis and across phyletic boundaries, thus, they ful6lled the usual criteria which distinguish MPF from upstream effecters which can also Induce meiotic maturation in G2arrested oocytes [24]. Monoclonal antibodies specific for thiophosphotylated proteins were also reported to recognize a component with MPF activity in Xenopw extracts treated with ATP-yS (Cyert et al., Dev Biof 1988, 129:209-216).Since highly purilied MPF (the Cdc2+5 complex) from Xenopuseggs does not incorporate radiolabel from y-3sS-ATP(Yamashitaand Maller,J Cell Biol 1988,107:717a),it appearsthat ATP-ySstabilizesthe MPF activity of a component which can be separated from p34cdc? This may account for many discrepancies in the literature between reports from different laboratories. For example, Gerhart et aI. (I Cell Biol 1984, 98:1247-1255), using homogenatestreated with ATP-yS,concluded that MPF reappears 35min after parthenogenetic activation in Xencpus eggs, whilst Capony et al (Deu Biol 1986, 117:1-12), using direct transfer of cytoplasm, first detected MPF 15-25min later when Cdc2 kinase activity began to increase 1201 (Dabauvalle et al, Cell 1988, 52:525-533). It may also explain why Dunphy and Newport (I Cell Biol 1988, IO6:2047-20561,using ATP-*IS,
of M-phase by maturation-promoting
factor
Dorke 271
readily detected MPF amplilication in cytosollc extracts prepared from cycloheximide-arrested Xenopus eggs, whilst My&e-Lye et al (J Cell Bioll988, 97:81+1) and Iabbe et al (1988), who did not use ATP-yS,failed to do so. It may also be worth emphasizing that the only evidence challenging the view that cyclin B acts stoichiometrically to activate Cdc2 klnase came from Murray et al [ 251, who Induced MPF activation in cyclin-depleted Xenqms extracts by adding ATP-ySand various amounts of exogenous cyclin; it was found that exogenous cyclin affectsthe rate but not the final level of MPF activity.Since HIK was not monitored, it is possible that a rate-limiting amount of Cdc2 kinase activitywas produced upon cyclin addition, which catalyzedthiophosphorylation and activation of an unknown component with MPF activity. This component could very well be an activator of Cdc2 dephosphotylation, or even Cdc2 phosphataseitself [24].
MPF inactivation Inhibition of MPF inactivation by m icroinjection of high concentrations of protease inhibitors suggestedthat cyclin degradationwas required for exit from M-phase(Picard et al, 1985). Direct evidence coniirming this view was provided by Murray et al. [25] who showed that a proteolysis-resistantmutant of cyclin B prevents MPF inactivation both in vitro and in vitro. Although Murray et al. could induce Ca*+ -dependent cyclin B degradation in a cell-free system prepared from metaphaseII-arrested Xenc@s oocytes, it is unlikely that cyclin degradation pe’ se is a Ca*+-dependent process. Indeed, specific degradation of cyclin and the resulting inactivation of MPF were readily observed in cell-free systemscontaining very high concentrations of ethyleneglycol-bis(~aminoethyl ether)-N,N,N’,N’-tetra-aceticacid (EGTA) [20,26]; addition of up to 1 m M free Ca*+ had no effect on cyclin stability [26]. Moreover, elevation of intracellular Ca*+ failed to Inactivate MPF in maturing star&h oocytes in uivo (Capony et al, 1986). In contrast, Ca*+ is required to inactivatea cytostatic factor (CSF), recently identified as the product of the c-mos protooncogene [ 27,281,which blocks the cyclin-degradatingpathway in metaphase II-arrested oocytes. Interestingly, ATP-Mg*+ was shown to be required for Induction of cyclin degradation in cell-free extracts [21,26]. This supports the view that cyclin degradation may involve a protein klnase whose relationship to Cdc2 kinase remains to be elucidated. The cascadeleading to cyclin degradationmay also be positively controlled by either type 1 or 2A protein phosphatasesbecauseokadaic acid stabilizesMPF activity and prevents cyclin degradation in star&h oocytes [ 181.
Conclusion The heterodimeric structure of MPF has now been elucidated and there is some agreementabout the events that control the periodic changesof its activity during the cell
272
Cell multiplication
cycle. Although much remains to be done before they can be described precisely,our knowledge of the molecular events that control m itotic processesis based on a firm foundation and a clearer picture of these eventsmust emerge in the near future. Recent studies have indicated that MPF controls m itotic events not only by regulating other enzymeslike pp6O”m [29,30] or control elements like elongation factor-l [31], but also by phosphorylating structural targets involved in the restructuring of Mphase cells (Peter et al, Cell 1990,in press; Belenguer et d, Mol Cell Biol1990, submitted; Peter et al., Cell 1990, submitted). MPF, now available in a pure state of high activity, can be used to dissect processessuch as nuclearenvelope breakdown (Peter et al, Cell 1990, submitted), spindle assembly [32] or inhibition of membrane-fusion events [33]. No doubt the next few years will bring a considerable advancein our understanding of how MPF kinase activity promotes m itotic events.
7. l
phase specific histone H, kinase. E M B O J 1989,8:227+2282. This paper shows a striking temporal correlation between HtK activation and the accumulation of a phosphoryiated form of cyciin.
8.
LIBBY JC, CWONV JP, CAPS
em
KAcilHADM, LEE JM, PICARDA, Dotter M: MPF from starfish oocytes at tirst meiotic metaphase is an heterodimer contaming one molecule of cdc2 and one molecule of cyclin B. E M B O J 1989, 8:30533058.
