The receptor for advanced glycation end-products (RAGE)-mediated cellular activation through the mitogen-activated protein kinase (MAPK) cascade, activation of NF-U UB and Rho family small G-proteins, cdc42/Rac, is implicated in the pathogenesis of in£ammatory disorders and tumor growth/metastasis. However, the precise molecular mechanisms for the initiation of cell signaling by RAGE remain to be elucidated. In this study, proteins which directly bind to the cytoplasmic C-terminus of RAGE were puri¢ed from rat lung extracts using an a⁄nity chromatography technique and identi¢ed to be extracellular signal-regulated protein kinase-1 and -2 (ERK-1/2). Their interactions were con¢rmed by immunoprecipitation of ERK-1/2 from RAGE-expressing HT1080 cell extracts with anti-RAGE antibody. Furthermore, the augmentation of kinase activity of RAGE-bound ERK upon the stimulation of cells with amphoterin was demonstrated by determining the phosphorylation level of myelin basic protein, an ERK substrate. In vitro binding studies using a series of C-terminal deletion mutants of human RAGE revealed the importance of the membrane-proximal cytoplasmic region of RAGE for the direct ERK^RAGE interaction. This region contained a sequence similar to the D-domain, a ERK docking site which is conserved in some ERK substrates including MAPK-interacting kinase-1/2, mitogen-and stress-activated protein kinase-1, and ribosomal S6 kinase. These data suggest that ERK may play a role in RAGE signaling through direct interaction with RAGE. ß
Smc5 and Smc6 proteins form a heterodimeric SMC (structural maintenance of chromosome) protein complex like SMC1-SMC3 cohesin and SMC2-SMC4 condensin, and they associate with non-SMC proteins Nse1 and Nse2 stably and Rad60 transiently. This multiprotein complex plays an essential role in maintaining chromosome integrity and repairing DNA double strand breaks (DSBs). This study characterizes a Schizosaccharomyces pombe mutant rad62-1, which is hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2 (a feature of recombination mutants). rad62-1 is hypersensitive to UV and gamma rays, epistatic with rhp51, and defective in repair of DSBs. rad62 is essential for viability and genetically interacts with rad60, smc6, and brc1. Rad62 protein physically associates with the Smc5-6 complex. rad62-1 is synthetically lethal with mutations in the genes promoting recovery from stalled replication, such as rqh1, srs2, and mus81, and those involved in nucleotide excision repair like rad13 and rad16. These results suggest that Rad62, like Rad60, in conjunction with the Smc5-6 complex, plays an essential role in maintaining chromosome integrity and recovery from stalled replication by recombination.Mutants in Schizosaccharomyces pombe rad2, Saccharomyces cerevisiae RAD27, and Escherichia coli polA, all of which are defective in processing Okazaki fragments, are synthetically lethal with mutations in recombination repair genes (8,11,23,24,35,37,40,43,44). In these mutants, double strand breaks (DSBs) are thought to be produced when replication forks encounter the single strand gaps or nicks remaining unsealed due to the inefficient processing of Okazaki fragments, so they require a highly efficient capacity to repair DSBs for survival. In an effort to identify novel genes involved in recombination repair, we isolated mutants that were hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2⌬ and cloned the genes by complementation of the MMS sensitivity. Genes identified in this way include rhp57 (44), rad60 (35), rad32, nbs1 (45), and fdh1, which encodes an F-box DNA helicase (our unpublished data). A recent genome-wide search for mutations synthetically lethal with rad27 in S. cerevisiae identified mutations in all of the well-studied recombination repair genes including RAD52, RAD50, MRE11, XRS2 RAD51, RAD55, RAD57, RAD54, SGS1, MUS81, and MMS4 (43). The screen also identified genes involved in checkpoint regulation (RAD9, RAD17, RAD24, and DDC1) and those related to regulation of chromatin structure (CAC2, ESC2, HST1, and HST3).We report here a novel gene, rad62, which was identified by isolating mutants that were hypersensitive to MMS and synthetically lethal with rad2 mutation and cloning the gene that complemented the MMS sensitivity of such a mutant. rad62 mutants show very similar phenotypes to those of rad60 mutants (7, 35) and rad18 mutants (30, 47). They are hypersensitive to UV, MMS, and gamma rays, epistatic with rhp51 with respect to the damage sensitivity, requir...
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