Ishihara Katsuya, Tsutsumi Kae, Kawane Shiho, Nakajima Motowo, Kasaoka Tatsuhiko
Discovery Biology, Tsukuba Research Institute, Novartis Pharma K.K., Okubo 8, Tsukuba-shi, Ibaraki 300-2611, Japan.
FEBS Lett. 2003 Aug 28;550(1-3):107-13. doi: 10.1016/s0014-5793(03)00846-9.
The receptor for advanced glycation end-products (RAGE)-mediated cellular activation through the mitogen-activated protein kinase (MAPK) cascade, activation of NF-kappaB and Rho family small G-proteins, cdc42/Rac, is implicated in the pathogenesis of inflammatory 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 purified from rat lung extracts using an affinity chromatography technique and identified to be extracellular signal-regulated protein kinase-1 and -2 (ERK-1/2). Their interactions were confirmed 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.
晚期糖基化终产物受体(RAGE)通过丝裂原活化蛋白激酶(MAPK)级联介导细胞活化,激活核因子κB和Rho家族小G蛋白(cdc42/Rac),这与炎症性疾病的发病机制以及肿瘤生长/转移有关。然而,RAGE引发细胞信号传导的确切分子机制仍有待阐明。在本研究中,利用亲和层析技术从大鼠肺提取物中纯化出直接与RAGE细胞质C末端结合的蛋白质,鉴定为细胞外信号调节蛋白激酶-1和-2(ERK-1/2)。通过用抗RAGE抗体从表达RAGE的HT1080细胞提取物中免疫沉淀ERK-1/2,证实了它们之间的相互作用。此外,通过测定ERK底物髓鞘碱性蛋白的磷酸化水平,证明了用双调蛋白刺激细胞后,与RAGE结合的ERK激酶活性增强。使用一系列人RAGE C末端缺失突变体进行的体外结合研究表明,RAGE膜近端细胞质区域对于ERK与RAGE的直接相互作用很重要。该区域包含一个与D结构域相似的序列,D结构域是一个ERK对接位点,在一些ERK底物中保守,包括与MAPK相互作用的激酶-1/2、丝裂原和应激激活蛋白激酶-1以及核糖体S6激酶。这些数据表明ERK可能通过与RAGE的直接相互作用在RAGE信号传导中发挥作用。