The first paper describing
9. l e
em
Of interest Of outstanding
l
interest
ARION D, MEIJER k B~UZUE~A l, BEACH D: cdc2 is a component of the M phase-specific histone H, kinase: evidence for identity with MPF. Ceil 1988, 55:371-378). Purified H, kinase from starfish oocytes contains p34d. The title is misleading because the kinase was not assayedfor MPF activity. 1.
l
2. l e
LABBE JC, PI~ARD A, PEAUCEUIER G, CAVADORE JC, NURSE P, DotiE M: Puritication of MPF from star&h: identification
as the Ht histone kinase p34cdeZ and a possible mechanism for its periodic activation. Cell 1989, 57:253263. MPF is negatively controlled by phosphoryiation croinjection of the conserved PSTAIR sequence.
and activated
by
mi-
LWGANTA GAUZIER J, L~HKA M, HOLUNGSWORTH R, MORENO S, NURSE P, MAUER J, S~LAFAN~ RA: Mammalian growthasH, histone kinase: a homolog of CDC28 protein sociated kinases controlling mitotic entry in yeast and frog cells. Mel Cell Bioll989, 93860-3868. XenqpusMPF catalyzes phosphoryiation of the same sites in H, as does the mammalian and yeast kinase.
3. l
l
Btuiw~v, I, D~A G, BEACH D: Activation of human CDC2 protein as a histone H, kinase is associated with complex formation with the p62 subunit. Proc Null Acad Sci USA 1989, 86:43624366.
Partial puriiication kinase from
and characterization Heia ceils.
of a cdc2-containing
Ht histone
role of cyclin B in meiosis I. J &II Biol 1989, 108:1431-1444.
WEST!ZM)(~RF JM, SWENSON KF, RUDERMAN JV: The
EDGARBA O’FARREUPH: Genetic control of ceU division patterns in the Dmsophflu embryo. Cell 1989, 57:177-187.
MORENO S, HAYLES J, NURSE P: Regulation of p34edc2 protein kinase during mitosis. Ceil 1989, 58:361-372. A sensitive assay that retains specificity in crude S. pornbe extracts is used to investigate the role of pl3m’, c&25+ and cdcZ3+ in regulating cdc2 kinase activity during ceU cycle. l
14. l
T, NURSE P, MALLER J: Dephosphorylation and activation of Xenopus p3@& protein kinase during the ceU cycle. N&we 1989, 33962ti29. GALIT~ER J, M A T S U K A W A
This
paper shows that maximal HtK activity of p34M correlates with its dephosphorylated state during the ceU cycle in Xen0pu.s oocytes. 15. l
WG, NRWPORTJW: Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase. Cell 1989, 58:181-191.
DUNPHV
This paper shows that tyrosine phosphoryiation of ~34~ ing interphase but absent during M-phase and suggests dephosphorylation is required for MPF activation.
16.
583193203. This paper shows that tyrosine dephosphotylation is one of a number of obiigatoty steps in the mitotic activation of cdc2 kinase. 17.
Goum K, NUKE P: Tyrosine phosphotylation of the tission yeast cdc2+ protein kinase regulates entry into mitosis. Nature 1989, 342:3945.
The single site of tyrosine site (Tyr 15). Substitution prematurely itIt0 mitosis.
level of both cyciin B mRNA and protein cycle. Cyciin B is associated with p34-
are regulated during at G2 but not Gl.
l
6.
J. BIUZUE~A I+ RUDERMAN J,
the ceil
is complexed with both cy clin A and B: evidence for proteolytic inactivation of MPF. GelI 1989, 56:829838. A paper suggestingassociationof both cyclic A and @in B with MPF. Evidence for proteolytic inactivation of MPF is not provided.
is high durthat tyrosine
MOIU AO, DRAETTA G, BEACH D, WANG JY: Reversible tyrosine phosphorylation of cd&? dephosphorylation accompanies activation during entry into mitosis. Cell 1989,
PINESJ, HEWER T: Isolation of a human cyclin cDNA evidence for cyclin mEHA and protein regulation in the cell cycle and for interaction with p34ede2. Cell 1989, 58:833846. This paper reports the sequenceof human cyclin B and shows that the 5. e.
l
of MPF.
13.
l e
DILVZ~TA G, LLKA F. WESTE~RF BEACH D: cdc2 protein kinase
structure
The sting gene is cloned and shown to be homologous with c&25. Mutations at the sft-ing locus cause cell cycle arrest in G2 of interphase 14.
l
4.
subunit
This paper gives the sequence of clam cyclin B and show that cyciin B microinjection induces entry of frog eggs into M-phase. It is suggested that fertiiization unmasks maternal cyclin B and generates active MPF.
and recommended
12. l
the complete
J, Btow J, Hum T: Translation of cycfin mRNA is necessary for extracts of activated Xenopus eggs to enter mitosis. Cell 1989, 56:947-956. MINSHUU
10. MURRAY AW, KtKXHNER MW: Cyclin synthesis drives the early me embryonic cell cycle. Nature 1989, 329275-285. This paper shows that destruction of the endogenous mRNA arrests extracts in interphase and that addition of exogenous cyclln B mRNA is suiflcient to produce multiple ceU cycles. 11.
references
D, C A V A W R E JC, DERANCOURT J,
Cutting cyclin B mRNA with antisense oiigonucleotide and endogenous RNAase H blocks entry into mitosis in a ceil-free egg extract.
l
Annotated reading
MEIJER I A~UON D, GotsrEy~ R, PINES J, BIUZUELA I HUNT T, BEACH D: Cyclin is a component of the sea urchin egg M-
18.
phosphoryiation is within the ATP-binding of Tyr 15 by phenyiaianine advances cells
PICARD A CAPONY JP, BRAUTIGAN DI Doa& M: Involvement of protein phosphatases 1 and 2A in the control of Mphase-promoting factor activity in star&h. J Cell Biol 1989,
1093347-3354. Okadaic acid microinjection triggers p34dc2 dephosphotyiation, activation and prevents cyclln from degeneration.
