1 CRBM, UPR 1086-CNRS, 1919 route de Mende, 34293 Montpellier Cedex 5,
France
2 ICRF, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK
(e-mail: doree{at}crbm.cnrs-mop.fr ; tim.hunt{at}cancer.org.uk )
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Summary |
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Cyclins were first recognized as proteins whose abundance oscillates during the early cell cycles of marine invertebrate eggs and their connection with MPF (maturation-promoting factor), the entity defined in frog and starfish oocytes whose activity controls entry into M phase, was far from clear at first. Indeed, it was a long time before MPF was shown to be a protein kinase, and direct proof that MPF is a heterodimer comprising one molecule of cyclin and one molecule of Cdc2 was finally obtained only when the Cdc2-associated component of purified starfish MPF was sequenced and found to be cyclin B. When this fundamental discovery was confirmed in vertebrates and mammalian members of the Cdc2 family were also shown to bind cyclins, Cdc2 became Cdk1, the first cyclin-dependent protein kinase.
Key words: Cell cycle, CDK, Cyclin, MPF, Oocyte, Protein kinase
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Introduction |
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Cyclins as MPF activators |
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The view that mitotic cyclins are in some way linked with oscillations in
MPF and mitotic kinase activities was strengthened when it was shown that
expression of clam cyclin A releases Xenopus oocytes from G2 arrest
and induces meiotic M phase in the absence of hormonal stimulation
(Swenson et al., 1986). Two
years later, Murray and Kirschner succeeded in producing `cycling' extracts of
frog eggs that perform multiple cell cycles in vitro
(Murray and Kirschner, 1989
).
They showed that, after destruction of endogenous cellular messenger RNAs,
which arrests the extracts in interphase, addition of exogenous cyclin mRNA is
sufficient to produce multiple cell cycles. Moreover, they observed that the
newly synthesized cyclin is degraded at the end of each mitosis
(Murray and Kirschner, 1989
).
The view that the synthesis of cyclin B is necessary for mitotic cell cycles
in cleaving Xenopus embryos became firmly established when Minshull
et al., who identified two cyclins as major translation products in cell-free
extracts, reported that antisense-mediated destruction of these mRNAs blocks
entry into mitosis (Minshull et al.,
1989a
).
Together, these findings demonstrated that cyclin is necessary for the appearance of MPF and that MPF disappears because of destruction of cyclin. Furthermore, they strongly suggested that the activity of MPF is intimately linked to a mitotic kinase activity whose oscillations share with MPF the same requirements for protein synthesis and degradation.
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Cdc2 as a component of MPF and the major mitotic kinase |
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In parallel with the investigations mentioned above, two other groups used
in vitro phosphorylation of histones as an assay to purify by conventional
column chromatography the major mitotic kinase from starfish oocytes
(Arion et al., 1988;
Labbé et al., 1988
).
Using PSTAIRE immunoblotting and/or binding to p13suc1, they
concluded that it was the Cdc2 kinase. Since Cdc2 appeared to be a component
of Xenopus MPF, Arion et al. suggested that Cdc2 kinase and MPF could
be the same entity, although they did not address this question
experimentally. However, this interpretation was difficult to reconcile with
the fact that, although enucleated starfish oocytes readily activate Cdc2
kinase in response to hormonal stimulation, they do not produce transferable
MPF activity (Kishimoto et al.,
1981
; Picard et al.,
1984
; Picard et al.,
1988
).
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From Cdc2 to Cdk1 |
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In the same year, it was reported that associated proteins could apparently
be removed from Cdc2 without loss of its histone kinase activity after
extensive purification from starfish oocytes
(Labbé et al., 1989a).
Although this suggested no absolute requirement for an activator protein
associated with Cdc2, cyclin could have undergone proteolysis during the
lengthy purification procedure, so that no fragment of consistent size peaking
with Cdc2 kinase activity would have been detected even when consecutive
fractions were silver-stained after analysis by SDS-PAGE in the final
preparation. Nor was autophosphorylation of any polypeptide detected in the
final peak of activity when the kinase was incubated with
[
-32P]ATP; yet, a cyclin fragment lacking the
phosphorylation sites could still have been associated with Cdc2.
