Phosphorylation of Human Rad9 Is Required for Genotoxin-activated Checkpoint Signaling

Abstract

Rad9, a key component of genotoxin-activated checkpoint signaling pathways, associates with Hus1 and Rad1 in a heterotrimeric complex (the 9-1-1 complex). Rad9 is inducibly and constitutively phosphorylated. However, the role of Rad9 phosphorylation is unknown. Here we identified nine phosphorylation sites, all of which lie in the carboxyl-terminal 119-amino acid Rad9 tail and examined the role of phosphorylation in genotoxin-triggered checkpoint activation. Rad9 mutants lacking a Ser-272 phosphorylation site, which is phosphorylated in response to genotoxins, had no effect on survival or checkpoint activation in Mrad9 ؊/؊ mouse ES cells treated with hydroxyurea (HU), ionizing radiation (IR), or ultraviolet radiation (UV). In contrast, additional Rad9 tail phosphorylation sites were essential for Chk1 activation following HU, IR, and UV treatment. Consistent with a role for Chk1 in S-phase arrest, HUand UV-induced S-phase arrest was abrogated in the Rad9 phosphorylation mutants. In contrast, however, Rad9 did not play a role in IR-induced S-phase arrest. Clonogenic assays revealed that cells expressing a Rad9 mutant lacking phosphorylation sites were as sensitive as Rad9؊/؊ cells to UV and HU. Although Rad9 contributed to survival of IR-treated cells, the identified phosphorylation sites only minimally contributed to survival following IR treatment. Collectively, these results demonstrate that the Rad9 phospho-tail is a key participant in the Chk1 activation pathway and point to additional roles for Rad9 in cellular responses to IR.

The effect of Hus1 on ionizing radiation sensitivity is associated with homologous recombination repair but is independent of nonhomologous end-joining

“…However, as shown here, Hus1 deficient cells show increased sensitivity to IR-induced killing when using the clonogenic assay that measures reproductive ability. These results support the model that Hus1 associates with Rad1 and Rad9 as a 9-1-1 complex to respond to IR-induced DNA damage because Rad9 deficient cells are hypersensitive to IR-induced killing (Roos-Mattjus et al, 2003;Hopkins et al, 2004). IRinduced DNA double strands breaks (DSBs) are the most severe threat for cell survival.…”

“…Rad9 is involved in IRinduced DNA damage response and Rad9 deficient cells are hypersensitive to IR-induced killing (Roos-Mattjus et al, 2003;Hopkins et al, 2004). It is reasoned, then, that Hus1 should also affect the sensitivity of mammalian cells to IR-induced killing.…”

The Rad9–Hus1–Rad1 (9–1–1) clamp activates checkpoint signaling via TopBP1

“…The tail is heavily phosphorylated, with multiple sites having been identified (Chen et al 2001;St. Onge et al 2001, 2003Roos-Mattjus et al 2003). Although these sites are not required to generate the 9-1-1 clamp or for genotoxin-triggered loading of the clamp onto chromatin, a Rad9 mutant in which all the sites were simultaneously mutated could not facilitate Chk1 phosphorylation (Roos-Mattjus et al 2003).…”

“…Onge et al 2001, 2003Roos-Mattjus et al 2003). Although these sites are not required to generate the 9-1-1 clamp or for genotoxin-triggered loading of the clamp onto chromatin, a Rad9 mutant in which all the sites were simultaneously mutated could not facilitate Chk1 phosphorylation (Roos-Mattjus et al 2003). Initial analyses of the phosphorylation sites revealed that Ser272, a site phosphorylated by ATM and ATR (Chen et al 2001), was not essential for Chk1 phosphorylation (Roos-Mattjus et al 2003).…”

“…Rad9 has two predicted domains (Rauen et al 2000;Venclovas and Thelen 2000;Roos-Mattjus et al 2003). The N-terminal two-thirds of the protein (∼270 amino acids) forms a PCNA-like structure that associates with Hus1 and Rad1 to yield the 9-1-1 clamp complex (Fig.…”