19. l
MPF
Nr;uur 1, CHARBONNEVJ M, GLJERRIER P: A requirement for protein phosphoryhtion in regulating the meiotic and mitotic ceU cycle in echinoderms. Da, Bioll989, 132:304-314.
Control This paper reports that 6dimethyiadenine, an inhibitor of protein phosphorylation, prevents sea-urchin embryos from entering mitosis. FEUX M, Pth’ESJ, HUNT T, KAttsENtlE: A post-ribosomal supematant from activated Xenopus eggs that displays posttranslationally regulated oscillation of its cdcz + mitotic kinase activity. EMBOJ 1989, 8:305+3070. This paper shows that both soluble and particulate material is required for oscillation of cdc2 kinase in o&o and that cyclin degradation requires ATP-Mg*+. 20.
00
21. l
SAGATAN, OSKARSSON M. COPEU\NDT, BRU~MAUGH J, VANDE WOUDE GF: Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocy-tes. Nature 1989, 335:519-525.
c-mar product is detected after. but not before, progesterone stimulation. Antisense microinjection blocks progesterone-induced meiotic maturation. SAGATA N, DAARI, ~SKARS~ON M, SHOWALTER SD, VANDEWOUDE GF: The product of the mos proto-oncogene as a candate ‘initiator’ for oocyte maturation. Science 1989, 245:64w6. ~39~ increasesbefore MPF activation. Microinjection of mas RNA activates MPF even in the absence of progesterone in Xen0pu.soocytes.
22. l
23. l
PAULESRS, BUCCIONER, MOSCHELRC, VANDEWOUDE GF, EPPIGJJ: Mouse Mos proto-oncogene product is present and functions during oogenesis. P m Nat1 Acad Sci USA 1989, 86:5395-5399.
p39- is present in prophase-arrestedoocytes Antisense microinjection prevents first-polar-body emission. M, IABBE JC, PIUW, A: M-phase promoting factor: its identification as the M phase-specific H, b&one kinase and its activation by dephosphorylation. J Cell Sci 1989, 12 (suppI): 3951. A component stabilized by thiophosphorylarion is separated from cdc2 kinase and shown to have MPF activity, 24.
DO&E
l
MURRAYAW, SOLOMONMJ, KIRSCHNER MW: The role of cy em clin synthesis and degradation in the control of maturation promoting factor activity. Nature 1989, 339:280-286. A proteolysis-resistantmutant of cyclin prevents MPF inactivation and the exit from mitosis both in vim and in vitro. 25.
LUCIA FC, RUDERMANJV: Control of programmed cyl e din destruction in a cell-free system. J cell Biol 1989, 109:189>1909. This paper describes a cell-free system that reproduces the normal pat. tern of cyclin A and cyclin B degradation. Cyciin degradation requires ATP-Mg*+ but not Ca*+. 26.
of M-phase by maturation-promoting
factor Do&
WATANABEN, VANDEWOUDE GF, ~KAWAY, SAGATAN: Spe cific proteolysis of the c-mos proto-oncogene product by calpain on fertilization of Xenopus eggs. Nature 1989, 342:505-511. p39”Qs has kinase activity and is degradated in vitro by an endogenous calpain.
27. l
SAGATAN, WATANABE N. VANDEWOUDEGF, IKAWAY: The G mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. Nature 1989, 342:512-518. Injection of mas RNA into 2-cell embryos induces cleavage arrest at metaphase. Antibodies to p39ma immunodeplete cytosol extracts of CSF activity.
28. l e
SHENOYS, CHOI JK, BAGRODLA S, COPEM TD, MALLERJI, S-IAUOWAYD: Purified maturation promoting factor phosphorylates pp60c-src by ~34 Edcz-associatedprotein kinase. cdl 1989, 51775-786. This paper suggeststhat ~p60~-~ is one of the targets for MPF action, although no activation of ppboG=was detected following phosphorylation by MPF. No phosphoryiation of ~34~ by ~p60’~ was observed. 29. l
MORGANDO, KAFUN JM, BISHOPJM, VARMUSHE: Mitosisspecific phosphorylation of p6OGm by p34&&-associated protein kinase. Cell 1989, 57:775-786. This paper shows that mitotic extracts from HeLa cells phosphorylate (but do not activate) baculovims-expressedpp6O”m at the same sites as those used during mitosis in vitro. Antibodies to p34a deplete mitotic extracts from pp6OC-mphosphorylating activity.
30. l
BEUE R, DERANCOURT J, POULHER, CAPONYJP, 02.0~ R, MULNER-~RILLON Ok A puriEed complex from Xenopus caytes contains a p47 protein, an In uluo substrate of MPF, and a p30 protein respectively homologous to elongation factors EF-la and EF-lfl. FEBS Left 1989, 255:101-104. This paper shows that elongation factor-la is a substrate of MPF both in rlivo and in vitra
31. l
VERDEF, LIBBYJC, Dot&~ M, KAPSEN-II E: Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature 1990, 343233-237. The pure M-phase-specificCdc2 kinase increasesmicrotubule turnover and reduces their lenght
32.
me
TUOMIKOSKI T, FELIXM4 DO&E M, GRUENBERG J: Inhibition of endocytic vesicle fusion in vitm by the cell-cycle control protein kinase cdc2. Nature 1989, 342:%2-944. Fusion of baby hamster kidney endocytic vesiclesis reduced in Xem+s mitotic cytosol when compared with interphase cytosol. This inhibition is reconstituted in interphase cytosol by adding the mitotic Cdc2 protein kinase.
