Division of Rheumatology, Goethe University Hospital Frankfurt am Main, 60590 Frankfurt am Main, Germany.
J Biol Chem. 2012 Jun 15;287(25):21214-23. doi: 10.1074/jbc.M112.347120. Epub 2012 Apr 27.
ADAM15, a disintegrin and metalloproteinase, is capable of counteracting genotoxic stress-induced apoptosis by the suppression of caspase-3 activation. A cell line expressing the membrane-bound ADAM15 without its cytoplasmic tail, however, lost this anti-apoptotic property, suggesting a crucial role of the intracellular domain as a scaffold for recruitment of survival signal-transducing kinases. Accordingly, an enhanced phosphorylation of FAK at Tyr-397, Tyr-576, and Tyr-861 was detected upon genotoxic stress by camptothecin in ADAM15-transfected T/C28a4 cells, but not in transfectants expressing an ADAM15 mutant without the cytoplasmic tail. Accordingly, a specific binding of the cytoplasmic ADAM15 domain to the C terminus of FAK could be shown by mammalian two-hybrid, pulldown, and far Western studies. In cells expressing full-length ADAM15, a concomitant activation of Src at Tyr-416 was detected upon camptothecin exposure. Cells transfected with a chimeric construct consisting of the extracellular IL-2 receptor α-chain and the cytoplasmic ADAM15 domain were IL-2-stimulated to prove that the ADAM15 tail can transduce a percepted extracellular signal to enhance FAK and Src phosphorylation. Our studies further demonstrate Src binding to FAK but not a direct Src interaction with ADAM15, suggesting FAK as a critical intracellular adaptor for ADAM15-dependent enhancement of FAK/Src activation. Moreover, the apoptosis induction elicited by specific inhibitors (PP2, FAK 14 inhibitor) of FAK/Src signaling was significantly reduced by ADAM15 expression. The newly uncovered counter-regulatory response to genotoxic stress in a chondrocytic survival pathway is potentially also relevant to apoptosis resistance in neoplastic growth.
去整合素金属蛋白酶 15（ADAM15）能够通过抑制半胱天冬酶-3 的激活来抵抗遗传毒性应激诱导的细胞凋亡。然而，表达没有细胞质尾巴的膜结合 ADAM15 的细胞系失去了这种抗凋亡特性，这表明细胞内结构域作为募集存活信号转导激酶的支架起着关键作用。因此，在用喜树碱处理 ADAM15 转染的 T/C28a4 细胞时，检测到遗传毒性应激后 FAK 的 Tyr-397、Tyr-576 和 Tyr-861 处的 FAK 磷酸化增强，但在表达没有细胞质尾巴的 ADAM15 突变体的转染子中没有检测到。因此，通过哺乳动物双杂交、拉下和远 Western 研究可以证明细胞质 ADAM15 结构域与 FAK 的 C 末端的特异性结合。在表达全长 ADAM15 的细胞中，在暴露于喜树碱时检测到Src 在 Tyr-416 处的伴随激活。用包含细胞外白细胞介素 2 受体 α 链和细胞质 ADAM15 结构域的嵌合构建体转染的细胞被白细胞介素 2 刺激，以证明 ADAM15 尾巴可以传递感知到的细胞外信号，以增强 FAK 和 Src 的磷酸化。我们的研究进一步表明 Src 与 FAK 结合，而不是 Src 与 ADAM15 的直接相互作用，这表明 FAK 是 ADAM15 依赖性增强 FAK/Src 激活的关键细胞内衔接蛋白。此外，通过 ADAM15 表达，特定的 FAK/Src 信号抑制剂（PP2、FAK14 抑制剂）诱导的细胞凋亡诱导明显减少。在软骨细胞存活途径中发现的这种新的遗传毒性应激的代偿性反应可能与肿瘤生长中的抗凋亡性有关。
