N. Rhind et P. Russell, TYROSINE PHOSPHORYLATION OF CDC2 IS REQUIRED FOR THE REPLICATION CHECKPOINT IN SCHIZOSACCHAROMYCES-POMBE, Molecular and cellular biology, 18(7), 1998, pp. 3782-3787
The DNA replication checkpoint inhibits mitosis in cells that are unab
le to replicate their DNA, as when nucleotide biosynthesis is inhibite
d by hydroxyurea. In the fission yeast Schizosaccharomyces pombe, gene
tic evidence suggests that this checkpoint involves the inhibition of
Cdc2 activity through the phosphorylation of tyrosine-15. On the contr
ary, a recent biochemical study indicated that Cdc2 is in an activated
state during a replication checkpoint, suggesting that phosphorylatio
n of Cdc2 on tyrosine-15 is not part of the replication checkpoint mec
hanism. We have undertaken biochemical and genetic studies to resolve
this controversy. We report that the DNA replication checkpoint in S.
pombe is abrogated in cells that carry the allele cdc2-Y15F, expressin
g an unphosphorylatable form of Cdc2. Furthermore, Cdc2 isolated from
replication checkpoint-arrested cells can be activated in vitro by Cdc
25, the tyrosine phosphatase responsible for dephosphorylating Cdc2 in
vivo, to the same extent as Cdc2 isolated from cdc25ts-blocked cells,
indicating that hydroxyurea treatment causes Cdc2 activity to be main
tained at a low level that is insufficient to induce mitosis. These st
udies show that inhibitory tyrosine-15 phosphorylation of Cdc2 is esse
ntial for the DNA replication checkpoint and suggests that Cdc25, and/
or one or both of Wee1 and Mik1, the tyrosine kinases that phosphoryla
te Cdc2, are regulated by the replication checkpoint.