Fleckenstein Diana S, Dirks Wilhelm G, Drexler Hans G, Quentmeier Hilmar
Department of Human and Animal Cell Cultures, DSMZ-German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1B, 38124 Braunschweig, Germany.
Leuk Res. 2003 Aug;27(8):687-94. doi: 10.1016/s0145-2126(02)00325-9.
p70S6K is an intracellular serine/threonine kinase that mediates cell cycle progression and gene transcription. Immunofluorescent staining shows in factor-dependent hematopoietic M-07e cells that p70S6K is localized both in the cytosol and, after cytokine stimulation, also in the nucleus. We hypothesized that the p70S6K might interact with a transcription factor in the nucleus or with other proteins in the cytosol besides the S6 protein. By screening a yeast two-hybrid HeLa cDNA library with full-length p70S6K cDNA as bait, we identified tumor necrosis factor receptor-associated factor (TRAF) 4 as a new binding partner for this kinase. TRAF4 is a member of the TRAF family of putative signal-transducing proteins. Members of this family are capable of negatively regulating apoptotic pathways by inducing the expression of genes that promote cell survival. Immunoprecipitation experiments showed that stimulation of receptors of the tumor necrosis factor (TNF) family induced the formation of TRAF4/p70S6K complexes. Transfection studies showed that TRAF4 functions in p70S6K activation: TNF induced phosphorylation of S6, the main intracellular substrate of the kinase, in cells stably expressing TRAF4, but not in TRAF4-negative cells. In addition to its role in p70S6K activation, we postulate an anti-apoptotic role for TRAF4, because the agonistic anti-Fas antibody CH-11 induced apoptosis in untransfected HEK-293 cells, but not in TRAF4-expressing HEK-293 cells.
(i) TNF-receptor activation leads to activation of the p70S6K; (ii) TRAF4 is a mediator in this TNF-induced signaling pathway; and (iii) TRAF4 inhibits Fas-induced apoptosis.
p70S6K是一种细胞内丝氨酸/苏氨酸激酶，介导细胞周期进程和基因转录。免疫荧光染色显示，在因子依赖性造血M-07e细胞中，p70S6K定位于细胞质中，细胞因子刺激后也定位于细胞核中。我们推测，p70S6K可能与细胞核中的转录因子相互作用，或者除了S6蛋白外，还与细胞质中的其他蛋白质相互作用。通过以全长p70S6K cDNA为诱饵筛选酵母双杂交HeLa cDNA文库，我们鉴定出肿瘤坏死因子受体相关因子（TRAF）4是该激酶的新结合伴侣。TRAF4是假定的信号转导蛋白TRAF家族的成员。该家族成员能够通过诱导促进细胞存活的基因表达来负调节凋亡途径。免疫沉淀实验表明，肿瘤坏死因子（TNF）家族受体的刺激诱导了TRAF4/p70S6K复合物的形成。转染研究表明，TRAF4在p70S6K激活中起作用：TNF诱导稳定表达TRAF4的细胞中该激酶的主要细胞内底物S6磷酸化，但在TRAF4阴性细胞中则不然。除了在p70S6K激活中的作用外，我们推测TRAF4具有抗凋亡作用，因为激动性抗Fas抗体CH-11在未转染的HEK-293细胞中诱导凋亡，但在表达TRAF4的HEK-293细胞中则不然。
（i）TNF受体激活导致p70S6K激活；（ii）TRAF4是该TNF诱导信号通路中的介质；（iii）TRAF4抑制Fas诱导的凋亡。
