Dipartimento di Medicina Molecolare e Biotecnologie Mediche, University of Naples Federico II, Naples, Italy.
J Biol Chem. 2013 Jun 14;288(24):17481-94. doi: 10.1074/jbc.M112.439422. Epub 2013 Apr 29.
The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [(35)S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.
受体酪氨酸激酶 AXL 在包括甲状腺癌在内的许多癌症类型中过表达，并且在肿瘤形成和进展中具有明确的作用。几种致癌受体酪氨酸激酶的正确折叠、成熟和活性需要 HSP90 伴侣。抗生素格尔德霉素或其衍生物 17-烯丙基-17-脱甲氧基格尔德霉素（17-AAG）通过抑制 HSP90 来抑制其客户蛋白的稳定性。在这里，我们表明 AXL 是 HSP90 的一种新型客户蛋白。17-AAG 诱导内源性或异位表达的 AXL 蛋白的时间和剂量依赖性下调，从而抑制 AXL 介导的信号转导和生物学活性。17-AAG 诱导的 AXL 下调特异性影响完全糖基化的成熟受体在细胞膜上的存在。通过使用生物素和 [(35)S]甲硫氨酸标记，我们表明 17-AAG 通过介导其在细胞内隔室中的降解来耗尽膜定位的 AXL，从而限制其在细胞表面的暴露。17-AAG 诱导 AXL 多泛素化和随后的蛋白酶体降解；在基础条件下，AXL 与 HSP90 共免疫沉淀。用 17-AAG 处理后，AXL 与共伴侣 HSP70 和泛素 E3 连接酶羧基末端的 HSC70 相互作用蛋白（CHIP）结合。过表达 CHIP，但不是无活性突变体 CHIP K30A，在用 17-AAG 处理时诱导 AXL 多泛素化物质的积累。AXL 对 17-AAG 的敏感性需要其细胞内结构域，因为 AXL 细胞内结构域缺失突变体对该化合物不敏感。活性 AXL 和激酶失活的 AXL 对 17-AAG 同样敏感，这意味着 AXL 对 17-AAG 的敏感性不需要受体磷酸化。总的来说，我们的数据阐明了 HSP90 抑制剂下调 AXL 的分子基础，并表明 HSP90 抑制在癌症治疗中可以通过抑制包括 AXL 在内的多种激酶发挥作用。