Microinjection of this highly purified Cdc2 kinase readily released oocytes
from G2 arrest in a variety of species. Surprisingly, it did so even in
starfish, even though the native Cdc2 kinase is not sufficient (in the absence
of nuclear material) to induce germinal vesicle breakdown in
non-hormone-stimulated starfish oocytes.
At that time, only a few protein kinases that contain regulatory subunits
had been identified, and the paradigm for such kinases was the cAMP-dependent
protein kinase (PKA), whose regulatory subunit had been shown to negatively
control the activity of the catalytic subunit. Thus, even for people convinced
that MPF was indeed a protein kinase, the positive role that cyclin seemed to
have in regulation of MPF activity did not imply that it was necessarily a
subunit of this kinase. In fact, no phenotype was associated with
overexpression of Cdc13 in fission yeast
(Booher and Beach, 1988).
The MPF kinase was, rather, believed to be kept inactive through
association with a hypothetical inhibitory subunit: binding of the cyclin was
proposed to dissociate the inhibitory subunit (anti-MPF) and activate the
kinase (Minshull et al.,
1989b) (Fig. 1). In
agreement with this view, an anti-MPF entity called INH (for inhibitor of MPF
amplification) had been characterized in Xenopus oocytes (Cyert and
Kirschner, 1988). In addition, negative control of Cdc2 kinase through
phosphorylation had been demonstrated in both starfish and Xenopus
oocytes (Labbé et al.,
1989a
; Dunphy and Newport,
1989
; Gautier et al.,
1989
), which was consistent with genetic studies in fission yeast
demonstrating that Wee1 kinase negatively controls Cdc2 kinase
(Russell and Nurse, 1987
).
Cyclin could thus control MPF activation by triggering dephosphorylation of
Cdc2 [that cyclin is already associated with Cdc2 prior to its
dephosphorylation by Cdc25c was demonstrated only two years later
(Gautier and Maller, 1991
;
Strausfeld et al., 1991
).
|
Identification of starfish MPF as a cyclin-BCdc2
heterodimer
Labbé et al. provided decisive evidence that cyclin B is a genuine
subunit of Cdc2 kinase when they reported purification to homogeneity of the
M-phase-specific kinase from starfish oocytes at first meiotic metaphase,
using a rapid three-step procedure based on affinity chromatography on
immobilized yeast protein p13suc1
(Labbé et al., 1989b).
Besides p34cdc2, the final peak of H1 kinase activity contained
only one other polypeptide, which had an apparent molecular weight of 47 kDa
and was identified as starfish cyclin B by direct microsequencing.
When the final preparation was incubated with [-32P]ATP,
cyclin B became extensively labeled; Cdc2 was labelled to a much lesser
extent, if at all. All the active fractions contained both Cdc2 and cyclin B,
and the latter was not detected outside the peak of activity. To strengthen
the view that both proteins were associated in a complex, Labbé et al.
separated Cdc2 from cyclin B by using an HPLC reverse-phase column developed
with an acetonitrile gradient
(Labbé et al., 1989b
).
Quantifying the relative absorbance of each protein by monitoring absorbance
at 220 nm, they observed a constant 1:1 stoichiometry of Cdc2 and cyclin B in
independent preparations and in consecutive fractions throughout the final
peak of kinase activity. These results demonstrated that the purified kinase
is a heterodimer containing one molecule of Cdc2 and one molecule of cyclin
B.
The purified kinase readily induced germinal vesicle breakdown and meiotic
maturation when injected into Xenopus oocytes, which was consistent
with the reported lack of zoological specificity of MPF
(Kishimoto et al., 1982).
Moreover, it was later shown to induce meiotic maturation in starfish oocytes
only when microinjected into the nucleus. When injected into the cytoplasm, it
underwent rapid inactivation, as does the native kinase when transferred from
enucleated donor oocytes (Picard et al.,
1991
). Thus the heterodimeric Cdc2 kinase behaved differently from
the earlier preparation (Labbé et
al., 1989a
) in which Cdc2 was the only major protein.
