In the fission yeast Schizosaccharomyces pombe, cdc2 function is required both in G1 to enter the cell cycle and in G2 to initiate mitosis. In higher eukaryotes, these functions appeared to be shared between several cdc2-like genes including CDK2. Temperature-sensitive mutations in S. pombe cdc2 that arrest the cell cycle in both G1 and G2 phases are not complemented by CDK2. We have used S. pombe to investigate what functions CDK2 can perform. We found that overexpression of the human homologue (HsCDK2) caused cell cycle arrest in G2/M showing that HsCDK2 interfered with mitotic events. Xenopus CDK2 (XlCDK2) overexpression did not cause cell cycle arrest and could rescue the G1 block but not the G2 block of a cdc2-M26 ts strain. A mutant XlCDK2-R33, which is inactive as a kinase, failed to rescue the G1 block, suggesting that the protein kinase activity of CDK2 is required to enter the cell cycle in these circumstances. We designed screens to select mutants that would require XlCDK2 expression for viability, hoping to isolate new gene functions interacting with, or that could be replaced by, XlCDK2 in G1, or new cdc2 mutants altered solely in their G1 role. From these screens several cell cycle mutants were selected that were XlCDK2-dependent. These were all cdc2 mutants altered only in their G2/M function. Therefore XlCDK2 can influence both the G1/S and G2/M transition points of cdc2 in S. pombe.

REFERENCES

Ayscough
K.
,
Hayles
J.
,
MacNeill
S. A.
,
Nurse
P.
(
1992
).
Cold-sensitive mutants of p34cdc2 that suppress a mitotic catastrophe phenotype in fission yeast.
Mol. Gen. Genet
232
,
344
350
Basi
G.
,
Schmid
E.
,
Maundrell
K.
(
1993
).
TATA box mutations in Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility.
Gene
123
,
131
136
Booher
R.
,
Beach
D. H.
(
1986
).
Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe.
Mol. Cell. Biol
6
,
3523
3530
Carr
A. M.
,
MacNeill
S. A.
,
Hayles
J.
,
Nurse
P.
(
1989
).
Molecular cloning and sequence analysis of mutant alleles of fission yeast cdc2 protein kinase gene: implications for cdc2+ protein structure and function.
Mol. Gen. Genet
218
,
41
49
Connell-Crowley
L.
,
Solomon
M. J.
,
Wei
N.
,
Harper
J. W.
(
1993
).
Phosphorylation independent activation of human cyclin-dependent kinase-2 by cyclin-A in vitro. Mol. Biol.
Cell
4
,
79
92
Costello
G.
,
Rodgers
L.
,
Beach
D.
(
1986
).
Fission yeast enters the stationary phase G0state from either mitotic G1or G2.
Curr. Genet
11
,
119
125
Desai
D.
,
Gu
Y.
,
Morgan
D. O.
(
1992
).
Activation of human cyclin-dependent kinases in vitro. Mol. Biol.
Cell
3
,
571
582
Devoto
S. H.
,
Mudryj
M.
,
Pines
J.
,
Hunter
T.
,
Nevins
J. R.
(
1992
).
A cyclin-A protein kinase complex possesses sequence-specific DNA binding activity: p33cdk2 is a component of the E2F-cyclin-A complex.
Cell
68
,
167
176
Dulic
V.
,
Lees
E.
,
Reed
S. I.
(
1992
).
Association of human cyclin E with a periodic G1-S phase protein kinase.
Science
257
,
1958
1961
Durkacz
B.
,
Carr
A.
,
Nurse
P.
(
1986
).
Transcription of the cdc2 cell cycle control gene of the fission yeast Schizosaccharomyces pombe.
EMBO J
5
,
369
373
Elledge
S. J.
,
Spottswood
M. R.
(
1991
).
A new human p34 protein kinase, CDK2, identified by complementation of a cdc28 mutation in Saccharomyces cerevisiae, is a homologue of Xenopus Eg1.
EMBO J
10
,
2653
2659
Fang
F.
