During the cell cycle, a checkpoint prevents the initiation of mitosis until S-phase is completed. The molecular mechanism may involve the RCC1 protein, which catalyses guanine nucleotide exchange on the Ras-related nuclear protein, Ran (or TC4). Genetic studies have suggested that RCC1 may be involved in sensing the replication state of DNA and controlling the activation of Cdc2/cyclin B protein kinase through Ran. In this report, we present direct biochemical evidence for the post-translational control of Cdc2/cyclin B activation by Ran. In a cell-free system of concentrated Xenopus egg extracts supplemented with nuclei, a mutant form of Ran (T24N) analogous to dominant inactive mutants of other Ras-related GTPases inhibits Cdc2/cyclin B activation in the presence of replicating nuclear DNA. This role for Ran is mediated through control of the tyrosine phosphorylation state of Cdc2 and appears to be distinct from other effects on nuclear import, nuclear formation and DNA replication. When extracts were supplemented with RCC1 protein prior to addition of Ran T24N, inhibition of Cdc2/cyclin B by Ran T24N was relieved. This suggests that Ran T24N may act in a dominant manner by sequestering RCC1 in an inactive form. In contrast to Ran T24N, a mutant of Ran (Q69L) defective in GTPase activity and hence locked in the GTP-bound state has no inhibitory effect on Cdc2/cyclin B activation. In the light of these results, we propose that generation of the GTP-bound form of Ran is required for Cdc2/cyclin B activation and entry into mitosis when this process is coupled to the progression of S-phase.

REFERENCES

Al-Khodairy
F.
,
Carr
A. M.
(
1992
).
DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe.
EMBO J
11
,
1343
1350
Bischoff
F. R.
,
Maier
G.
,
Tilz
G.
,
Ponstingl
H.
(
1990
).
A 47-kDa human nuclear protein recognized by antikinetochore autoimmune sera is homologous with the protein encoded by RCC1, a gene implicated in onset of chromosome condensation.
Proc. Nat. Acad. Sci. USA
87
,
8617
8621
Bischoff
F. R.
,
Ponstingl
H.
(
1991
).
Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1.
Nature
354
,
80
82
Bischoff
F. R.
,
Ponstingl
H.
(
1991
).
Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide.
Proc Nat. Acad. Sci. USA
88
,
10830
10834
Bischoff
F. R.
,
Klebe
C.
,
Kretschmer
J.
,
Wittinghofer
A.
,
Ponstingl
H.
(
1994
).
Ran-GAP1 induces GTPase activity of nuclear Ras-related Ran.
Proc. Nat. Acad. Sci. USA
91
,
2587
2591
Blow
J. J.
,
Laskey
R. A.
(
1986
).
Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.
Cell
47
,
577
587
Clarke
P. R.
,
Leiss
D.
,
Pagano
M.
,
Karsenti
E.
(
1992
).
Cyclin A-and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts.
EMBO J
11
,
1751
1761
Clarke
P. R.
,
Hoffmann
I.
,
Draetta
G.
,
Karsenti
E.
(
1993
).
Dephosphorylation of cdc25-C by a type-2A protein phosphatase: specific regulation during the cell cycle in Xenopus egg extracts.
Mol. Biol. Cell
4
,
397
411
Coutavas
E.
,
Ren
M.
,
Oppenheim
J. D.
,
D'Eustachio
P.
,
Rush
M. G.
(
1993
).
Characterization of proteins that interact with the cell-cycle regulatory protein Ran/TC4.
Nature
366
,
585
587
Dasso
M.
,
Newport
J. W.
(
1990
).
Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus.
Cell
61
,
811
823
Dasso
M.
,
Nishitani
H.
,
Kornbluth
S.
,
Nishimoto
T.
,
Newport
J. W.
(
1992
).
RCC1, a regulator of mitosis, is essential for DNA replication.
Mol. Cell. Biol
12
,
3337
3345
Drivas
G. T.
,
Shih
A.
,
Coutavas
E.
,
Rush
M. G.
,
D'Eustachio
P.
(
1990
).
Characterization of four novel ras -like genes expressed in a human teratocarcinoma cell line.
Mol. Cell. Biol
10
,
1793
1798
Enoch
T.
,
Nurse
P.
(
1991
).
Coupling M phase and S phase: controls maintaining the dependence of mitosis on chromosome replication.
Cell
65
,
921
923
Enoch
T.
,
Carr
A. M.
,
Nurse
P.
(
1992
).
Fission yeast genes involved in coupling mitosis to completion of DNA replication.
Genes Dev
6
,
2035
2046
Farnsworth
C. L.
,
Feig
L. A.
(
1991
).
