RESUMO
Soluble inositol polyphosphates represent a variegate class of signalling molecules essential for the function of disparate cellular processes. Recently, the phytic acid derivate inositol pyrophosphate, InsP(7) (PP-IP(5) or IP(7)) has been shown to pyro-phosphorylate proteins in a kinase independent way. To begin to understand the functional importance of this new phosphorylation mechanism, a source of cold and radiolabelled InsP(7) is indispensable. However, cold InsP(7) is expensive to buy, and labelled InsP(7) is not commercially available. Here we provide a protocol to synthesise and purify InsP(7) to a level of purity required for in vivo and in vitro experiments. We begin by purifying recombinant mouse inositol hexakisphosphate kinase (IP6K1) from Escherichia coli. With purified IP6K1, we produce cold InsP(7) and 5beta[(32)P] InsP(7) that we subsequently use in vitro experiments to phosphorylate proteins extracts from different species.
Assuntos
Fosfatos de Inositol/isolamento & purificação , Fosfatos de Inositol/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/isolamento & purificação , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Animais , Escherichia coli/genética , Camundongos , Radioisótopos de Fósforo/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Inositol pyrophosphates are involved in a variety of cellular functions, but the specific pathways and/or downstream targets remain poorly characterized. In the present study we use Saccharomyces cerevisiae mutants to examine the potential roles of inositol pyrophosphates in responding to cell damage caused by ROS (reactive oxygen species). Yeast lacking kcs1 [the S. cerevisiae IP6K (inositol hexakisphosphate kinase)] have greatly reduced IP7 (diphosphoinositol pentakisphosphate) and IP8 (bisdiphosphoinositol tetrakisphosphate) levels, and display increased resistance to cell death caused by H2O2, consistent with a sustained activation of DNA repair mechanisms controlled by the Rad53 pathway. Other Rad53-controlled functions, such as actin polymerization, appear unaffected by inositol pyrophosphates. Yeast lacking vip1 [the S. cerevisiae PP-IP5K (also known as IP7K, IP7 kinase)] accumulate large amounts of the inositol pyrophosphate IP7, but have no detectable IP8, indicating that this enzyme represents the physiological IP7 kinase. Similar to kcs1Delta yeast, vip1Delta cells showed an increased resistance to cell death caused by H2O2, indicating that it is probably the double-pyrophosphorylated form of IP8 [(PP)2-IP4] which mediates the H2O2 response. However, these inositol pyrophosphates are not involved in directly sensing DNA damage, as kcs1Delta cells are more responsive to DNA damage caused by phleomycin. We observe in vivo a rapid decrease in cellular inositol pyrophosphate levels following exposure to H2O2, and an inhibitory effect of H2O2 on the enzymatic activity of Kcs1 in vitro. Furthermore, parallel cysteine mutagenesis studies performed on mammalian IP6K1 are suggestive that the ROS signal might be transduced by the direct modification of this evolutionarily conserved class of enzymes.
Assuntos
Peróxido de Hidrogênio/farmacologia , Fosfatos de Inositol/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Fosfatos de Inositol/genética , Fosfatos de Inositol/metabolismo , Fosfatos de Inositol/farmacologia , Camundongos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiologia , Organismos Geneticamente Modificados , Fleomicinas/farmacologia , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Inositol pyrophosphates are unique signaling molecules implicated in the regulation of diverse cellular processes. Two new studies by Mulugu et al. (2007) and Lee et al. (2007) extend the biological and metabolic diversity of this class of molecules. They identify yeast Vip1 as a new inositol pyrophosphate synthase and show that the products of Vip1 activity regulate a cyclin/cyclin-dependent kinase complex.