When antibodies against recombinant starfish cyclin B became available
(Strausfeld et al., 1991), it
could be shown that cyclin B undergoes proteolysis during the gel filtration
step of the first purification procedure, generating numerous polypeptide
fragments, none of which is present in sufficient amounts to be detected by
silver staining in the final preparation. At least part of the cyclin box
escapes proteolysis, owing to its secondary structure. Since only the
N-terminal domain of cyclin contains phosphorylation sites, the final
preparation failed to autophosphorylate Cdc2-bound cyclin fragments. Moreover,
because this truncated cyclin lacks the N-terminal CRS (cytoplasmic retention
signal) and NES (nuclear exclusion signal) of cyclin B, the kinase complex
rapidly translocates into the nucleus of recipient oocytes and escapes
cytoplasmic inactivation when microinjected into G2-arrested oocytes. This
probably accounts for its paradoxically high MPF activity in starfish.
Towards a CDK family of protein kinases
One year later, identification of cyclin-BCdc2 as MPF was extended
to Xenopus oocytes: using specific antibodies against
Xenopus cyclins, Gautier et al. showed that their highly purified MPF
preparation prepared from oocytes at second metaphase (see above) contained a
mixture of cyclin-B1Cdc2 and cyclin-B2Cdc2 (Gautier et al.,
1990). No cyclin-ACdc2 was detected, even though oocytes produce cyclin
A in the second meiotic cell cycle. In 1990 and 1991, close relatives of Cdc2
were identified in Drosophila and Xenopus
(Lehner and O'Farrell, 1990;
Paris et al., 1991
). Neither
of these could rescue cell cycle arrest caused by mutations in cdc2
or CDC28 in yeasts, but Fang and Newport showed that selective
depletion of the Xenopus cdc2-related protein (known as Eg-1 at that
time) can suppress DNA replication in Xenopus egg extracts, a finding
at variance with depletion of Cdc2 (Fang
and Newport, 1991
). Moreover, it was shown that a close relative
of Eg-1 in humans can rescue G1 arrest in CDC28 budding yeast mutants
but not G2 arrest in mutants of fission yeast cdc2
(Ninomiya-Tsugi et al., 1991
;
Elledge and Spottswood, 1991
).
This demonstrated that both Cdc2 relatives have different functions in the
control of the cell cycle. Both were shown to bind to cyclin A. When it became
evident that mammalian Cdc2 homologs can also bind to cyclins
(Pines and Hunter, 1990
), a
new convention for naming these kinases was established by consensus at the
Cold Spring Harbor Symposium on the Cell Cycle in 1991: kinases that are
associated with cyclins would be called `cyclin-dependent kinases', or CDKs.
And so Cdc2 became Cdk1...
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References |
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Arion, D., Meijer, L., Brizuela, L. and Beach, D. (1988). Cdc2 is a component of the M-phase specific histone H1 kinase: evidence for identity with MPF. Cell 55,371 -378.[Medline]
Booher, R. and Beach, D. (1988). Involvement of cdc13+ in mitotic control in Schizosaccharomyces pombe: possible interaction of the gene product with microtubules. EMBO J. 7,2321 -2327.[Abstract]
Booher, R. N., Alfa, C. E., Hyams, J. S. and Beach, D. H. (1989). The fission yeast cdc2/cdc13/suc1 protein kinase: regulation of catalytic activity and nuclear localisation. Cell 58,489 -496.