Dominant inhibitory mutations in the Mg2+-binding site of RasHprevent its activation by GTP.
Mol. Cell. Biol
11
,
4822
4829
Feig
L. A.
,
Cooper
G. M.
(
1988
).
Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.
Mol. Cell. Biol
8
,
3235
3243
Glotzer
M.
,
Murray
A. W.
,
Kirschner
M. W.
(
1991
).
Cyclin is degraded by the ubiquitin pathway.
Nature
349
,
132
138
Gould
K. L.
,
Nurse
P.
(
1989
).
Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis.
Nature
342
,
39
45
Gurdon
J. B.
(
1976
).
Injected nuclei in frog oocytes: Fate, enlargement, and chromatin dispersal.
J. Embryol. Exp. Morph
36
,
523
540
Hartwell
L. H.
,
Weinert
T. A.
(
1989
).
Checkpoints: controls that ensure the order of cell cycle events.
Science
246
,
629
634
Hunt
T.
(
1991
).
Cyclins and their partners: from a simple idea to complicated reality.
Semin. Cell Biol
2
,
213
222
Hutchison
C. J.
,
Cox
R.
,
Drepaul
R. S.
,
Gomperts
M.
,
Ford
C. C.
(
1987
).
Periodic DNA synthesis in cell-free extracts of Xenopus eggs.
EMBO J
6
,
2003
2010
John
J.
,
Sohman
R.
,
Feuerstein
J.
,
Linke
R.
,
Wittinghofer
A.
,
Goody
R.
(
1990
).
Kinetics of interaction of nucleotides with nucleotide-free H-ras p21.
Biochemistry
29
,
6058
6065
John
J.
,
Rensland
H.
,
Schlichting
i.
,
Vetter
I.
,
Borasio
G. D.
,
Goody
R. S.
,
Wittinghofer
A.
(
1993
).
Kinetic and structural analysis of the Mg2+-binding site of the guanine nucleotide binding protein p21H-ras.
J. Biol. Chem
268
,
923
929
Kharbanda
S.
,
Saleem
A.
,
Datta
R.
,
Yuan
Z.-M.
,
Weichselbaum
R.
,
Kufe
D.
(
1994
).
Ionizing radiation induces rapid tyrosine phosphorylation of p34cdc2.
Cancer Res
54
,
1412
1414
Kinoshita
N.
,
Yamano
H.
,
Niwa
H.
,
Yoshida
T.
,
Yanagida
M.
(
1993
).
Negative regulation of mitosis by the fission yeast protein phosphatase ppa2.
Genes Dev
7
,
1059
1071
Klebe
C.
,
Nishimoto
T.
,
Wittinghofer
F.
(
1993
).
Functional expression in Escherichia coli of the mitotic regulator proteins p24ranand p45rcc1and fluorescence measurements of their interaction.
Biochemistry
32
,
11923
11928
Kornbluth
S.
,
Dasso
M.
,
Newport
J.
(
1994
).
Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression.
J. Cell Biol
125
,
705
719
Kornbluth
S.
,
Sebastian
B.
,
Hunter
T.
,
Newport
J.
(
1994
).
Membrane localization of the kinase which phosphorylates p34cdc2on threonine 14.
Mol. Biol. Cell
5
,
273
282
Kumagai
A.
,
Dunphy
W. G.
(
1991
).
The cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system.
Cell
64
,
903
914
Kumagai
A.
,
Dunphy
W. G.
(
1992
).
Regulation of the cdc25 protein during the cell cycle in Xenopus extracts.
Cell
70
,
139
151
Lock
R. B.
(
1992
).
Inhibition of p34 cdc2 kinase activation, p34 cdc2 tyrosine dephosphorylation, and mitotic progression in Chinese hamster ovary cells exposed to etoposide.
Cancer Res
52
,
1817
22
Lounsbury
K. M.
,
Beddow
A. L.
,
Macara
I. G.
(
1994
).
A family of proteins that stabilize the Ran/TC4 GTPase in its GTP-bound conformation.
J. Biol. Chem
269
,
11285
11290
Matsumoto
T.
,
Beach
D.
(
1991
).
Premature initiation of mitosis in yeast lacking RCC1 or an interacting GTPase.
Cell
66
,
347
360
Mechali
M.
,
Harland
R. M.
(
1982
).
DNA synthesis in a cell-free system from Xenopus eggs: priming and elongation on single-stranded DNA in vitro.
Cell
30
,
93
101
Melchior
F.
,
Paschal
B.
,
Evans
J.
,
Gerace
L.
(
1993
).
Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor.
J. Cell Biol
123
,
1649
1659
Moore
M. S.
,
Blobel
G.
(
1993
).
The GTP-binding protein Ran/TC4 is required for protein import into the nucleus.
