RESUMO
We have developed stable cell lines expressing green fluorescent protein fusion proteins containing polyglutamine repeats of various lengths under tetracycline control. The expression of the expanded (43Q) repeat protein resulted in aggregate formation in a time-dependent fashion. The accumulation of aggregates did not induce apoptosis, although the survival of these cells was critically dependent on the presence of serum and growth factors. However, the expression of 43Q expanded protein strongly activated the ataxia telangiectasia mutated kinase/ATM and Rad3-related kinase (ATM/ATR)-dependent DNA damage response, as shown by selective phosphorylation of ATM substrates. This activation was dependent on 43 CAG protein expression, reversible and sensitive to caffeine and reducing agents. Similarly, we found phosphorylated ATM substrates in fibroblasts from Huntington's disease or SCA-2 patients. Oxidative stress induced accumulation of ATM/ATR phosphorylated protein in HD and SCA-2 patients, but not in normal controls. Furthermore, a significant phosphorylation of H2AX was shown by fibroblasts from patients. We conclude that polyglutamine induces ATM/ATR-dependent DNA damage response through accumulation of reactive oxygen species. ATM activation can be used to monitor the disease in vivo.
Assuntos
Dano ao DNA , Fibroblastos/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia , Cafeína/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA , Ativação Enzimática , Fibroblastos/química , Fibroblastos/patologia , Proteínas de Fluorescência Verde , Histonas/metabolismo , Doença de Huntington/patologia , Proteínas Luminescentes/metabolismo , Células PC12 , Peptídeos/genética , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ataxias Espinocerebelares/patologia , Estresse Fisiológico/fisiopatologia , Especificidade por Substrato , Fatores de Tempo , Expansão das Repetições de Trinucleotídeos/genética , Proteínas Supressoras de TumorRESUMO
A-kinase anchor proteins (AKAPs) assemble multi-enzyme signaling complexes in proximity to substrate/effector proteins, thus directing and amplifying membrane-generated signals. S-AKAP84 and AKAP121 are alternative splicing products with identical NH(2) termini. These AKAPs bind and target protein kinase A (PKA) to the outer mitochondrial membrane. Tubulin was identified as a binding partner of S-AKAP84 in a yeast two-hybrid screen. Immunoprecipitation and co-sedimentation experiments in rat testis extracts confirmed the interaction between microtubules and S-AKAP84. In situ immunostaining of testicular germ cells (GC2) shows that AKAP121 concentrates on mitochondria in interphase and on mitotic spindles during M phase. Purified tubulin binds directly to S-AKAP84 but not to a deletion mutant lacking the mitochondrial targeting domain (MT) at residues 1-30. The MT is predicted to form a highly hydrophobic alpha-helical wheel that might also mediate interaction with tubulin. Disruption of the wheel by site-directed mutagenesis abolished tubulin binding and reduced mitochondrial attachment of an MT-GFP fusion protein. Some MT mutants retain tubulin binding but do not localize to mitochondria. Thus, the tubulin-binding motif lies within the mitochondrial attachment motif. Our findings indicate that S-AKAP84/AKAP121 use overlapping targeting motifs to localize signaling enzymes to mitochondrial and cytoskeletal compartments.
