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Differential binding of ceramide to MEKK1 in glomerular endothelial and mesangial cells

机译:神经酰胺与肾小球内皮细胞和系膜细胞中MEKK1的差异结合

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Previously, we have shown that ceramide is able to directly bind to and activate c-Raf and to trigger the downstream classical mitogen-activated protein kinase (MAPK/ERK) cascade in glomerular mesangial cells [Proc. Natl. Acad. Sci. USA 93 (1996) 6959]. In this study, we show that ceramide acts differently in glomerular endothelial cells in that treatment of endothelial cells with exogenous ceramide leads to a potent activation of the stress-activated protein kinase (SAPK/JNK) cascade but not to an activation of the classical ERIC cascade. A similar effect was observed with the inflammatory cytokines TNFα and IL-1β which activate a sphingomyelinase and thereby increase intracellular ceramide levels. The activation of JNKs as shown by c-Jun phosphorylation assays was paralleled by increased phosphorylation of the two JNK isoforms, p45 and p54. In addition, also the activator of JNKs, SEK1, was found to be increasingly phosphorylated by exogenous ceramide as well as by TNFα. In contrast, dihydroceramide had no effect on JNK or SEK1 phosphorylation. To see whether ceramide directly binds to MEKK1, which is the c-Raf analog in the SAPK cascade, a radioiodinated photoaffinity labeling analogue of ceramide, (N-[3-[[[2-(~(125)I)iodo-4-[3-(trifluoromethyl)-3//-diazirin-3-yl]benzyl]oxy]-carbonyl] propanoyl]-D-erythro-sphingosine) ([~(125)I]TID-ceramide) was used. Stimulation of endothelial cells with this [~(125)I]TID-ceramide for 5 min followed by a short photolysis defined MEKK1 as a direct target of ceramide. With the same method, protein kinase C-α (PKC-α) was identified as a ceramide target. In contrast, no binding to c-Raf or the MEKK1 activator p65-PAK could be detected. A direct binding of ceramide to MEKK1 was also confirmed by affinity chromatography using a ceramide-coupled sepharose column. Furthermore, the ceramide-activated SAPK/JNK cascade is clearly involved in the mechanism of apoptosis, since in the presence of a JNK inhibitor, ceramide-induced DNA fragmentation is significantly reduced. In summary, we have shown that ceramide potently activates the SAPK cascade but not the ERK cascade in endothelial cells, which contrasts to mesangial cells where ceramide activates the ERK pathway and has only a minor effect on the SAPK cascade. Regarding the direct target of ceramide binding and action in endothelial cells, we identified MEKK1 as a further member of the growing family of ceramide-activated protein kinases.
机译:以前,我们已经证明神经酰胺能够直接结合并激活c-Raf并触发肾小球系膜细胞中下游的经典丝裂原活化蛋白激酶(MAPK / ERK)级联[Proc。 Natl。学院科学USA 93(1996)6959]。在这项研究中,我们表明神经酰胺在肾小球内皮细胞中的作用不同,因为外源性神经酰胺对内皮细胞的处理会导致应激激活的蛋白激酶(SAPK / JNK)级联的有效激活,而不是经典ERIC的激活级联。炎性细胞因子TNFα和IL-1β激活了鞘磷脂酶,从而增加了细胞内神经酰胺的水平,观察到了类似的效果。如c-Jun磷酸化分析所示,JNK的激活与两种JNK同种型p45和p54的磷酸化增加平行。此外,还发现JNK的激活剂SEK1被外源性神经酰胺以及TNFα磷酸化程度更高。相反,二氢神经酰胺对JNK或SEK1磷酸化没有影响。要查看神经酰胺是否直接结合MEKK1,MEKK1是SAPK级联中的c-Raf类似物,是神经酰胺的放射性碘标记的光亲和性标记类似物,(N- [3-[[[2-(〜(125)I)iodo-4使用-[[3-(三氟甲基)-3 //-二叠氮基-3-基]苄基]氧基]-羰基]丙酰基] -D-赤型-鞘氨醇([〜(125)I] TID-神经酰胺)。用此[〜(125)I] TID-神经酰胺刺激内皮细胞5分钟,然后进行短暂的光解,将MEKK1定义为神经酰胺的直接靶标。使用相同的方法,蛋白激酶C-α(PKC-α)被确定为神经酰胺靶标。相反,未检测到与c-Raf或MEKK1激活剂p65-PAK的结合。还通过使用神经酰胺偶联的琼脂糖凝胶柱的亲和色谱法证实了神经酰胺与MEKK1的直接结合。此外,神经酰胺激活的SAPK / JNK级联显然参与细胞凋亡的机制,因为在存在JNK抑制剂的情况下,神经酰胺诱导的DNA断裂显着减少。总而言之,我们已经表明,神经酰胺能有效激活内皮细胞中的SAPK级联反应,而不激活ERK级联反应,这与肾小球系膜细胞的神经酰胺激活ERK途径而对SAPK级联反应影响很小。关于神经酰胺在内皮细胞中的结合和作用的直接靶点,我们确定MEKK1是神经酰胺激活的蛋白激酶的增长家族的另一个成员。

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