The onset of mitosis is controlled by the cyclin dependent kinase Cdc2p. Cdc2p activity is controlled through the balance of phosphorylation and dephosphorylation of tyrosine-15 (Y15) by the Wee1p kinase and Cdc25p phosphatase. In the fission yeast Schizosaccharomyces pombe, detection of DNA damage in G(2) activates a checkpoint that prevents entry into mitosis through the maintenance of Y15 phosphorylation of Cdc2p, thus ensuring DNA repair precedes chromosome segregation. The protein kinase Chk1p is the endpoint of this checkpoint pathway. We have previously reported that overexpression of Chk1p causes a wee1(+)-dependent G(2) arrest, and this or irradiation leads to hyperphosphorylation of Wee1p. Moreover, Chk1p directly phosphorylates Wee1p in vitro. These data suggested that Wee1p is a key target of Chk1p action in checkpoint control. However, cells lacking wee1(+) are checkpoint proficient and sustained Chk1p overexpression arrests cell cycle progression independently of Wee1p. Therefore, up-regulation of Wee1p alone cannot enforce a checkpoint arrest. Chk1p can also phosphorylate Cdc25p in vitro. These phosphorylation events are thought to promote the interaction with 14–3-3 proteins the cytoplasmic retention of the 14–3-3/Cdc25p complexes. However, we show here that the G(2) DNA damage checkpoint is intact in cells that regulate mitotic entry independently of Cdc25p. Further, these cells are still sensitive to Chk1p-mediated arrest, and so down-regulation of Cdc25p is also insufficient to regulate checkpoint arrest. Conversely, inactivation of both wee1(+) and cdc25(+)abolishes checkpoint control. We also show that activation of the G(2) DNA damage checkpoint induces a transient increase in Wee1p levels. We conclude that the G(2) DNA damage checkpoint simultaneously signals via both up-regulation of Wee1p and down-regulation of Cdc25p, thus providing a double-lock mechanism to ensure cell cycle arrest and genomic stability.

Al-Khodairy
F.
,
Carr
A. M.
(
1992
).
DNA repair mutants defining G2checkpoint pathways in Schizosaccharomyces pombe.
EMBO J
11
,
1343
1350
Baber-Furnari
B. A.
,
Rhind
N.
,
Boddy
M. N.
,
Shanahan
P.
,
Lopez-Girona
A.
,
Russell
P.
(
2000
).
Regulation of mitotic inhibitor mik1 helps to enforce the DNA damage checkpoint.
Mol. Biol. Cell
11
,
1
11
Barbet
N. C.
,
Carr
A. M.
(
1993
).
Fission yeast wee1 protein kinase is not required for DNA damage-dependent mitotic arrest.
Nature
364
,
824
827
Blasina
A.
,
de Weyer
I. V.
,
Laus
M. C.
,
Luyten
W.
,
Parker
A. E.
,
McGowan
C. H.
(
1999
).
A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase.
Curr. Biol
9
,
1
10
Bunz
F.
,
Dutriaux
A.
,
Lengauer
C.
,
Waldman
T.
,
Zhou
S.
,
Brown
J. P.
,
Sedivy
J. M.
,
Kinzler
K. W.
,
Vogelstein
B.
(
1999
).
Requirement for p53 and p21 to sustain G2arrest after DNA damage.
Science
282
,
1497
1501
Chen
L.
,
Liu
T. H.
,
Walworth
N. C.
(
1999
).
Association of Chk1 with 14–3-3 proteins is stimulated by DNA damage.
Genes Dev
13
,
675
685
Christensen
P. U.
,
Bentley
N. J.
,
Martinho
R. G.
,
Nielsen
O.
,
Carr
A. M.
(
2000
).
Mik1 levels accumulate in S phase and may mediate an intrinsic link between S phase and mitosis.
Proc. Nat. Acad. Sci. USA
97
,
2579
2584
Dalal
S. N.
,
Schweitzer
C. M.
,
Gan
J.
,
DeCaprio
J. A.
(
1999
).
Cytoplasmic localization of human cdc25C during interphase requires an intact 14–3-3 binding site.
Mol. Cell Biol
19
,
4465
4479
Fantes
P.