33. l
273
of M-phase
by maturation-promoting
factor
M. Do&e Centre
de Recherches
sur la Biochimie Current
des Macromolecules, Opinion
in Cell Biology
Introduction The alternation between interphase and M-phaseis controlled by periodic changesin the activity of maturationpromoting factor (MPF). Recently,MPF was purikd from Xenopuseggs(khka et al, Proc NatlAcudSci USA1988, 85:3009-3013) and was shown to contain a p34 protein homologue of the yeast cell-cycle-controlgene c&2 (Gamier et al, Cell 1988, 54:433-439;Dunphy et al, Cell 1988,54:423-431>.Moreover, evidencewas obtained that cyclins, a family of proteins whose levels follow a sawtooth oscillation during the cell cycle, may control MPF activation in both marine invertebrates (Swenson et al., Cell 1986, 47867-870; Pines and Hunt, EMBO J 1987, 6:2987-2327;Hagan et al, J Cell Sci 1988,91:587-595).In this review, I discuss recent progress which clariIies the subunit structure of MPF and sheds some light on the mechanisms responsible for its periodic changes in activity during meiotic maturation and early embryogenesis in animal cells. The accompanyingreview by Glover (this issue, pp 258-261) will specikally focus on Drasophila.
MPF: identification Mitotic
and subunit structure
cdc 2 kinases
Arion et al. [l] substantially puriiied an H, histone kinase (HrK) from starfish oocytes, that had been shown to peak at M-phaseduring meiosis and early cleavagein echoderms (Picard et al, De0 Biol 1985, 109:311-320, and Dev Growth Dfl&. 1987, 291:93-103) and amphibians (Cicirelli et al, J Biol them 1988, 263:2009-2019; Labbe et al, Dev Biol 1988, 127:157-169). The iinal preparation contained CdQ, shown by immunoblotting and binding to p13=‘, a yeast protein which interacts physically with cdc2. Demonstration that the M-phase-specilic HrK corresponds to MPF came from Iabbe et al. [2] who also purified H,K from stariish oocytes (Labbk et al, Nature 1988, 335:251-254). The iinal preparation (specik activity, 0.5 l.rmol of phosphate per min/mg of enzyme to Hr histone) contained Cdc2 as the single major protein. MPF activity was found to co-migrate with H,K through-
CNRS and INSERM, Montpellier,
France
1990, 2:269-273
out purilication (no ATP-Crswas used at any time to ‘stabilize’MPF, see below). The mammalian growth-associated H1K, an enzyme whose activity is sharply elevatedat mitosis, was purified from Novikoff rat hepatoma cells [3]. The final preparation (spectic activity, 0.5 urn01of phosphate per min/mg of enzyme) also contained Cdc2. In addition, it was found to catalyzephosphorylation of the same sites in Hr as did Xen0pu.sMPF.An H,K containing Cdc2 was also purikd from human cervical carcinoma (HeIa) cells arrested at M-phase by nocodazole treatment [4]. The kinase was probably substantially purikl, although its ~%ralspeciiic activity was lower than star&h MPF by severalorders of magnitude. MpF is a heterodimer
containing
cyclin
and cdc2
Although starfish p34a retaining both H, kinase and MPF activities could be separated from other proteins following extensive purikation by conventional column chromatography,various lines of evidencesuggestedthat it formed part of a multiprotein complex in the living cell or when directly assayedin crude extracts. A 62 kD protein, subsequently identiiied as cyclin B [ 51, was found to coimmunoprecipitate with p34a from G2 but not Gl HeIa cells (Draetta and Beach, Cell 1988, 54:17-26X In the clam, Spisuh, anticyclin B immunoprecipitates were found to have H,K activity [ 61. Moreover, cyclin B coeluted with the main peak of H,K activity after gel iiltration of crude extracts, although a second Hr kinase peak of lower apparent M, was detected, which did not contain any cyclin. Cyclin B and cdc2 were also found to coelute with H,K activity at the last step, after its partial purification from sea-urchin mitotic extracts [7]. A cyclin ACdc2 complex was also reported to be a minor component of the M-phase-specificHrK in early clam embryos [6]. However, at variancewith anticyclin B reactivity, anticyclin A reactivity ran ahead of H,K activity on a sizing column, suggestingthat most cyclin A associates with proteins other than Cdc2. Using an improved procedure based on pl3aQephar ose a&inky chromatography, Iabbe et al [8] purilkd MPF to apparent homogeneity and with high recovery
Abbreviations CSF-cytostatic factor; H,K-H, histone kinase; MPF-maturation-promoting factor; mRNA-messenger RNA; PSTAIR+onserved ECVPSTAIREISLLKE region of cdc2.
