RESUMEN
Human MOK2 is a DNA-binding transcriptional repressor. Previously, we identified nuclear lamin A/C proteins as protein partners of hsMOK2. Furthermore, we found that a fraction of hsMOK2 protein was associated with the nuclear matrix. We therefore suggested that hsMOK2 interactions with lamin A/C and the nuclear matrix may be important for its ability to repress transcription. In this study, we identify JNK-associated leucine zipper and JSAP1 scaffold proteins, two members of c-Jun N-terminal kinase (JNK)-interacting proteins family as partners of hsMOK2. Because these results suggested that hsMOK2 could be phosphorylated, we investigated the phosphorylation status of hsMOK2. We identified Ser38 and Ser129 of hsMOK2 as phosphorylation sites of JNK3 kinase, and Ser46 as a phosphorylation site of Aurora A and protein kinase A. These three serine residues are located in the lamin A/C-binding domain. Interestingly, we were able to demonstrate that the phosphorylation of hsMOK2 interfered with its ability to bind lamin A/C.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Lamina Tipo A/metabolismo , Técnicas del Sistema de Dos Híbridos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Aurora Quinasas , Sitios de Unión , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Células HeLa , Humanos , Lamina Tipo A/genética , Microscopía Fluorescente , Proteína Quinasa 10 Activada por Mitógenos/metabolismo , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Serina/genética , Serina/metabolismo , Dedos de ZincRESUMEN
BACKGROUND INFORMATION: hsMOK2 (human MOK2) is a DNA-binding transcriptional repressor. For example, it represses the IRBP (interphotoreceptor retinoid-binding protein) gene by competing with the CRX (cone-rod homeobox protein) transcriptional activator for DNA binding. Previous studies have shown an interaction between hsMOK2 and nuclear lamin A/C. This interaction could be important to explain hsMOK2 ability to repress transcription. RESULTS: In the present study, we have tested whether missense pathogenic mutations of lamin A/C, which are located in the hsMOK2-binding domain, could affect the interaction with hsMOK2. We find that none of the tested mutations is able to disrupt hsMOK2 binding in vitro or in vivo. However, we observe an aberrant cellular localization of hsMOK2 into nuclear aggregates when pathogenic lamin A/C mutant proteins are expressed. CONCLUSIONS: These results indicate that pathogenic mutations in lamin A/C lead to sequestration of hsMOK2 into nuclear aggregates, which may deregulate MOK2 target genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Lamina Tipo A , Mutación Missense , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The human and murine MOK2 proteins are factors able to recognize both DNA and RNA through their zinc finger motifs. This dual affinity of MOK2 suggests that MOK2 might be involved in transcription and post-transcriptional regulation of MOK2 target genes. The IRBP gene contains two MOK2-binding elements, a complete 18 bp MOK2-binding site located in intron 2 and the essential core MOK2-binding site (8 bp of conserved 3'-half-site) located in the IRBP promoter. We have demonstrated that MOK2 can bind to the 8 bp present in the IRBP promoter and repress transcription from this promoter by competing with the CRX activator for DNA binding. In this study, we identify a novel interaction between lamin A/C and hsMOK2 by using the yeast two-hybrid system. The interaction, which was confirmed by GST pull-down assays and co-immunolocalization studies in vivo, requires the N-terminal acidic domain of hsMOK2 and the coiled 2 domain of lamin A/C. Furthermore, we show that a fraction of hsMOK2 protein is associated with the nuclear matrix. We therefore suggest that hsMOK2 interactions with lamin A/C and the nuclear matrix may be important for its ability to repress transcription.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Lamina Tipo A/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Núcleo Celular/química , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Células HeLa , Humanos , Lamina Tipo A/química , Lamina Tipo A/genética , Matriz Nuclear/química , Matriz Nuclear/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Factores de Transcripción/química , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: Chk1 kinase is activated by phosphorylation at serine-345 by Rad3 checkpoint kinase and is required for DNA damage checkpoint in late S and G2 phase of S. pombe cell cycle. We studied the ability of two chk1 mutants, chk1-1 and chk1-2, to undergo phosphorylation and to delay cell cycle progression in response to different types of DNA lesions. RESULTS: Both the Chk1-1 and Chk1-2 mutant proteins are phosphorylated to various extents when DNA is damaged in early G2 phase of cell cycle by either UV irradiation or gamma irradiation. However, chk1-2 mutant does not delay cell cycle progression in a dose dependent manner specifically upon gamma irradiations. This defect is not associated with an important loss of survival. Furthermore, both chk1 mutants survive to Camptothecin treatment despite undetectable Chk1-1 or Chk1-2 phosphorylated forms. We show that both mutant proteins are not phosphorylated in cds1 devoid cells treated with ribonucleotide reductase inhibitor hydroxyurea or when the replisome is affected by a thermosensitive mutation in DNA polymerase delta. This inability is associated with the loss of checkpoint function. We found that an increased level of Crb2/Rhp9 protein specifically complements the defect of the chk1-1 mutant allowing Chk1-1 phosphorylation upon treatment with hydroxyurea of dcds1 cells. CONCLUSIONS: Mutants chk1-1 and chk1-2 behave differently according to the type of lesion generated on DNA.