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1.
Nat Cell Biol ; 4(12): 998-1002, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12447382

RESUMO

53BP1 is a conserved nuclear protein that is implicated in the DNA damage response. After irradiation, 53BP1 localizes rapidly to nuclear foci, which represent sites of DNA double strand breaks, but its precise function is unclear. Using small interference RNA (siRNA), we demonstrate that 53BP1 functions as a DNA damage checkpoint protein. 53BP1 is required for at least a subset of ataxia telangiectasia-mutated (ATM)-dependent phosphorylation events at sites of DNA breaks and for cell cycle arrest at the G2-M interphase after exposure to irradiation. Interestingly, in cancer cell lines expressing mutant p53, 53BP1 was localized to distinct nuclear foci and ATM-dependent phosphorylation of Chk2 at Thr 68 was detected, even in the absence of irradiation. In addition, Chk2 was phosphorylated at Thr 68 in more than 50% of surgically resected lung and breast tumour specimens from otherwise untreated patients [corrected]. We conclude that the constitutive activation of the DNA damage checkpoint pathway may be linked to the high frequency of p53 mutations in human cancer, as p53 is a downstream target of Chk2 and ATM.


Assuntos
Proteínas de Transporte/genética , Ciclo Celular/genética , Dano ao DNA/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Fosfoproteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Genes p53 , Humanos , Mutação , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
2.
Cancer Res ; 65(17): 7533-40, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16140914

RESUMO

The response of eukaryotic cells to DNA damage includes the activation of phosphatidylinositol-3 kinase-related kinases (PIKK), such as ATM, ATR, and DNA-dependent protein kinase (DNA-PK). These three kinases have very similar substrate specificities in vitro, but in vivo, their substrates overlap only partially. Several in vivo substrates of ATM and ATR have been identified and almost all of them are involved in DNA damage-induced cell cycle arrest and/or apoptosis. In contrast, few in vivo substrates of DNA-PK have been identified. These include histone H2AX and DNA-PK itself. We identify here valosin-containing protein (VCP) as a novel substrate of DNA-PK and other PIKK family members. VCP is phosphorylated at Ser784 within its COOH terminus, a region previously shown to target VCP to specific intracellular compartments. Furthermore, VCP phosphorylated at Ser784 accumulated at sites of DNA double-strand breaks (DSBs). VCP is a protein chaperone that unfolds and translocates proteins. Its phosphorylation in response to DNA damage and its recruitment to sites of DNA DSBs could indicate a role of VCP in DNA repair.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/fisiologia , Adenosina Trifosfatases , Sequência de Aminoácidos , Anticorpos/farmacologia , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2 , DNA de Neoplasias/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Transfecção , Proteína com Valosina
4.
Cancer Res ; 63(24): 8586-91, 2003 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-14695167

RESUMO

53BP1 and NFBD1/MDC1 are recruited rapidly to sites of DNA double-strand breaks (DSBs), where they are hypothesized to function downstream of the ataxia-telangiectasia mutated (ATM) checkpoint kinase as "mediators" of DNA DSB signaling. To test this hypothesis, we suppressed 53BP1 and NFBD1/MDC1 expression by small interference RNA and monitored ATM autophosphorylation at Ser(1981) as a marker for ATM activation. Suppression of NFBD1/MDC1 led to decreased ATM activation and phosphorylation of ATM substrates. This phenotype was identical to that observed in cells with defective Nbs1 function and is consistent with recent observations identifying NFBD1/MDC1 as a component of the Mre11-Rad50-Nbs1 protein complex. In cells with wild-type Nbs1, suppression of 53BP1 expression had no effect on ATM activation but was associated with increased recruitment of NFBD1/MDC1 and Nbs1 to sites of DNA breaks, suggesting that decreased 53BP1 function might be compensated for by increased NFBD1/MDC1 and Nbs1 activity. Indeed, in cells with mutant Nbs1, suppression of 53BP1 led to decreased ATM activation and phosphorylation of ATM substrates. We conclude that DNA DSBs activate ATM through at least two independent pathways involving 53BP1 and NFBD1/MDC1-Nbs1, respectively.


Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular/fisiologia , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares/fisiologia , Fosfoproteínas , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2 , Proteínas de Ligação a DNA/antagonistas & inibidores , Ativação Enzimática , Fibroblastos , Células HeLa , Humanos , Proteínas Nucleares/antagonistas & inibidores , Fosforilação , RNA Interferente Pequeno/genética , Transativadores/antagonistas & inibidores , Transfecção , Proteínas Supressoras de Tumor , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
5.
DNA Repair (Amst) ; 3(8-9): 945-52, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15279780

RESUMO

p53 Binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DNA damage checkpoint proteins with C-terminal BRCT domains and is most likely the human ortholog of the budding yeast Rad9 protein, the first cell cycle checkpoint protein to be described. 53BP1 localizes rapidly to sites of DNA double strand breaks (DSBs) and its initial recruitment to these sites has not been shown to be dependent on any other protein. Initially, 53BP1 was thought to be a mediator of DNA DSB signaling, but now it has been shown to function upstream of ataxia-telangiectasia mutated (ATM), in one of at least two parallel pathways leading to ATM activation in response to DNA damage. Currently, only a single tudor and two BRCT domains are recognized in 53BP1; however, their precise functional role is not understood. Elucidating the function of 53BP1 will be critical to understanding how cells recognize DNA DSBs and how ATM is activated.


Assuntos
Dano ao DNA , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Quinase do Ponto de Checagem 2 , DNA/genética , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Biológicos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Fosfoproteínas/genética , Conformação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
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