,
Newport
J. W.
(
1991
).
Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes.
Cell
66
,
731
742
Fleig
U. N.
,
Nurse
P.
(
1991
).
Expression of a dominant negative allele of cdc2 prevents activation of the endogenous p34cdc2 kinase.
Mol. Gen. Genet
226
,
432
440
Fleig
U. N.
,
Gould
K. L.
,
Nurse
P.
(
1992
).
A dominant negative allele ofp34cdc2 shows altered phosphoamino acid content and sequesters p56cdc13 cyclin.
Mol. Cell. Biol
12
,
2295
2301
Forsburg
S.
,
Nurse
P.
(
1991
).
Cell cycle regulation in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Annu. Rev. Cell Biol
7
,
227
256
Gabrielli
B. G.
,
Roy
L. M.
,
Maller
J. L.
(
1993
).
Requirement for cdk2 in Cytostatic Factor-mediated metaphase II arrest.
Science
259
,
1766
1769
Gu
Y.
,
Rosenblatt
J.
,
Morgan
D. O.
(
1992
).
Cell cycle regulation of cdk2 activity by phosphorylation of Thr160 and Tyr15.
EMBO J
11
,
3995
4005
Hirai
T.
,
Yamashita
M.
,
Yoshikuni
M.
,
Tokumoto
T.
,
Kajiura
H.
,
Sakai
N.
,
Nagahama
Y.
(
1992
).
Isolation and characterisation of Godfish cdk2, a cognate variant of the cell cycle regulator cdc2.
Dev. Biol
152
,
113
120
Hofer
F.
,
Hollerstein
H.
,
Janner
F.
,
Minet
M.
,
Thuriaux
P.
,
Leupold
U.
(
1979
).
The genetic fine structure of nonsense suppressors in Schizosaccharomyces pombe.
Curr. Genet
1
,
45
61
Koff
A.
,
Cross
F.
,
Fisher
A.
,
Schumacher
J.
,
Le Guellec
K.
,
Philippe
M.
,
Roberts
J. M.
(
1991
).
Human cyclin E, a new cyclin that interacts with two members of the CDC2 gene family.
Cell
66
,
1217
1228
Koff
A.
,
Giordano
A.
,
Desai
D.
,
Yamashita
K.
,
Wade Harper
J.
,
Elledge
S.
,
Nishimoto
T.
,
Morgan
D. O.
,
Robert Franza
B.
,
Roberts
J. M.
(
1992
).
Formation and activation of a cyclin E-cdk2 complex during the G1phase of the human cell cycle.
Science
257
,
1689
1694
Krek
W.
,
Marks
J.
,
Schmitz
N.
,
Nigg
E. A.
,
Simanis
V.
(
1992
).
Vertebrate p34cdc2 phosphorylation sites mutants: effects upon cell cycle progression in the fission yeast Schizosaccharomyces pombe.
J. Cell Sci
102
,
43
53
Lee
M. G.
,
Nurse
P.
(
1987
).
Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2.
Nature
327
,
31
35
Lees
E.
,
Faha
B.
,
Dulic
V.
,
Reed
S. I.
,
Harlow
E.
(
1992
).
CyclinE/cdk2 and cyclin A/cdk2 kinases associate with p107 and E2F in a temporally distinct manner.
Genes Dev
6
,
1874
1885
MacNeill
S.
,
Creanor
J.
,
Nurse
P.
(
1991
).
Isolation, characterisation and molecular cloning of new mutant alleles in the fission yeast p34cdc2 establishing the dependency of S-phase on mitosis.
Mol. Gen. Genet
229
,
109
118
MacNeill
S.
,
Nurse
P.
(
1993
).
Mutational analysis of the fission yeast p34cdc2 protein kinase gene.
Mol. Gen. Genet
236
,
415
426
Maundrell
K.
(
1993
).
Thiamine-repressible expression vectors pREP and pRIP for fission yeast.
Gene
123
,
127
130
Mendenhall
M. D.
,
Richardson
H. E.
,
Reed
S. I.
(
1988
).