(
1979
).
Epistatic gene interactions in the control of division in fission yeast.
Nature
279
,
428
430
Forbes
K. C.
,
Humphrey
T.
,
Enoch
T.
(
1998
).
Suppressors of cdc25p overexpression identify two pathways that influence the G2/M checkpoint in fission yeast.
Genetics
150
,
1361
1375
Furnari
B.
,
Rhind
N.
,
Russell
P.
(
1997
).
Cdc25 mitotic inducer targeted by chk1 DNA damage checkpoint kinase.
Science
277
,
1495
1497
Furnari
B.
,
Blasina
A.
,
Boddy
M. N.
,
McGowan
C. H.
,
Russell
P.
(
1999
).
Cdc25 inhibited in vivo and in vitro by checkpoint kinases Cds1 and Chk1.
Mol. Biol. Cell
10
,
833
845
Gould
K. L.
,
Moreno
S.
,
Tonks
N. K.
,
Nurse
P.
(
1990
).
Complementation of the mitotic activator, p80cdc25, by a human protein-tyrosine phosphatase.
Science
250
,
1573
1576
Greenwood
E.
,
Nishitani
H.
,
Nurse
P.
(
1998
).
Cdc18p can block mitosis by two independent mechanisms.
J. Cell Sci
111
,
3101
3108
Kumagai
A.
,
Guo
Z.
,
Emani
K. H.
,
Wang
S. X.
,
Dunphy
W. G.
(
1998
).
The Xenopus chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control in cell-free extracts.
J. Cell Biol
142
,
1559
1569
Kumagai
A.
,
Yakowec
P. S.
,
Dunphy
W. G.
(
1998
).
14-3-3 proteins act as negative regulators of the mitotic inducer Cdc25 in Xenopus egg extracts.
Mol. Biol. Cell
9
,
345
354
Kumagai
A.
,
Dunphy
W. G.
(
1999
).
Binding of 14–3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25.
Genes Dev
13
,
1067
1072
Leu
J. Y.
,
Roeder
G. S.
(
1999
).
The pachytene checkpoint in S. cerevisiae depends on Swe1-mediated phosphorylation of the cyclin-dependent kinase Cdc28.
Mol. Cell
4
,
805
814
Lopez-Girona
A.
,
Furnari
B.
,
Mondesert
O.
,
Russell
P.
(
1999
).
Nuclear localization of Cdc25 is regulated by DNA damage and a 14–3-3 protein.
Nature
397
,
172
175
Lundgren
K.
,
Walworth
N.
,
Booher
R.
,
Dembski
M.
,
Kirschner
M.
,
Beach
D.
(
1991
).
mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2.
Cell
64
,
1111
1122
Martinho
R. G.
,
Lindsay
H. D.
,
Flaggs
G.
,
DeMaggio
A. J.
,
Hoekstra
M. F.
,
Carr
A. M.
,
Bentley
N. J.
(
1998
).
Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.
EMBO J
17
,
7239
7249
Michael
W. M.
,
Newport
J.
(
1998
).
Coupling of mitosis to the completion of S phase through Cdc34-mediated degradation of Wee1 [published erratum appears in Science 1999 Jan 1;283(5398):35].
Science
282
,
1886
1889
Millar
J. B. A.
,
Lenaers
G.
,
Russell
P.
(
1992
).
Pyp3 PTPase acts as a mitotic inducer in fission yeast.
EMBO J
11
,
4933
4941
Moreno
S.
,
Klar
A.
,
Nurse
P.
(
1991
).
Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.
Meth. Enzymol
194
,
795
723
Murakami
H.
,
Nurse
P.
(
1999
).
Meiotic DNA replication checkpoint control in fission yeast.
Genes Dev
13
,
2581
2593
Nurse
P.
(
1975
).
Genetic control of cell size at cell division in yeast.
Nature
256
,
547
555
O'Connell
M. J.
,
Norbury
C.
,
Nurse
P.
(
1994
).
Premature chromatin condensation upon accumulation of NIMA.
EMBO J
13
,
4926
4937
O'Connell
M. J.
,
Raleigh
J. M.
,
Verkade
H. D.
,
Nurse
P.