Nature
365
,
661
663
Murray
A. W.
,
Kirschner
M. W.
(
1989
).
Cyclin synthesis drives the early embryonic cell cycle.
Nature
339
,
275
280
Murray
A. W.
(
1992
).
Creative blocks: cell-cycle checkpoints and feedback controls.
Nature
359
,
599
604
Nasmyth
K.
,
Hunt
T.
(
1993
).
Dams and sluices.
Nature
366
,
634
635
Newport
J.
(
1987
).
Nuclear reconstitution in vitro: Stages of assembly around protein-free DNA.
Cell
48
,
205
217
Nishitani
H.
,
Ohtsubo
M.
,
Yamashita
K.
,
Iida
H.
,
Pines
J.
,
Yasudo
H.
,
Shibata
Y.
,
Hunter
T.
,
Nishimoto
T.
(
1991
).
Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis.
EMBO J
10
,
1555
1564
Nurse
P.
(
1990
).
Universal control mechanism regulating onset of M-phase.
Nature
344
,
503
508
O'Connor
P. M.
,
Ferris
D. K.
,
Pagano
M.
,
Draetta
G.
,
Pines
J.
,
Hunter
T.
,
Longo
D. L.
,
Kohn
K. W.
(
1993
).
G2 delay induced by nitrogen mustard in human cells affects cyclin A/cdk2 and cyclin B1/cdc2-kinase complexes differently.
J. Biol. Chem
268
,
8298
8308
Ohtsubo
M.
,
Okazaki
H.
,
Nishimoto
T.
(
1989
).
The RCC1 protein, a regulator for the onset of chromosome condensation locates in the nucleus and binds to DNA.
J. Cell Biol
109
,
1389
1397
Pai
F. E.
,
Kabsch
W.
,
Krengel
U.
,
Holmes
K. C.
,
John
J.
,
Wittinghofer
A.
(
1989
).
Structure of the guanine-nucleotide binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation.
Nature
341
,
209
214
Pines
J.
(
1993
).
Cyclins and cyclin-dependent kinases: take your partners.
Trends Biochem. Sci
18
,
195
197
Polakis
P.
,
McCormick
F.
(
1993
).
Structural requirements for the interaction of p21ras with GAP, exchange factors, and its biological effector target.
J. Biol. Chem
268
,
9157
9160
Ren
M.
,
Drivas
G.
,
D'Eustachio
P.
,
Rush
M. G.
(
1993
).
Ran/TC4: asmall nuclear GTP-binding protein that regulates DNA synthesis.
J. Cell Biol
120
,
313
323
Ren
M.
,
Coutavas
E.
,
D'Eustachio
P.
,
Rush
M. G.
(
1994
).
Effects of mutant Ran/TC4 proteins on cell cycle progression.
Mol. Cell. Biol
14
,
4216
4224
Sazer
S.
,
Nurse
P.
(
1994
).
A fission yeast RCC1-related protein is required for the mitosis to interphase transition.
EMBO J
13
,
606
615
Schweighofer
F.
,
Cai
H.
,
Chevallier-Multon
M. C.
,
Fath
I.
,
Cooper
G.
,
Tocque
B.
(
1993
).
The Saccharomyces cerevisiae SDC25 C-domain gene product overcomes the dominant inhibitory activity of Ha-Ras Asn-17.
Mol. Cell. Biol
13
,
39
43
Sheehan
M. A.
,
Mills
A. D.
,
Sleeman
A. M.
,
Laskey
R. A.
,
Blow
J. J.
(
1988
).
Steps in the assembly of replication competent nuclei in a cell-free system from Xenopus eggs.
J. Cell Biol
106
,
1
12
Smythe
C.
,
Newport
J. W.
(
1992
).
Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34cdc2.
Cell
68
,
787
797
Solomon
M. J.
,
Glotzer
M.
,
Lee
T. H.
,
Philippe
M.
,
Kirschner
M. W.
(
1990
).
Cyclin activation of p34cdc2.
Cell
63
,
1013
1024
Tang
Z.
,
Coleman
T. R.
,
Dunphy
W. G.
(
1993
).
Two distinct mechanisms for the negative regulation of the wee1 protein kinase.
EMBO J
12
,
3427
3436
Tsao
Y. P.
,
D'Arpa
P.
,
Liu
L. F.
(
1992
).
The involvement of active DNA synthesis in camptothecin-induced G2 arrest: altered regulation of p34cdc2/cyclin B.
Cancer Res
52
,
1823
1829
Weinert
T.
,
Lydall
D.
(
1993
).
Cell cycle checkpoints, genetic instability and cancer.
Semin. Cancer Biol
4
,
129
140
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