A Role for the Phosphorylation of hRad9 in Checkpoint Signaling

Abstract

The integrity of the human genome is preserved by signal transduction pathways called checkpoints, which delay progression through the cell cycle when DNA damage is present. Three checkpoint proteins, hRad9, hRad1, and hHus1, form a proliferating cell nuclear antigen-like, heterotrimeric complex that has been proposed to function in the initial detection of DNA structural abnormalities. hRad9 is highly modified by phosphorylation, in a constitutive manner and in response to both DNA damage and cell cycle position. Here we present evidence that Cell cycle checkpoints are signal transduction pathways that maintain the proper order of cell cycle events (1). Several checkpoints preserve the integrity of DNA by sensing genetic anomalies and delaying progression through the cell cycle so that enough time is provided for these anomalies to be corrected. The contribution of checkpoints to human health is illustrated by a growing list of checkpoint genes that are mutated in cancer and cancer predisposition syndromes (2-8).The hRad9 protein is the human homologue of Schizosaccharomyces pombe Rad9, a member of the checkpoint Rad family of proteins. In fission yeast, the checkpoint rad genes (rad1 ϩ , rad3 ϩ , rad9 ϩ , rad17 ϩ , rad26 ϩ , and hus1 ϩ ) are required for the S phase (DNA replication) and G 2 (DNA damage) checkpoints (9 -13). Yeasts lacking these genes fail to inactivate Cdc2 and enter premature, lethal mitosis when challenged with agents that inhibit DNA synthesis or damage DNA (14, 15). Like its yeast counterpart, hRad9 forms a ring-shaped, heterotrimeric complex with the hRad1 and hHus1 proteins (16 -18). Each member of the hRad9-hRad1-hHus1 complex (also known as the 9-1-1 complex), shares sequence homology with PCNA, 1 a homotrimer that encircles DNA and tethers DNA polymerase ␦ during DNA synthesis (19). PCNA is loaded onto DNA by the pentameric protein complex replication factor C (RFC), which is composed of one large subunit and four smaller subunits (20). In a manner analogous to PCNA and RFC, 9-1-1 is loaded onto DNA by a complex between hRad17 and the four smallest subunits of RFC (21). Since DNA damage induces hRad17-dependent association of 9-1-1 with chromatin, the 9-1-1 complex is believed to be involved in the direct recognition of DNA lesions during the initial stages of the checkpoint response (22). Also involved in this recognition are two phosphatidylinositol 3-kinase-related kinases, ATM and ATR, that regulate several cell cycle transitions and are central components of the cell checkpoint machinery (23). Even though these kinases appear to respond to different types of DNA lesions, they share a long list of common checkpoint substrates, including hRad17 (24 -26) and hRad9 (27). In fission yeast, Rad3 (which shares homology with both ATR and ATM) requires Rad9, Rad1, Hus1, and Rad17 to phosphorylate certain substrates (28). Similarly, in human cells, phosphorylation of hRad17 by ATR requires hHus1 (22). These findings support a model in which the 9-1-1 complex recruits substrates for ATM or...

Rad9 Protein Contributes to Prostate Tumor Progression by Promoting Cell Migration and Anoikis Resistance

“…Stable silencing of Rad9 did not increase cell death as judged by the trypan viability assay (supplemental Fig. S3B) and, in agreement with published reports (19,20), neither did it affect cell cycle phase distribution (supplemental Fig. S3C).…”

Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response: Figure 1.

“…This interaction between Rad9 and Rad4(TopBP1) is required for checkpoint activation (Furuya et al 2004), raising the possibility that 9-1-1 may bring TopBP1 to ATR-ATRIP after ATR phosphorylation of Rad9. Whether this proposed mechanism operates in Xenopus extracts or human cells is unclear (St Onge et al 2003;Lupardus and Cimprich 2006). While 9-1-1 and TopBP1 are important regulators of the ATR-ATRIP kinase, they are not required for some of the early events during the DNA damage response.…”

“…The colocalization of ATR-ATRIP, Rad17, and 9-1-1 complexes on damaged DNA may provide a mechanism to up-regulate the kinase activity of ATR-ATRIP and to bring about the phosphorylation of a subset of ATR substrates including Rad17 and Rad9. Phosphorylated Rad17 and Rad9 may function to recruit downstream signaling proteins such as Claspin and TopBP1 (St Onge et al 2003;Wang et al 2006), both of which are also phosphorylated by ATR-ATRIP. Once phosphorylated, TopBP1 is transformed into a potent stimulator of ATR-ATRIP, and it may trigger a "forward-feeding" loop of signaling (Hashimoto et al 2006;Kumagai et al 2006).…”

Cyclin-Dependent Kinase 2–Dependent Phosphorylation of ATRIP Regulates the G2-M Checkpoint Response to DNA Damage

“…Inhibition of CDK activity promotes cell cycle arrest and provides time for DNA repair. However, it is becoming increasingly clear that the cell cycle and CDK activity actually regulate many aspects of the DNA damage response (27)(28)(29)(30)(31)(33)(34)(35). We have now defined a requirement for CDK-dependent phosphorylation of ATRIP to promote the G 2 -M checkpoint response.…”

“…The resection of the end to yield ssDNA promotes ATR activation (31)(32)(33). Third, CDKs phosphorylate the COOH terminus of Rad9, and this phosphorylation is important for checkpoint signaling (34). Fourth, inhibition of CDK activity can cause a loss of Chk1 expression in some cell types (35).…”