Cyert, M. S. and Kirscher, M. W. (1988). Regulation of MPF activity in vitro. Cell 53,185 -195.[Medline]
Dorée, M., Peaucellier, G. and Picard, A. (1983). Activity of the maturation-promoting factor and the extent of protein phosphorylation oscillate simultaneously during meiotic maturation of starfish oocytes. Dev. Biol. 99,489 -501.[Medline]
Draetta, G., Luca, F., Westendorf, J., Brizuela, L., Ruderman, J. and Beach, D. (1989). Cdc2 kinase is complexed with both cyclin A and B, evidence for proteolytic inactivation of MPF. Cell 56,829 -838.[Medline]
Dunphy, W. G. and Newport, J. W. (1989). Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase. Cell 58,181 -191.[Medline]
Elledge, S. J. and Spottswood, M. R. (1991). A new human p34 protein kinase, CDK2, identified by complementation of a cdc28 mutation in Saccharomyces cerevisiae, is a homolog of Xenopus Eg1. EMBO J. 10,2653 -2659.[Abstract]
Evans, T., Rosenthal, E., Youngblom, J., Distel, D. and Hunt, T. (1983). Cyclin, a protein specified by maternal mRNA that is destroyed at each cleavage division. Cell 33,389 -396.[Medline]
Fang, F. and Newport, J. (1991). Evidence that the G1-S and G2-M transition are controlled by different cdc2 proteins in higher eukaryotes. Cell 66,731 -740.[Medline]
Gautier, J. and Maller, J. L. (1991). Cyclin B in Xenopus oocytes: implication for the mechanism of pre-MPF activation. EMBO J. 10,177 -182.[Abstract]
Gautier, J., Norbury, C., Lohka, M., Nurse, P. and Maller, J. (1988). Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+. Cell 54,433 -439.[Medline]
Gautier, J., Matsukawa, T., Nurse, P. and Maller, J. (1989). Dephosphorylation and activation of Xenopus p34cdc2 protein kinase during the cell cycle. Nature 339,626 -629.[Medline]
Gerhart, J. C., Wu, M. and Kirschner, M. (1984). Cell cycle dynamics of an M-phase specific cytoplasmic factor in Xenopus laevis oocytes and eggs. J. Cell Biol. 98,1247 -1255.[Abstract]
Guerrier, P., Moreau, M. and Dorée, M. (1977). Hormonal control of meiosis in starfish: stimulation of protein phosphorylation induced by 1-methyladenine. Mol. Cell. Endocrinol. 7,137 -150.[Medline]
Hultin, T. (1961). The effect of puromycin on protein metabolism and cell division in fertilized sea urchin eggs. Experientia 17,410 -411.[Medline]
Kishimoto, T., Hirai, S. and Kanatani, H. (1981). Role of germinal vesicle in producing maturation-promoting factor in starfish oocytes. Dev. Biol. 81,177 -181.[Medline]
Kishimoto, T., Kuriyama, R., Kondo, H. K. and Kanatani, H. (1982). Generality of the action of various maturation-promoting factors. Exp. Cell Res. 137,121 -126.[Medline]
Labbé, J. C., Lee, M. G., Nurse, P., Picard, A. and Dorée, M. (1988). Activation at M-phase of a protein kinase encoded by a starfish homolog of the cell cycle control gene cdc2+. Nature 335,251 -254.[Medline]
Labbé, J. C., Picard, A., Peaucellier, G., Cavadore, J. C., Nurse, P. and Dorée, M. (1989a). Purification of MPF from starfish: identification as the H1 histone kinase p34cdc2 ad a possible mechanism for its periodic activation. Cell 57,253 -263.[Medline]
Labbé, J. C., Capony, J. P., Caput, D., Cavadore, J. C., Derancourt, J., Kaghad, M., Lelias, J. M., Picard, A. and Dorée, M. (1989b). MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule of cyclin B. EMBO J. 8,3053 -3058.[Abstract]
Lee, M. and Nurse, P. (1987). Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature 327,31 -35.[Medline]
Lehner, C. F. and O'Farrell, P. H. (1990). Drosophila cdc2 homologs: a functional homolog is coexpressed with its cognate variant. EMBO J. 9,3573 -3581.[Abstract]
