DNA damage-dependent and -independent phosphorylation of the hRad9 checkpoint protein

Cell cycle checkpoints are regulatory mechanisms that maintain genomic inte grity by preventing cell cycle progression when genetic anomalies are prese nt. The hRad9 protein is the human homologue of Schizosaccharomyces pombe R ad9, a checkpoint protein required for preventing the onset of mitosis if D NA damage is present or if DNA replication is incomplete. Genetic and bioch emical analyses indicate that hRad9 is a component of the checkpoint respon se in humans and has possible roles in regulating the cell cycle, apoptosis , and DNA repair. Previous studies indicate that hRad9 is modified by phosp horylation, both in the absence of exogenous stress and in response to vari ous genotoxins. In this study, we report the mapping of several sites of co nstitutive phosphorylation of hRad9 to (S/T)PX(R/P) sequences near the C te rminus of the protein. We also demonstrate that a serine to alanine mutatio n at residue 272 abrogates an ionizing radiation (IR)-induced phosphorylati on of hRad9 and further show that phosphorylation at (S/T)P sites is not a prerequisite for IR-induced phosphorylation of serine 272. Finally, we repo rt that hRad9 undergoes cell cycle-regulated hyper-phosphorylation in G(2)/ M that is enhanced by IR but distinct from that on serine 272. Unlike the I R-induced phosphorylation at serine 272, this event is dependent on serine 277 and threonine 292, two C-terminal (S/T)P sites in hRad9.