RESUMEN
Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross-links. It was reported earlier that FANCD2 co-localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid-S to G2 phase within sites containing single-stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring-like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11-processed DNA double-strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas Nucleares/metabolismo , Ácido Anhídrido Hidrolasas , Proteínas de Ciclo Celular/genética , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/análisis , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Células HeLa , Humanos , Proteína Homóloga de MRE11 , Microscopía Electrónica , Proteínas Nucleares/genética , Unión Proteica , Estabilidad Proteica , ARN Interferente Pequeño/genéticaRESUMEN
LCPTP (leucocyte-phosphotyrosine phosphatase) is a 42kDa protein tyrosine phosphatase expressed predominantly in haematopoietic cells which has been implicated in the early stages of the T cell receptor signalling pathway. The substrates of LCPTP have been shown to include MAP kinase family members, but it remains unclear whether LCPTP is found in stable constitutive association with these enzymes, or associates transiently during dephosphorylation. Here we report on LCPTP/MAP kinase interactions in CD3-stimulated Jurkat T cells. Pull-downs from Jurkat T cells using a recombinant GST-LCPTP substrate-trap protein, but not wild-type LCPTP show a clear specific association with both ERK1 and ERK2. In Jurkat cells overexpressing LCPTP, a small fraction of cell ERK1 can be immunoprecipitated in stable association with LCPTP. However, in both unstimulated and anti-CD3 antibody stimulated Jurkat T cells, we were unable to demonstrate any constitutive interaction between endogenous LCPTP and any MAP kinase family members. We propose that both ERK1 and ERK2 interact transiently with LCPTP as substrates for the phosphatase rather than as constitutive protein partners.
Asunto(s)
Leucocitos/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , AMP Cíclico/análisis , Humanos , Células Jurkat , Proteína Quinasa 3 Activada por Mitógenos , Datos de Secuencia Molecular , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
BACKGROUND: Fanconi anemia (FA) is a complex recessive genetic disease characterized by progressive bone marrow failure (BM) and a predisposition to cancer. We have previously shown using the Fancc mouse model that the progressive BM failure results from a hematopoietic stem cell defect suggesting that function of the FA genes may reside in primitive hematopoietic stem cells. METHODS: Since genes involved in stem cell differentiation and/or maintenance are usually regulated at the transcription level, we used a semiquantitative RT-PCR method to evaluate FA gene transcript levels in purified hematopoietic stem cells. RESULTS: We show that most FA genes are highly expressed in primitive CD34-positive and negative cells compared to lower levels in more differentiated cells. However, in CD34- stem cells the Fancc gene was found to be expressed at low levels while Fancg was undetectable in this population. Furthermore, Fancg expression is significantly decreased in Fancc -/- stem cells as compared to wild-type cells while the cancer susceptibility genes Brca1 and Fancd1/Brac2 are upregulated in Fancc-/- hematopoietic cells. CONCLUSIONS: These results suggest that FA genes are regulated at the mRNA level, that increased Fancc expression in LTS-CD34+ cells correlates with a role at the CD34+ differentiation stage and that lack of Fancc affects the expression of other FA gene, more specifically Fancg and Fancd1/Brca2, through an unknown mechanism.
RESUMEN
The function of the Fanconi anemia group C protein (FANCC) is still unknown, though many studies point to a role in damage response signaling. Unlike other known FA proteins, FANCC is mainly localized to the cytoplasm and is thought to act as a messenger of cellular damage rather than an effector of repair. FANCC has been shown to interact with several cytoplasmic and nuclear proteins and to delay the onset of apoptosis through redox regulation of GSTP1. We investigated the fate and function of FANCC during apoptosis. Here we show that FANCC undergoes proteolytic modification by a caspase into a predominant 47-kDa ubiquitinated protein fragment. Lack of proteolytic modification at the putative cleavage site delays apoptosis but does not affect MMC complementation. These results suggest that FANCC function is regulated through proteolytic processing.