Lohka, M. J., Hayer, M. K. and Maller, J. L. (1988). Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. Proc. Natl. Acad. Sci. USA 85,3009 -3015.[Abstract]
Maller, J., Wu, M. and Gerhart, J. C. (1977). Changes in protein phosphorylation accompanying maturation of Xenopus laevis oocytes. Dev. Biol. 58,295 -312.[Medline]
Masui, Y. and Markert, C. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool. 177,129 -146.[Medline]
Meijer, L., Arion, D., Golsteyn, R., Pines, J., Brizuela, L., Hunt, T. and Beach, D. (1989). Cyclin and p34cdc2 are associated with M-phase specific histone H1 kinase in fertilized sea urchin eggs. EMBO J. 8,2275 -2282.[Abstract]
Minshull, J., Blow, J. J. and Hunt, T. (1989a). Translation of cyclin mRNA is necessary for extracts of activated Xenopus eggs to enter mitosis. Cell 56,947 -956.[Medline]
Minshull, J., Pines, J., Golsteyn, R., Standart, N., Mackie, S., Colman, A., Blow, J., Ruderman, J. V., Wu, M. and Hunt, T. (1989b). The role of cyclin synthesis, modification and destruction in the control of cell division. J. Cell Sci. Suppl. 12,77 -97.[Medline]
Moreno, S., Hayles, S. and Nurse, P. (1989). Regulation of p34cdc2 protein kinase during mitosis. Cell 58,361 -370.[Medline]
Murray, A. W. and Kirschner, M. (1989). Cyclin synthesis drives the early embryonic cell cycle. Nature 339,275 -280.[Medline]
Ninomiya-Tsugi, J., Nomoto, S., Yasuda, H., Reed, S. I. and Matsumoto, K. (1991). Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation. Proc. Natl. Acad. Sci. USA 88,9006 -9010.[Abstract]
Paris, J., le Guellec, R., Couturier, A., le Guellec, K., Omilli, F., Camonis, J., MacNeill, S. and Philippe, M. (1991). Cloning by differential screening of a Xenopus cDNA coding for a protein highly homologous to cdc2. Proc. Natl. Acad. Sci. USA 88,1039 -1043.[Abstract]
Picard, A. and Dorée, M. (1984). The role of the germinal vesicle material in producing maturation-promoting factor (MPF), as revealed by the removal and transplantation of nuclear material in starfish oocytes. Dev. Biol. 104,357 -365.[Medline]
Picard, A., Peaucellier, G., le Bouffant, F., le Peuch, C. and Dorée, M. (1985). Role of protein synthesis and proteases in production and inactivation of maturation-promoting activity during meiotic maturation of starfish oocytes. Dev. Biol. 109,311 -320.[Medline]
Picard, A., Harricane, M. C., Labbé, J. C. and Dorée, M. (1988). Germinal vesicle components are not required for the cell-cycle oscillator of the early starfish embryo. Dev. Biol. 128,121 -128.[Medline]
Picard, A., Labbé, J. C., Barakat, H., Cavadore, J. C. and Dorée, M. (1991). Okadaic acid mimics a nuclear component required for cyclin B-cdc2 kinase microinjection to drive starfish oocytes into M-phase. J. Cell Biol. 115,337 -344.[Abstract]
Pines, J. and Hunter, T. (1990). Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B. Nature 346,760 -764.[Medline]
Russell, P. and Nurse, P. (1987). Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog. Cell 49,559 -567.[Medline]
Strausfeld, U., Labbé, J. C., Fesquet, D., Cavadore, J. C., Picard, A., Sadu, K., Russell, P. and Dorée, M. (1991). Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein. Nature 351,242 -245.[Medline]
Swenson, K. I., Farrell, K. M. and Ruderman, J. V. (1986). The clam embryo protein cyclin A induces entry into M-phase and the resumption of meiosis in Xenopus oocytes. Cell 47,861 -870.[Medline]
Wagenaar, E. B. (1983). The timing of synthesis of proteins required for mitosis in the cell cycle of the sea urchin embryo. Exp. Cell Res. 144,393 -403.[Medline]
Wasserman, W. J. and Masui, Y. (1975). Effects of cycloheximide on a cytoplasmic factor initiating meiotic naturation in Xenopus. Exp Cell Res. 91,381 -388.[Medline]