Current
Science Ltd ISSN 0955*74
269
270
Cell multidication
(50%). At variancewith their previous results, they found the m itotic kinase (specific activity, 8 urn01of phosphate per m in/mg of protein to HI histone) to consist of a complex formed by a 1: 1 stoichiometric associationof Cdc2 with cyclin B, identified by direct m icrosequencing.Their previous Ending was explained if one assumedthat, during the alternative procedure which comprised six steps of conventional chromatography extended over 2 days, either the stoichiometric complex became disrupted or the cyclin component underwent proteol@s. In any case, the previous result indicates that an intact cyclin subunit is not required for Cdc2 kinase activity. Probably, cyclin B is required in vim for Cdc2 kinase activation. Whether MPF also exists as a cyclin A-CdQ complex during later development remains to be investigated since MPF was purified to homogeneity from oocytes at first meiotic metaphaseonly, which do not contain cyclin A
[61. Cyclin B and MPF activation p34a is stable during meiosis and early embryogenesis (Labti et al, 1988) [6,7]. However, protein synthesisis required at each cell cycle for MPF activation (Wagenaar, Exp Gdf Res 1983, 144:39-l; Gerhart et al, Cytobias 1985, 43~335-347;Picard et al, 1985 and 1987; Karsenti et al, Dev Bioll987, 119442453) making cyclin an attractive candidate for an MPF-activatorprotein. This was demonstratedby showing that speciEcdestruction of endogenous cyclin B messengerRNA (mRNA) arrests frogegg extracts in interphase [9] and that addition of exogenous cyclin B mRNA to similar extracts is sufficient to produce multiple cell cycles [lo]. There is a delay in viva between the time after which protein synthesis is no longer required and the time when kinase activation occurs. This suggeststhat, although cydin B accumulation is required for MPF activation, it is not the only factor involved in the activation process. Post-translationalmodification of either cyclin or Cdc2 may be required for complex formation. Human cells in early Gl synthesizecyclin B, but it is not associatedwith Cdc2 151.In the clam, it was proposed [ 111that maternal cyclin B is masked and the interaction with Cdc2 prevented until fertilization. Alternatively, part of the Cdc2 pool may be associated,through its conserved EGVPSTAIREISLLKE region (called PSTAIR),with an inhibitory molecule whose normal function is to prevent formation of the active cyclin B-Cdc2 complex. Indeed, m icroinjection of the PSTAIRpeptide was sufhcient to activate MPF in star&h oocytes [2], and it was found to accelerate its action in [email protected] (Gauthier et al, 1988). Further evident that cyclin may interact with Cdc2 through its PSTAIRregion came from Pines and Hunter [ 51, who showed that cyclin B does not coprecipitate with antibodies to the PSTAIRsequence.Formation of the cyclin B-Cdc2 complex is also not sufficient to activate MPF. Indeed the complex can be isolated from G2-arrested oocytes, yet it does not contain active MPF (IabM and I.e Bouffant, personal communication). Thus, other steps
in the pathway that leads to MPF activation may be subject to regulation.They may involve the product of homologues of the c&25+ gene,which acts in yeast as a dosedependent activator of the m itosis-inducing function of c&+ [ 12,131(Russeland Nurse, cell 1986,451:145-153).
Control of MPF activity by phosphorylation and dephosphorylation reactions Despite some early reports which emphasized the correlation between increased phosphoryfation of p34a and activation of its catalytic activity during the cell cycle (Draetta and Beach, 1988; Draetta et aL, Nature 1988, 336:73%743) there are good evidencesthat p34d dephosphoryfation controls MPF activation. In starfish, it was shown that while p34a is phosphorylated, its protein kinase activity is inhibited, preventing entry into Mphase, but once p34C&Qis dephosphorylated,its protein kinase activity increasesand M-phaseis initiated [ 21. Similar correlations were observed in Xenopus eggs [ 14,151, mammalian cells in culture [ 161 and yeast [ 181. Moreover, Cdc2 kinasecould be activatedin vitm simply by removing ATP from crude extracts [2] (Labb+ et al, 1988). Dephosphorylation of tyrosine accompaniescdc2 kinase activation during entry into m itosis [ 15-171. In yeast, the site of tyrosine phosphotylation has been mapped to tyrosine 15, a residue within the presumptive ATP-binding site. Substitution of this tyrosine by pheny&nine advanced cells prematurely into m itosis [ 171.In mouse 3T3 fibroblasts, inhibition of tyrosine dephosphoryfation by exposure of cells to vanadatewas found to arrest them reversiblyat G2. Nonetheless,specific dephosphoryfation of tyrosine residuesby phosphotyrosine phosphatase1B failed to activateCdc2 kinase in interphasic extracts on tyrosine, but also on threonine and serine residues,which undergo dephosphoryfation at the time of Cdc2 activation. The corresponding phosphataseis not known, but it is neither type 1 nor type 2A phosphatasebecauseit is not sensitive to okadaic acid [18] (Felix et al, EMBO J 1990, in press). Besides p34dCa dephosphoryfation, cyclin B phosphoryfation may be involved in Cdc2 kinase activation under physiological conditions. Indeed, both events occur almost simultaneouslyduring the first m itotic cell cycle in the sea urchin [7]. However, cyclin B in an excellent substrate for Cdc2 kinase, at least in vitro [8]. Thus, it is diKicult to conclude from temporal correlation alone that cyclin phosphorytation is causal for kinase activation. Nonetheless, other reports indicate that a phosphoryfation event is required for activation of the cdc2-cyclic B complex. Entry of sea-urchin eggs into m itotic metaphasewas shown to be reversibly prevented by 6dimethyfadenine, an inhibitor of protein phosphorylation [ 191.Although other interpretations are possible (see below), Cdc2 kinase activation in oocytes m icroinjected with okadaic acid [18] (Chris et al, FEBS Lett 1989,245:91-94) or a-naphthyiphosphate,a broad-spectrum phosphataseinhibitor (Pondaven and Meijer, Ezap
Control
Cd Res1986,163:447-; Iabbe et aA, Dev GrcrwLbD$ fZ 1988,30:197-207) may also reflect cyclin phosphorylation resulting from inhibition of the reverse reaction. According to this view, MPF would be subject to both negative control (at the level of its Cdc2 subunit) and positive control (at the level of its cyclin B subunit) by phosphotylation. The report by Felix et al [20] that cellfree extracts, prepared from activatedXen~us eggs,display one cycle of Cdc2 kinase activation even in the absence of ATP-Mg*+ and in the presence of 6dimethyladenenine shows that cyclin B phosphotylation, if required, is not the linal event triggering Cdc2 kinase activation. The initial activation of MPF following hormonal stimulation, at least in amphibians, requires translation of the c-mos protooncogene [ 20-231. Nonetheless,serial transfer of MPF remains effective even in the absenceof protein synthesis(Wassermanand Masui, E&0 Cell Res1975, 91:381-388; Dome, Exp Cell Res 1982, 139:127-133). Thus, expression of c-mosdoes not appear to be a compulsory event for MPF activation.
