Depletion of Guanine Nucleotides Leads to the Mdm2-Dependent Proteasomal Degradation of Nucleostemin
Depletion of Guanine Nucleotides Leads to the Mdm2-Dependent Proteasomal Degradation of Nucleostemin
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DOI：
10.1158/0008-5472.can-08-3413
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发表时间：
2009-04-01
期刊：
影响因子：
11.2
通讯作者：
Mitchell, Beverly S.
中科院分区：
文献类型：
--
作者：
Huang, Min;Itahana, Koji;Mitchell, Beverly S.
核干细胞蛋白是细胞增殖的正调节因子，在多种干细胞、肿瘤和肿瘤细胞细颗粒中高度表达。该蛋白以GTP依赖的方式穿梭于核仁和细胞核之间。AVN-944（从头嘌呤合成酶IMP脱氢酶的抑制剂）选择性耗尽细胞内鸟嘌呤核苷酸，导致肿瘤细胞系中核干细胞蛋白快速消失，这一效应不会发生在其他两种核仁蛋白（核磷蛋白或核仁素）中。内源性核干细胞蛋白质被MG 132完全稳定，MG 132是26 S蛋白酶体的抑制剂，表达的增强型绿色荧光蛋白标记的核干细胞蛋白质的水平也是如此，野生型蛋白质和在G（1）GTP结合位点含有突变的蛋白质都是如此。Nutlin-3a是一种破坏E3泛素连接酶Mdm 2与p53结合的小分子，在鸟嘌呤核苷酸耗尽时稳定核干细胞蛋白，siRNA介导的Mdm 2表达敲低和Mdm 2显性阴性形式的过表达也是如此。无论是多柔比星还是放线菌素D，都不会导致核干细胞蛋白从核仁中释放，导致核干细胞蛋白降解。我们的结论是，nucleostemin是一个目标MDM 2介导的泛素化和降解时，不绑定到GTP。由于这种效果不发生与其他化疗药物，诱导的nucleostemin蛋白降解的IMP脱氢酶抑制剂或其他小分子，破坏GTP结合的肿瘤细胞可能会提供一种新的方法来治疗某些肿瘤性疾病。[Cancer Res 2009;69（7）：3004-12]
Nucleostemin is a positive regulator of cell proliferation and is highly expressed in a variety of stem cells, tumors, and tumor cell fines. The protein shuttles between the nucleolus and the nucleus in a GTP-dependent fashion. Selective depletion of intracellular guanine nucleotides by AVN-944, an inhibitor of the de novo purine synthetic enzyme, IMP dehydrogenase, leads to the rapid disappearance of nucleostemin protein in tumor cell lines, an effect that does not occur with two other nucleolar proteins, nucleophosmin or nucleolin. Endogenous nucleostemin protein is completely stabilized by MG132, an inhibitor of the 26S proteasome, as are the levels of expressed enhanced green fluorescent protein-tagged nucleostemin, both wild-type protein and protein containing mutations at the G(1) GTP binding site. Nutlin-3a, a small molecule that disrupts the binding of the E3 ubiquitin ligase, Mdm2, to p53, stabilizes nucleostemin protein in the face of guanine nucleotide depletion, as does siRNA-mediated knockdown of Mdm2 expression and overexpression of a dominant-negative form of Mdm2. Neither Doxorubicin nor Actinomycin D, which cause the release of nucleostemin from the nucleolus, results in nucleostemin degradation. We conclude that nucleostemin is a target for Mdm2-mediated ubiquitination and degradation when not bound to GTP. Because this effect does not occur with other chemotherapeutic agents, the induction of nucleostemin protein degradation in tumor cells by IMP dehydrogenase inhibition or by other small molecules that disrupt GTP binding may offer a new approach to the treatment of certain neoplastic diseases. [Cancer Res 2009;69(7):3004-12]