(
1997
).
Chk1 is a wee1 kinase in the G2DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation.
EMBO J
16
,
545
554
Ouyang
B.
,
Li
W.
,
Pan
H.
,
Meadows
J.
,
Hoffmann
I.
,
Dai
W.
(
1999
).
The physical association and phosphorylation of Cdc25C protein phosphatase by Prk.
Oncogene
18
,
6029
6036
Peng
C. Y.
,
Graves
P. R.
,
Thoma
R. S.
,
Wu
Z.
,
Shaw
A. S.
,
Piwnica-Worms
H.
(
1997
).
Mitotic and G2checkpoint control: regulation of 14–3-3 protein binding by phosphorylation of Cdc25C on serine-216.
Science
277
,
1501
1505
Peng
C. Y.
,
Graves
P. R.
,
Ogg
S.
,
Thoma
R. S.
,
Byrnes
M. J.
3rd
,
Wu
Z.
,
Stephenson
M. T.
,
Piwnica-Worms
H.
(
1998
).
C-TAK1 protein kinase phosphorylates human Cdc25C on serine 216 and promotes 14–3-3 protein binding.
Cell Growth Differ
9
,
197
208
Rhind
N.
,
Furnari
B.
,
Russell
P.
(
1997
).
Cdc2 tyrosine phosphorylation is required for the DNA damage checkpoint in fission yeast.
Genes Dev
11
,
504
511
Russell
P.
,
Nurse
P.
(
1986
).
cdc25+ functions as an inducer in the mitotic control of fission yeast.
Cell
45
,
145
153
Russell
P.
,
Nurse
P.
(
1987
).
Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog.
Cell
49
,
559
567
Sanchez
Y.
,
Wong
C.
,
Thoma
R. S.
,
Richman
R.
,
Wu
Z.
,
Piwnica-Worms
H.
,
Elledge
S. J.
(
1997
).
Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25 [see comments].
Science
277
,
1497
1501
Sheldrick
K. S.
,
Carr
A. M.
(
1993
).
Feedback controls and G2checkpoints: fission yeast as a model system.
BioEssays
15
,
775
781
Shiozaki
K.
,
Russell
P.
(
1995
).
Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast.
Nature
378
,
739
743
Sveiczer
A.
,
Novak
B.
,
Mitchison
J. M.
(
1996
).
The size control of fission yeast revisited.
J. Cell Sci
109
,
2947
2957
Sveiczer
A.
,
Novak
B.
,
Mitchison
J. M.
(
1999
).
Mitotic control in the absence of cdc25 mitotic inducer in fission yeast.
J. Cell Sci
112
,
1085
1092
Toyoshima
F.
,
Moriguchi
T.
,
Wada
A.
,
Fukuda
M.
,
Nishida
E.
(
1998
).
Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2checkpoint.
EMBO J
17
,
2728
2735
Uetz
P.
,
Giot
L.
,
Cagney
G.
,
Mansfield
T. A.
,
Judson
R. S.
,
Knihjt
J. R.
, et al. 
(
2000
).
A comphrensive analysis of protein-protein interactions in Saccharomyces cerevisiae.
Nature
403
,
623
627
Walworth
N.
,
Davey
S.
,
Beach
D.
(
1993
).
Fission yeast chk1 protein kinase links the rad checkpoint pathway to cdc2.
Nature
363
,
368
371
Ye
X. S.
,
Fincher
R. R.
,
Tang
A.
,
Osmani
S. A.
(
1996
).
The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2in Aspergillus nidulans.
EMBO J
16
,
182
192
Zeng
Y.
,
Forbes
K. C.
,
Wu
Z.
,
Moreno
S.
,
Piwnica-Worms
H.
,
Enoch
T.
(
1998
).
Replication checkpoint requires phosphorylation of the phosphatase cdc25 by cds1 or chk1.
Nature
395
,
507
–.
Zeng
Y.
,
Piwnica-Worms
H.
(
1999
).
DNA damage and replication checkpoints in fission yeast require nuclear exclusion of the Cdc25 phosphatase via 14–3-3 binding.
Mol. Cell Biol
19
,
7410
7419
This content is only available via PDF.