Evidence for active factors monitored and distinct from cdc2 kinase
as MPF
Active factors monitored as MPF in biological assaysmay not invariably have been Cdc2 kinase itself. In concordance with this view, two p&s of MPF activity could be separated from Xfznopuseggs after treating a O-33% ammonium sulfate fraction with ATP-US.The first peak correspond to the Cdc2 kinase. The second one neither contained significant H,K nor any material which cross reacted with antibodies to Cdc2 [24]. Both peaks were active in the absence of protein synthesis and across phyletic boundaries, thus, they ful6lled the usual criteria which distinguish MPF from upstream effecters which can also Induce meiotic maturation in G2arrested oocytes [24]. Monoclonal antibodies specific for thiophosphotylated proteins were also reported to recognize a component with MPF activity in Xenopw extracts treated with ATP-yS (Cyert et al., Dev Biof 1988, 129:209-216).Since highly purilied MPF (the Cdc2+5 complex) from Xenopuseggs does not incorporate radiolabel from y-3sS-ATP(Yamashitaand Maller,J Cell Biol 1988,107:717a),it appearsthat ATP-ySstabilizesthe MPF activity of a component which can be separated from p34cdc? This may account for many discrepancies in the literature between reports from different laboratories. For example, Gerhart et aI. (I Cell Biol 1984, 98:1247-1255), using homogenatestreated with ATP-yS,concluded that MPF reappears 35min after parthenogenetic activation in Xencpus eggs, whilst Capony et al (Deu Biol 1986, 117:1-12), using direct transfer of cytoplasm, first detected MPF 15-25min later when Cdc2 kinase activity began to increase 1201 (Dabauvalle et al, Cell 1988, 52:525-533). It may also explain why Dunphy and Newport (I Cell Biol 1988, IO6:2047-20561,using ATP-*IS,
of M-phase by maturation-promoting
factor
Dorke 271
readily detected MPF amplilication in cytosollc extracts prepared from cycloheximide-arrested Xenopus eggs, whilst My&e-Lye et al (J Cell Bioll988, 97:81+1) and Iabbe et al (1988), who did not use ATP-yS,failed to do so. It may also be worth emphasizing that the only evidence challenging the view that cyclin B acts stoichiometrically to activate Cdc2 klnase came from Murray et al [ 251, who Induced MPF activation in cyclin-depleted Xenqms extracts by adding ATP-ySand various amounts of exogenous cyclin; it was found that exogenous cyclin affectsthe rate but not the final level of MPF activity.Since HIK was not monitored, it is possible that a rate-limiting amount of Cdc2 kinase activitywas produced upon cyclin addition, which catalyzedthiophosphorylation and activation of an unknown component with MPF activity. This component could very well be an activator of Cdc2 dephosphotylation, or even Cdc2 phosphataseitself [24].
MPF inactivation Inhibition of MPF inactivation by m icroinjection of high concentrations of protease inhibitors suggestedthat cyclin degradationwas required for exit from M-phase(Picard et al, 1985). Direct evidence coniirming this view was provided by Murray et al. [25] who showed that a proteolysis-resistantmutant of cyclin B prevents MPF inactivation both in vitro and in vitro. Although Murray et al. could induce Ca*+ -dependent cyclin B degradation in a cell-free system prepared from metaphaseII-arrested Xenc@s oocytes, it is unlikely that cyclin degradation pe’ se is a Ca*+-dependent process. Indeed, specific degradation of cyclin and the resulting inactivation of MPF were readily observed in cell-free systemscontaining very high concentrations of ethyleneglycol-bis(~aminoethyl ether)-N,N,N’,N’-tetra-aceticacid (EGTA) [20,26]; addition of up to 1 m M free Ca*+ had no effect on cyclin stability [26]. Moreover, elevation of intracellular Ca*+ failed to Inactivate MPF in maturing star&h oocytes in uivo (Capony et al, 1986). In contrast, Ca*+ is required to inactivatea cytostatic factor (CSF), recently identified as the product of the c-mos protooncogene [ 27,281,which blocks the cyclin-degradatingpathway in metaphase II-arrested oocytes. Interestingly, ATP-Mg*+ was shown to be required for Induction of cyclin degradation in cell-free extracts [21,26]. This supports the view that cyclin degradation may involve a protein klnase whose relationship to Cdc2 kinase remains to be elucidated. The cascadeleading to cyclin degradationmay also be positively controlled by either type 1 or 2A protein phosphatasesbecauseokadaic acid stabilizesMPF activity and prevents cyclin degradation in star&h oocytes [ 181.
Conclusion The heterodimeric structure of MPF has now been elucidated and there is some agreementabout the events that control the periodic changesof its activity during the cell
272
Cell multiplication
cycle. Although much remains to be done before they can be described precisely,our knowledge of the molecular events that control m itotic processesis based on a firm foundation and a clearer picture of these eventsmust emerge in the near future. Recent studies have indicated that MPF controls m itotic events not only by regulating other enzymeslike pp6O”m [29,30] or control elements like elongation factor-l [31], but also by phosphorylating structural targets involved in the restructuring of Mphase cells (Peter et al, Cell 1990,in press; Belenguer et d, Mol Cell Biol1990, submitted; Peter et al., Cell 1990, submitted). MPF, now available in a pure state of high activity, can be used to dissect processessuch as nuclearenvelope breakdown (Peter et al, Cell 1990, submitted), spindle assembly [32] or inhibition of membrane-fusion events [33]. No doubt the next few years will bring a considerable advancein our understanding of how MPF kinase activity promotes m itotic events.
7. l
phase specific histone H, kinase. E M B O J 1989,8:227+2282. This paper shows a striking temporal correlation between HtK activation and the accumulation of a phosphoryiated form of cyciin.
8.
LIBBY JC, CWONV JP, CAPS
em
KAcilHADM, LEE JM, PICARDA, Dotter M: MPF from starfish oocytes at tirst meiotic metaphase is an heterodimer contaming one molecule of cdc2 and one molecule of cyclin B. E M B O J 1989, 8:30533058.
