In S phase checkpoint assays, values were expressed as mean S

In S phase checkpoint assays, values were expressed as mean S.D. sites and demonstrated that phosphorylation of both was rapidly induced by radiation. These phosphorylations were abolished by a specific inhibitor of ATM and were dependent on ATM and the Mre11-Rad50-Nbs1 complex. As found for Ser(P)1981, ATM phosphorylated at Ser367and Ser2996localized to sites of DNA damage induced by radiation, but ATM recruitment was not dependent on phosphorylation at these sites. Phosphorylation at Ser367and Ser2996was functionally important because mutant forms of ATM were defective in correcting the S phase checkpoint defect and restoring radioresistance in ataxia-telangiectasia cells. These data provide further support for the importance of autophosphorylation in the activation and function of ATMin vivo. Keywords:DNA Damage, Mass Spectrometry (MS), Post-translational Modification, Protein Phosphorylation, Serine Threonine Protein Kinase, Signal Transduction, ATM Kinase, Ataxia-telangiectasia == Introduction == DNA double strand breaks (DSB)4arise during normal physiological cell processes, such as immunoglobulin and T cell receptor gene rearrangements (1), but also arise in response to exposure to a variety of DNA-damaging agents that, if left unrepaired, lead to cell death or genomic instability, cancer, and other pathologies (2). DNA DSB are repaired in mammalian cells by non-homologous end joining and homologous recombination that predominate at different stages of the cell cycle (3). The Mre11-Rad50-Nbs1 (MRN) complex is a primary sensor of DNA DSB, and there is evidence that it participates in both forms of DSB repair (47). Hypomorphic mutations in members of this complex give rise to disorders characterized by sensitivity to agents that generate DNA DSB, cell cycle checkpoint defects, genome instability, and neurological abnormalities (810). These syndromes overlap in their clinical and cellular phenotypes with ataxia-telangiectasia (A-T) defective in ATM kinase (11). ATM is a member of the phosphoinositide 3-kinase-like kinase (PIKK) family of proteins and is primarily activated by DNA DSB to signal to both the DNA repair machinery and the cell cycle checkpoints (12,13). Considerable progress 3AC has been made in understanding the mechanism of ATM activation (1417). Although the nature of the initial stimulus for activation remains undefined, it seems likely that relaxation of chromatin structure is sufficient to initiate activation (14). ATM is constitutively present as an inactive Rabbit Polyclonal to Histone H3 (phospho-Ser28) dimer that monomerizes as part of the activation process, facilitated by both autophosphorylation and acetylation (14,18). More recently, Sunet al.(19) have shown that after DNA damage, casein kinase 2 phosphorylates and releases HP1 from chromatin, which enables the recruitment of the ATM-Tip60 complex to MRN at the break site. This facilitates interaction between the chromodomain of Tip60 and the unbound histone H3 K9me3, leading to acetylation and activation of the kinase activity of ATM. The initial activation of ATM appears to be incomplete because it relies on recruitment to the site of the break by the MRN complex for full activation (15). This is supported by a variety ofin vitroandin vivostudies that demonstrate MRN dependence on the activation of ATM (2023). In addition, although ATM can be activated in cells deficient in Nbs1, it fails to localize to nuclear foci at sites of DNA damage in these cells (24). Once activated, ATM can then phosphorylate a multitude of substrates that participate in different cellular processes, including DNA repair and cell cycle control (25). A cascade of reactions is initiated, starting with the phosphorylation of H2AX (H2AX), leading to chromatin modification that assists in signaling reactions and DNA repair (26). Recent data suggest that dephosphorylation at Tyr142is a prerequisite for H2AX phosphorylation and that this alteration plays a central role in the assembly of the DNA damage response complex (27). Assembly of DNA damage response proteins at the break site is consolidated by phosphorylation and ubiquitylation (13). ATM phosphorylates a number of these proteins, including MDC1, Nbs1, 53BP1, and 3AC BRCA1. Phosphorylation of MDC1 by a second enzyme, casein kinase-2, is responsible for 3AC the retention of the MRN complex on chromatin (28,29). Phosphorylation of MDC1 at ATM consensus sites facilitates interaction with the RING finger ubiquitin ligase, RNF8, which in turn ubiquitylates H2A and H2AX, leading to the accumulation of 53BPI, BRCA1, and other proteins at the site of damage (3032). It is now evident that this accumulation is consolidated by the involvement of a second ubiquitin ligase, RNF168, that also ubiquitylates H2A and H2AX (33,34). In addition to activation by DNA DSB recent data show that ATM is also an important sensor of reactive oxygen species in human.