RESUMEN
Ionizing radiation induces autophosphorylation of the ataxia-telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A-like activity in vitro. OA did not induce gamma-H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double-strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A-like protein phosphatase activity. Moreover, we show that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM autophosphorylation and activity in vivo.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ataxia Telangiectasia , Proteínas de la Ataxia Telangiectasia Mutada , Dominio Catalítico , Proteínas de Ciclo Celular/química , Línea Celular , Ensayo Cometa , Proteínas de Unión al ADN/química , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de la radiación , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes , Eliminación de Gen , Genes Dominantes , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Hidrazinas , Microscopía Confocal , Modelos Biológicos , Ácido Ocadaico/farmacología , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/efectos de los fármacos , Fosfoproteínas Fosfatasas/genética , Fosforilación , Pruebas de Precipitina , Proteína Fosfatasa 2 , Proteínas Serina-Treonina Quinasas/química , Estructura Terciaria de Proteína , Radiación Ionizante , Serina/química , Transducción de Señal , Proteínas Supresoras de Tumor/química , Técnicas del Sistema de Dos HíbridosRESUMEN
The human Tousled-like kinases 1 and 2 (TLK) have been shown to be active during S phase of the cell cycle. TLK activity is rapidly suppressed by DNA damage and by inhibitors of replication. Here we report that the signal transduction pathway, which leads to transient suppression of TLK activity after the induction of double-strand breaks (DSBs) in the DNA, is dependent on the presence of a functional ataxia-telangiectasia-mutated kinase (ATM). Interestingly, we have discovered that rapid suppression of TLK activity after low doses of ultraviolet (UV) irradiation or aphidicolin-induced replication block is also ATM-dependent. The nature of the signal that triggers ATM-dependent downregulation of TLK activity after UVC and replication block remains unknown, but it is not due exclusively to DSBs in the DNA. We also demonstrate that TLK suppression is dependent on the presence of a functional Nijmegan Breakage Syndrome protein (NBS1). ATM-dependent phosphorylation of NBS1 is required for the suppression of TLK activity, indicating a role for NBS1 as an adaptor or scaffold in the ATM/TLK pathway. ATM does not phosphorylate TLK directly to regulate its activity, but Chk1 does phosphorylate TLK1 GST-fusion proteins in vitro. Using Chk1 siRNAs, we show that Chk1 is essential for the suppression of TLK activity after replication block, but that ATR, Chk2 and BRCA1 are dispensable for TLK suppression. Overall, we propose that ATM activation is not linked solely to DSBs and that ATM participates in initiating signaling pathways in response to replication block and UV-induced DNA damage.