Dominant negative protein kinase mutations that confer a G1arrest phenotype.
Proc. Nat. Acad. Sci. USA
85
,
4426
4430
Meyerson
M.
,
Faha
B.
,
Su
L. K.
,
Harlow
E.
,
Tsai
L. H.
(
1991
).
The cyclin-dependent kinase family.
Cold Spring Harbor Symp. Quant. Biol
56
,
177
186
Moreno
S.
,
Hayles
J.
,
Nurse
P.
(
1989
).
Regulation of p34cdc2 protein kinase during mitosis.
Cell
58
,
361
372
Moreno
S.
,
Klar
A.
,
Nurse
P.
(
1991
).
Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.
Meth. Enzymol
194
,
795
823
Ninomiya-Tsuji
J.
,
Nomoto
S.
,
Yasuda
H.
,
Reed
S. I.
,
Matsumoto
K.
(
1991
).
Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation.
Proc. Nat. Acad. Sci. USA
88
,
9006
9010
Nurse
P.
(
1975
).
Genetic control of cell size at cell division in yeast.
Nature
256
,
547
551
Nurse
P.
(
1990
).
Universal control mechanism regulating onset of M-phase.
Nature
344
,
503
508
Nurse
P.
,
Bissett
Y.
(
1981
).
Gene required in G1for commitment to cell cycle and in G2for control of mitosis in fission yeast.
Nature
292
,
558
560
Pagano
M.
,
Draetta
G.
,
Jansen-Durr
P.
(
1992
).
Association of cdk2 kinase with the transcription factor E2F during S-phase.
Science
255
,
1144
1147
Pagano
M.
,
Pepperkok
R.
,
Lukas
J.
,
Baldin
V.
,
Ansorge
W.
,
Bartek
J.
,
Draetta
G.
(
1993
).
Regulation of the cell-cycle by the cdk2 protein-kinase in cultured human fibroblasts.
J. Cell Biol
121
,
101
111
Paris
J.
,
Le Guellec
R.
,
Couturier
A.
,
LeGuellec
K.
,
Omilli
F.
,
Camonis
J.
,
MacNeill
S.
,
Philippe
M.
(
1991
).
Cloning by differential screening and characterization at the RNA level of a Xenopus cDNA coding for a protein highly homologous to cdc2.
Proc. Nat. Acad. Sci. USA
88
,
1039
1043
Peeper
D. S.
,
Parker
L. L.
,
Ewen
M. E.
,
Toebes
M.
,
Hall
F. L.
,
Xu
M.
,
Zantenna
A.
,
Vandereb
A. J.
,
Piwnica-Worms
H.
(
1993
).
A-type and B-type cyclins differentially modulate substrate-specificity of cyclin cdk complexes.
EMBO J
12
,
1947
1954
Pines
J.
(
1993
).
Cyclins and cyclin-dependent kinases: take your partners.
Trends Biochem. Sci
18
,
195
197
Reed
S. I.
(
1992
).
The role of p34 kinases in the G1to S-phase transition.
Annu. Rev. Cell Biol
8
,
529
561
Taylor
S. S.
(
1989
).
cAMP-dependent protein kinase.
J. Biol. Chem
264
,
8443
8446
Th'ng
J. P. H.
,
Wright
P. S.
,
Hamaguchi
J.
,
Lee
M. G.
,
Norbury
C. J.
,
Nurse
P.
,
Bradbury
E. M.
(
1990
).
The FT210 cell line is a mouse G2phase mutant with a temperature-sensitive CDC2 gene product.
Cell
63
,
313
324
Tsai
L. H.
,
Harlow
E.
,
Meyerson
M.
(
1991
).
Isolation of the human cdk2 gene that encodes the cyclin A and adenvirus E1A associated p33 kinase.
Nature
353
,
174
177
Xiong
Y.
,
Zhang
H.
,
Beach
D.
(
1992
).
D-type cyclins associate with multiple protein-kinases and the DNA-replication and repair factor PCNA.
Cell
71
,
505
514
This content is only available via PDF.