News and Publications

Regulation of the Cell Cycle

P. Russell, M. Boddy, J.-M. Brondello, B. Furnari, F. Gaits, J. Kanoh, A. Lopez-Girona, O. Mondesert, B. Moser, N. Rhind, K. Shiozaki, M. Shiozaki

The cell cycle consists of 2 phases: the S phase of DNA synthesis and the M phase of mitosis. These are separated by gap phases in the order G₁ S G₂ M. We use the fission yeast Schizosaccharomyces pombe to study regulation of the cell cycle. We have detected a network of protein kinases and phosphatases that control mitosis. The kinase Cdc2 is at the center of this network. Cdc2 is regulated by inhibitory phosphorylation on tyrosine-15. This residue is phosphorylated by the proteins Wee1 and Mik1 and dephosphorylated by Cdc25. Studies of fission yeast have paved the way for analysis of mitotic control systems in human cells.

Checkpoints in the cell cycle delay mitosis until DNA is fully replicated and repaired. In fission yeast, the DNA-damage checkpoint is enforced by the protein kinase Chk1. Our recent studies established that Chk1 inhibits dephosphorylation of Cdc2 on tyrosine-15 in vivo. This finding suggested that Cdc25 might be a potential target of Chk1. This hypothesis was confirmed by experiments that indicated that Cdc25 and Chk1 interact in vivo and that Chk1 phosphorylates Cdc25 in vitro. In fission yeast, Chk1 activity requires Rad3, a kinase that is closely related to human ATM, a protein that is mutated in patients with ataxia-telangiectasia. Persons deficient in ATM are profoundly sensitive to irradiation, indicating that ATM is required for checkpoint or repair functions. Thus, the damage checkpoint discovered in fission yeast appears to be conserved in humans.

Other results established that phosphorylation of Cdc2 on tyrosine-15 is essential for the checkpoint that prevents mitosis when DNA replication is inhibited. Curiously, Rad3 is required for this checkpoint, but Chk1 is not. This puzzle was solved by our studies that showed that 2 protein kinases, Chk1 and Cds1, enforce the replication checkpoint. Cds1 is a protein that binds to and phosphorylates Wee1. It is also required for the large increase of Mik1 that occurs in cells arrested at the replication checkpoint. The replication checkpoint is intact in cds1 or chk1 single mutants but abolished in cds1 chk1 double mutants, indicating that the 2 kinases jointly enforce the checkpoint.

In addition, an intra--S phase checkpoint slows the rate of DNA replication in response to DNA damage. We found that this checkpoint requires Rad3 and Cds1 but not Chk1. These experiments also revealed that this checkpoint differentiates between different types of DNA damage.

We are also examining the mechanisms involved in responses activated by stress. In fission yeast, we detected a stress-activated protein kinase cascade that is analogous to a similar cascade in humans. Like its human counterparts, the fission yeast kinase Spc1 is activated by many forms of stress, including osmotic, oxidative, or heat shock; nutrient limitation; and ultraviolet irradiation. It is unknown how cells sense and respond to such a diverse array of environmental changes. In some of our most recent studies, we discovered that heat stress activates Spc1 by a novel mechanism that bypasses an upstream element of the kinase cascade.

Upon activation, Spc1 translocates into the nucleus where it phosphorylates the transcription factor Atf1. Little is known about the regulation of nuclear translocation of stress-activated protein kinases. We discovered that nuclear translocation of Spc1 requires phosphorylation by the upstream kinase Wis1. Moreover, Spc1 is retained in the nucleus through its interaction with Atf1. These findings provide new information about the regulation of stress signal transduction in fission yeast that may be applicable to similar signal transduction systems in humans.

PUBLICATIONS

Boddy, M.N., Furnari, B., Mondesert, O., Russell, P. Replication checkpoint enforced by kinases Cds1 and Chk1. Science 280:909, 1998.

Furnari, B., Rhind, N., Russell, P. Cdc25 mitotic inducer targeted by Chk1 DNA damage checkpoint kinase. Science 277:1495, 1997.

Gaits, F., Degols, G., Shiozaki, K., Russell, P. Phosphorylation and association with the transcription factor Atf1 regulate localization of Spc1/Sty1 stress-activated kinase in fission yeast. Genes Dev. 12:1464, 1998.

Lopez-Girona, A., Mondesert, O., Leatherwood, J., Russell, P. Negative regulation of Cdc18 DNA replication protein by Cdc2. Mol. Biol. Cell 9:63, 1998.

Rhind, N., Russell, P. Cdc2 activation is inhibited by tyrosine phosphorylation during a replication checkpoint in Schizosaccharomyces pombe. Mol. Cell. Biol., in press.

Rhind, N., Russell, P. The Schizosaccharomyces pombe S-phase checkpoint differentiates between different types of DNA damage. Genetics, in press.

Shiozaki, K., Shiozaki, M., Russell, P. Heat stress activates fission yeast Spc1/Sty1 MAPK by a MEKK-independent mechanism. Mol. Biol. Cell, in press.