The first paper describing
9. l e
em
Of interest Of outstanding
l
interest
ARION D, MEIJER k B~UZUE~A l, BEACH D: cdc2 is a component of the M phase-specific histone H, kinase: evidence for identity with MPF. Ceil 1988, 55:371-378). Purified H, kinase from starfish oocytes contains p34d. The title is misleading because the kinase was not assayedfor MPF activity. 1.
l
2. l e
LABBE JC, PI~ARD A, PEAUCEUIER G, CAVADORE JC, NURSE P, DotiE M: Puritication of MPF from star&h: identification
as the Ht histone kinase p34cdeZ and a possible mechanism for its periodic activation. Cell 1989, 57:253263. MPF is negatively controlled by phosphoryiation croinjection of the conserved PSTAIR sequence.
and activated
by
mi-
LWGANTA GAUZIER J, L~HKA M, HOLUNGSWORTH R, MORENO S, NURSE P, MAUER J, S~LAFAN~ RA: Mammalian growthasH, histone kinase: a homolog of CDC28 protein sociated kinases controlling mitotic entry in yeast and frog cells. Mel Cell Bioll989, 93860-3868. XenqpusMPF catalyzes phosphoryiation of the same sites in H, as does the mammalian and yeast kinase.
3. l
l
Btuiw~v, I, D~A G, BEACH D: Activation of human CDC2 protein as a histone H, kinase is associated with complex formation with the p62 subunit. Proc Null Acad Sci USA 1989, 86:43624366.
Partial puriiication kinase from
and characterization Heia ceils.
of a cdc2-containing
Ht histone
role of cyclin B in meiosis I. J &II Biol 1989, 108:1431-1444.
WEST!ZM)(~RF JM, SWENSON KF, RUDERMAN JV: The
EDGARBA O’FARREUPH: Genetic control of ceU division patterns in the Dmsophflu embryo. Cell 1989, 57:177-187.
MORENO S, HAYLES J, NURSE P: Regulation of p34edc2 protein kinase during mitosis. Ceil 1989, 58:361-372. A sensitive assay that retains specificity in crude S. pornbe extracts is used to investigate the role of pl3m’, c&25+ and cdcZ3+ in regulating cdc2 kinase activity during ceU cycle. l
14. l
T, NURSE P, MALLER J: Dephosphorylation and activation of Xenopus p3@& protein kinase during the ceU cycle. N&we 1989, 33962ti29. GALIT~ER J, M A T S U K A W A
This
paper shows that maximal HtK activity of p34M correlates with its dephosphorylated state during the ceU cycle in Xen0pu.s oocytes. 15. l
WG, NRWPORTJW: Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase. Cell 1989, 58:181-191.
DUNPHV
This paper shows that tyrosine phosphoryiation of ~34~ ing interphase but absent during M-phase and suggests dephosphorylation is required for MPF activation.
16.
583193203. This paper shows that tyrosine dephosphotylation is one of a number of obiigatoty steps in the mitotic activation of cdc2 kinase. 17.
Goum K, NUKE P: Tyrosine phosphotylation of the tission yeast cdc2+ protein kinase regulates entry into mitosis. Nature 1989, 342:3945.
The single site of tyrosine site (Tyr 15). Substitution prematurely itIt0 mitosis.
level of both cyciin B mRNA and protein cycle. Cyciin B is associated with p34-
are regulated during at G2 but not Gl.
l
6.
J. BIUZUE~A I+ RUDERMAN J,
the ceil
is complexed with both cy clin A and B: evidence for proteolytic inactivation of MPF. GelI 1989, 56:829838. A paper suggestingassociationof both cyclic A and @in B with MPF. Evidence for proteolytic inactivation of MPF is not provided.
is high durthat tyrosine
MOIU AO, DRAETTA G, BEACH D, WANG JY: Reversible tyrosine phosphorylation of cd&? dephosphorylation accompanies activation during entry into mitosis. Cell 1989,
PINESJ, HEWER T: Isolation of a human cyclin cDNA evidence for cyclin mEHA and protein regulation in the cell cycle and for interaction with p34ede2. Cell 1989, 58:833846. This paper reports the sequenceof human cyclin B and shows that the 5. e.
l
of MPF.
13.
l e
DILVZ~TA G, LLKA F. WESTE~RF BEACH D: cdc2 protein kinase
structure
The sting gene is cloned and shown to be homologous with c&25. Mutations at the sft-ing locus cause cell cycle arrest in G2 of interphase 14.
l
4.
subunit
This paper gives the sequence of clam cyclin B and show that cyciin B microinjection induces entry of frog eggs into M-phase. It is suggested that fertiiization unmasks maternal cyclin B and generates active MPF.
and recommended
12. l
the complete
J, Btow J, Hum T: Translation of cycfin mRNA is necessary for extracts of activated Xenopus eggs to enter mitosis. Cell 1989, 56:947-956. MINSHUU
10. MURRAY AW, KtKXHNER MW: Cyclin synthesis drives the early me embryonic cell cycle. Nature 1989, 329275-285. This paper shows that destruction of the endogenous mRNA arrests extracts in interphase and that addition of exogenous cyclln B mRNA is suiflcient to produce multiple ceU cycles. 11.
references
D, C A V A W R E JC, DERANCOURT J,
Cutting cyclin B mRNA with antisense oiigonucleotide and endogenous RNAase H blocks entry into mitosis in a ceil-free egg extract.
l
Annotated reading
MEIJER I A~UON D, GotsrEy~ R, PINES J, BIUZUELA I HUNT T, BEACH D: Cyclin is a component of the sea urchin egg M-
18.
phosphoryiation is within the ATP-binding of Tyr 15 by phenyiaianine advances cells
PICARD A CAPONY JP, BRAUTIGAN DI Doa& M: Involvement of protein phosphatases 1 and 2A in the control of Mphase-promoting factor activity in star&h. J Cell Biol 1989,
1093347-3354. Okadaic acid microinjection triggers p34dc2 dephosphotyiation, activation and prevents cyclln from degeneration.
