RESUMEN
The Ku antigen (70- and 80-kDa subunits) is a regulatory subunit of DNA-dependent protein kinase (DNA-PK) that promotes the recruitment of the catalytic subunit of DNA-PK (DNA-PKcs) to DNA ends and to specific DNA sequences from which the kinase is activated. Ku and DNA-PKcs plays essential roles in double-stranded DNA break repair and V(D)J recombination and have been implicated in the regulation of specific gene transcription. In a yeast two-hybrid screen of a Jurkat T cell cDNA library, we have identified a specific interaction between the 70-kDa subunit of Ku heterodimer and the homeodomain of HOXC4, a homeodomain protein expressed in the hematopoietic system. Unexpectedly, a similar interaction with Ku was observed for several additional homeodomain proteins including octamer transcription factors 1 and 2 and Dlx2, suggesting that specific binding to Ku may be a property shared by many homeodomain proteins. Ku-homeodomain binding was mediated through the extreme C terminus of Ku70 and was abrogated by amino acid substitutions at Lys595/Lys596. Ku binding allowed the recruitment of the homeodomain to DNA ends and dramatically enhanced the phosphorylation of homeodomain-containing proteins by DNA-PK. These results suggest that Ku functions as a substrate docking protein for signaling by DNA-PK to homeodomain proteins from DNA ends.
Asunto(s)
Antígenos Nucleares , ADN Helicasas , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Transcripción Genética , Hormona Adrenocorticotrópica/genética , Secuencia de Aminoácidos , Animales , Pollos , Clonación Molecular , Cricetinae , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Humanos , Células Jurkat , Autoantígeno Ku , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Factor 2 de Transcripción de Unión a Octámeros , Fosforilación , Unión Proteica , Biosíntesis de Proteínas , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Garrapatas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , XenopusRESUMEN
NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids. Although Ku is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (Kd = 0.84 +/- 0.24 nM) than DNA end binding. Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence. Sequences with similarity to NRE1 competed efficiently for NRE1 binding. Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends. Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation. Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527. This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.