Threonine-11, phosphorylated by Rad3 and atm in vitro, is required for activation of fission yeast checkpoint kinase Cds1.

Inheritance of complete and accurate copies of the genome is the singular goal of cell division. Precise genome duplication is an intrinsically difficult process that can be strained further by external agents that interfere with DNA replication or damage DNA. Genome surveillance mechanisms exist to cope with these problems (16, 21). These systems serve two primary purposes. One purpose is to prevent mitosis when DNA replication is interrupted or DNA is damaged. These cell cycle checkpoints actively couple the onset mitosis to the completion of DNA replication and repair. The other purpose of genome surveillance mechanisms is to regulate various repair and replication systems that help cells survive replicational stress and DNA damage. The fission yeast Schizosaccharomyces pombe has served as a valuable model system for the discovery and investigation of genome integrity checkpoint mechanisms (35). Genetic studies of fission yeast have uncovered a group of genes that are required for arresting cell division when DNA is damaged or when replication is inhibited with the drug hydroxyurea (HU). The products of these checkpoint RAD genes include Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1 (13). Other proteins such as Cut5 (also known as Rad4) and Rfc3, which are necessary for DNA replication, are also important for both the DNA replication (S-M) and G2-M DNA damage checkpoints (38, 40). The most intriguing checkpoint protein may be Rad3, a very large protein (2,386 amino acids) that is related to phosphatidylinositol kinases (PIKs) (6). Other PIK-like proteins include human ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3 related) and DNA-dependent protein kinase (20, 39, 43). Although related to phosphatidylinositol 3-kinases, these enzymes function as protein kinases in vivo. In common with fission yeast Rad3, ATM is required for arrest in G2 phase of the cell cycle in response to DNA damage caused by ionizing radiation and for slowed replication of damaged DNA (16, 24, 37). ATM is thought to control G2 arrest in part by activating Cds1 (also known as Chk2) (7, 8, 26), the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases. ATR has been implicated in the checkpoint response to UV damage and the inhibition of DNA replication (11, 43). Recently, Chk1 phosphorylation was found to be ATR dependent, suggesting that Chk1 is regulated by ATR (19, 25). Rad3 and the other checkpoint Rad proteins control two downstream protein kinases in fission yeast. When DNA is damaged during G2 phase, Chk1 becomes phosphorylated in a Rad3-dependent manner (42). Chk1 prevents the onset of mitosis by regulation of Cdc25 and Mik1, two proteins that control the inhibitory phosphorylation of the cyclin-dependent kinase Cdc2 (2, 17, 18, 34, 36). The significance of Chk1 phosphorylation is uncertain, but it correlates with the requirement for Chk1 to arrest cell division in response to DNA damage. It is unknown if Chk1 is a direct physiological substrate of Rad3. Fission yeast Cds1 becomes phosphorylated and activated by a Rad3-dependent mechanism when DNA is damaged during S phase or when DNA replication is interrupted with HU or mutations of several essential genes (9, 24). Cds1 enforces the S-M checkpoint by regulating Cdc25 and Mik1 (9, 17). In cds1 mutants treated with HU, the onset of mitosis is prevented by Chk1, but these cells are inviable (9, 10, 24). This fact demonstrates that Cds1 has replicational stress recovery functions that are distinct from its cell cycle checkpoint activity. How Cds1 is regulated is unknown. Fission yeast Cds1 and its homologs are recognizable by similar kinase domains, an N-terminal Ser-Gln/Thr-Gln (SQ/TQ) cluster domain, and a forkhead-associated (FHA) domain (8, 26). SQ and TQ sequences are the preferred sites of phosphorylation by ATM in p53, c-Abl, Brca1, and Nbs1 (3, 4, 12, 14, 23). FHA domains are believed to act as protein-protein interaction domains and in some instances can bind to phosphorylated partners (22, 41). Budding yeast Rad53 is unique in possessing a second C-terminal FHA domain (1). Several findings link Rad3 to Cds1. As mentioned above, Rad3 is necessary for phosphorylation and activation of Cds1 in vivo (9, 24). Active Cds1 associates with overproduced Rad3 in vivo (30). Furthermore, Cds1 associates with Rad26 when both proteins are overproduced, and Rad26 forms a protein complex with Rad3 (15, 24). These findings suggested that Rad3 might directly activate Cds1 in vivo. Experiments designed to test this hypothesis are described in this report. We show that Rad3 and human ATM phosphorylate fission yeast Cds1 at threonine-11. Threonine-11 forms part of a conserved TQ motif. We report that threonine-11 is crucially important for Cds1 activation and function in vivo. These studies provide strong support for the model that Rad3 directly controls Cds1 activity by phosphorylating Cds1 at threonine-11.