19. l
MPF
Nr;uur 1, CHARBONNEVJ M, GLJERRIER P: A requirement for protein phosphoryhtion in regulating the meiotic and mitotic ceU cycle in echinoderms. Da, Bioll989, 132:304-314.
Control This paper reports that 6dimethyiadenine, an inhibitor of protein phosphorylation, prevents sea-urchin embryos from entering mitosis. FEUX M, Pth’ESJ, HUNT T, KAttsENtlE: A post-ribosomal supematant from activated Xenopus eggs that displays posttranslationally regulated oscillation of its cdcz + mitotic kinase activity. EMBOJ 1989, 8:305+3070. This paper shows that both soluble and particulate material is required for oscillation of cdc2 kinase in o&o and that cyclin degradation requires ATP-Mg*+. 20.
00
21. l
SAGATAN, OSKARSSON M. COPEU\NDT, BRU~MAUGH J, VANDE WOUDE GF: Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocy-tes. Nature 1989, 335:519-525.
c-mar product is detected after. but not before, progesterone stimulation. Antisense microinjection blocks progesterone-induced meiotic maturation. SAGATA N, DAARI, ~SKARS~ON M, SHOWALTER SD, VANDEWOUDE GF: The product of the mos proto-oncogene as a candate ‘initiator’ for oocyte maturation. Science 1989, 245:64w6. ~39~ increasesbefore MPF activation. Microinjection of mas RNA activates MPF even in the absence of progesterone in Xen0pu.soocytes.
22. l
23. l
PAULESRS, BUCCIONER, MOSCHELRC, VANDEWOUDE GF, EPPIGJJ: Mouse Mos proto-oncogene product is present and functions during oogenesis. P m Nat1 Acad Sci USA 1989, 86:5395-5399.
p39- is present in prophase-arrestedoocytes Antisense microinjection prevents first-polar-body emission. M, IABBE JC, PIUW, A: M-phase promoting factor: its identification as the M phase-specific H, b&one kinase and its activation by dephosphorylation. J Cell Sci 1989, 12 (suppI): 3951. A component stabilized by thiophosphorylarion is separated from cdc2 kinase and shown to have MPF activity, 24.
DO&E
l
MURRAYAW, SOLOMONMJ, KIRSCHNER MW: The role of cy em clin synthesis and degradation in the control of maturation promoting factor activity. Nature 1989, 339:280-286. A proteolysis-resistantmutant of cyclin prevents MPF inactivation and the exit from mitosis both in vim and in vitro. 25.
LUCIA FC, RUDERMANJV: Control of programmed cyl e din destruction in a cell-free system. J cell Biol 1989, 109:189>1909. This paper describes a cell-free system that reproduces the normal pat. tern of cyclin A and cyclin B degradation. Cyciin degradation requires ATP-Mg*+ but not Ca*+. 26.
of M-phase by maturation-promoting
factor Do&
WATANABEN, VANDEWOUDE GF, ~KAWAY, SAGATAN: Spe cific proteolysis of the c-mos proto-oncogene product by calpain on fertilization of Xenopus eggs. Nature 1989, 342:505-511. p39”Qs has kinase activity and is degradated in vitro by an endogenous calpain.
27. l
SAGATAN, WATANABE N. VANDEWOUDEGF, IKAWAY: The G mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. Nature 1989, 342:512-518. Injection of mas RNA into 2-cell embryos induces cleavage arrest at metaphase. Antibodies to p39ma immunodeplete cytosol extracts of CSF activity.
28. l e
SHENOYS, CHOI JK, BAGRODLA S, COPEM TD, MALLERJI, S-IAUOWAYD: Purified maturation promoting factor phosphorylates pp60c-src by ~34 Edcz-associatedprotein kinase. cdl 1989, 51775-786. This paper suggeststhat ~p60~-~ is one of the targets for MPF action, although no activation of ppboG=was detected following phosphorylation by MPF. No phosphoryiation of ~34~ by ~p60’~ was observed. 29. l
MORGANDO, KAFUN JM, BISHOPJM, VARMUSHE: Mitosisspecific phosphorylation of p6OGm by p34&&-associated protein kinase. Cell 1989, 57:775-786. This paper shows that mitotic extracts from HeLa cells phosphorylate (but do not activate) baculovims-expressedpp6O”m at the same sites as those used during mitosis in vitro. Antibodies to p34a deplete mitotic extracts from pp6OC-mphosphorylating activity.
30. l
BEUE R, DERANCOURT J, POULHER, CAPONYJP, 02.0~ R, MULNER-~RILLON Ok A puriEed complex from Xenopus caytes contains a p47 protein, an In uluo substrate of MPF, and a p30 protein respectively homologous to elongation factors EF-la and EF-lfl. FEBS Left 1989, 255:101-104. This paper shows that elongation factor-la is a substrate of MPF both in rlivo and in vitra
31. l
VERDEF, LIBBYJC, Dot&~ M, KAPSEN-II E: Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature 1990, 343233-237. The pure M-phase-specificCdc2 kinase increasesmicrotubule turnover and reduces their lenght
32.
me
TUOMIKOSKI T, FELIXM4 DO&E M, GRUENBERG J: Inhibition of endocytic vesicle fusion in vitm by the cell-cycle control protein kinase cdc2. Nature 1989, 342:%2-944. Fusion of baby hamster kidney endocytic vesiclesis reduced in Xem+s mitotic cytosol when compared with interphase cytosol. This inhibition is reconstituted in interphase cytosol by adding the mitotic Cdc2 protein kinase.
33. l
273
