Profiling of UV-induced ATM/ATR signaling pathways

Abstract

To ensure survival in the face of genomic insult, cells have evolved complex mechanisms to respond to DNA damage, termed the DNA damage checkpoint. The serine/threonine kinases ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) activate checkpoint signaling by phosphorylating substrate proteins at SQ/TQ motifs. Although some ATM/ATR substrates (Chk1, p53) have been identified, the lack of a more complete list of substrates limits current understanding of checkpoint pathways. Here, we use immunoaffinity phosphopeptide isolation coupled with mass spectrometry to identify 570 sites phosphorylated in UV-damaged cells, 498 of which are previously undescribed. Semiquantitative analysis yielded 24 known and 192 previously uncharacterized sites differentially phosphorylated upon UV damage, some of which were confirmed by SILAC, Western blotting, and immunoprecipitation/ Western blotting. ATR-specific phosphorylation was investigated by using a Seckel syndrome (ATR mutant) cell line. Together, these results provide a rich resource for further deciphering ATM/ATR signaling and the pathways mediating the DNA damage response.DNA damage ͉ mass spectrometry ͉ phosphorylation M aintaining the integrity of the genome is of utmost importance for cellular survival. For this reason, cells have evolved complex mechanisms to inhibit cell cycle progression in response to genomic insult, termed the DNA damage checkpoint (1). Activating checkpoint mechanisms gives cells time to repair or bypass the damage using specialized DNA polymerases or, in cases of high levels of damage, to activate apoptotic pathways (2). Elucidating pathways involved in checkpoint activation and maintenance continues to be an active area of research.A family of phosphoinositol-3-phosphate kinase-like kinases are critical to the proper function of the DNA damage checkpoint. The two central kinases involved are ataxia telangiectasiamutated (ATM) and ATM and Rad3-related (ATR). This kinase family also includes DNA-dependent protein kinase (DNA-PK) and a more recently discovered member of the family, SMG1 (3). These kinases are activated in response to DNA damage and subsequently phosphorylate targets responsible for such diverse activities as blocking cell cycle progression, coordinating DNA repair activities, and affecting transcription of DNA damage response genes. ATR is activated in response to a variety of damaging agents: UV light, alkylating agents such as methyl methanesulfonate (MMS), and chemical inhibitors of DNA replication such as aphidicolin and hydroxyurea (4, 5). ATM, however, is primarily involved in the response to double-strand breaks, such as those caused by gamma irradiation (IR) (6). Deficiency in ATM/R, as well as other components of the DNA damage checkpoint, has been found to cause debilitating diseases such as ataxia telangiectasia (ATM mutants), Fanconi's anemia, Seckel syndrome (ATR mutants), and the avoidance of checkpoint activation to allow cancer progression.In response to DNA damage, ATM/R phosphorylate checkpoint k...

Tumor suppressor and deubiquitinase BAP1 promotes DNA double-strand break repair

“…Thus, BAP1 might be involved in the DNA damage response by coordinating H2A ubiquitination. Notably, proteomic studies revealed BAP1 among phosphorylated proteins during DNA damage (15). Nonetheless, the role of BAP1 in the DNA damage response and more generally, the mechanism of tumor suppression exerted by this DUB remain unclear.…”

Resposta a danos no DNA após exposição à luz ultravioleta: apagando o fogo antes do incêndio celular

“…O número em parênteses corresponde ao ano de publicação do trabalho e foi adaptada da revisão de Ljungman, 2000. 1 -(Muller 1927); 2 - (Hollaender e Claus, 1936); 3 - (Kelner 1949); 4 - (Watson e Crick, 1953); 5 - (Kanazir e Errera, 1954);6 -(RupertT et al, 1958);7 -(Beukers e Berends, 1960); 8 - (Masters e Pardee, 1962); 9 - (Boyce e Howard-Flanders, 1964;Setlow e Carrier, 1964); 10 - (Holliday 1964); 11 - (Rupp e Howard-Flanders, 1968); 12 -(J E Cleaver 1968); 13 - (Lindahl 1974); 14 - (Witkin 1974);15 -(Wagner e Meselson, 1976); 16 - (Lane e Crawford, 1979;Linzer e Levine, 1979); 17 - (Wilson et al, 1982); 18 - (Mellon et al, 1987);19 -(Kastan et al 1991);20 -(Kastan et al 1992); 21 - (Walworth et al, 1993); 22 - (Fishel et al 1993);23 -(van der Kemp et al 1996); 24 - (Rogakou et al 1998); 25 - (Matsuoka et al, 1998);26 -(Masutani et al 1999); 27 - (Tibbetts et al 1999);28 -(de Boer et al 2002); 29 - (Kannouche et al, , 2004); 30 - (Bartkova et al 2005;Gorgoulis et al 2005); 31 - (Matsuoka et al 2007;Stokes et al 2007); 32 - ( Sugasawa et al 2009) .…”

“…O fosforiloma de ATR revelou 570 alvos de fosforilação após irradiação com luz UV, sendo 498 nunca antes descritos (Stokes et al, 2007). Entre esses alvos, muitos alvos envolvidos com a replicação do DNA (MCMs, RPA, RFC, TopBP1 e DNA polimerases como pol η), reforçando um papel importante no controle de origem de replicação, estabilidade da forquilha e recuperação da replicação.…”

The Atr Protein Kinase Controls UV-Dependent Upregulation of p16INK4A Through Inhibition of Skp2-Related Polyubiquitination/Degradation

“…Posttranslational modifications are of great importance during the cellular DNA damage response (DDR). Indeed, different protein kinases responsible for phosphorylating a plethora of proteins are involved (1,2). Furthermore, tremendous work during the last 2 decades showed the crucial role of protein ubiquitination in the cellular DDR (3).…”