Hepatitis C virus NS2 protein is phosphorylated by the protein kinase CK2 and targeted for degradation to the proteasome
Hepatitis C virus NS2 protein is phosphorylated by the protein kinase CK2 and targeted for degradation to the proteasome
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DOI：
10.1128/jvi.79.5.2700-2708.2005
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发表时间：
2005-03-01
影响因子：
5.4
通讯作者：
Erdtmann, L
中科院分区：
文献类型：
--
作者：
Franck, N;Le Seyec, J;Erdtmann, L
丙型肝炎病毒（HCV）非结构2 （NS2）蛋白是一种疏水跨膜蛋白，参与细胞凋亡抑制和基因转录调节等多种功能。我们在这里研究了NS2蛋白的周转，发现NS2在不同的细胞系中被蛋白酶体迅速降解，如在原代人肝细胞中。由于翻译后修饰可以影响蛋白质周转，我们在NS2中寻找潜在的磷受体位点。计算序列分析结合NS2点突变体的筛选表明，丝氨酸残基168是降解的关键。在寻找NS2蛋白激酶的过程中，我们通过序列分析发现丝氨酸残基168是共识酪蛋白激酶2 （CK2）识别位点（S/TXXE）的一部分。该基序高度保守，因为它可以在所有HCV基因型的NS2初级共识序列中找到。为了验证CK2是否参与NS2的磷酸化，我们通过体外激酶实验证明CK2磷酸化NS2，只要这个CK2基序是保守的。有趣的是，当CK2基序被单点突变修饰时，NS2对蛋白质降解产生抗性。此外，姜黄素抑制CK2活性降低了体外NS2磷酸化，稳定了HepG2细胞中NS2的表达。最后，我们在Huh-7.5复制子细胞中发现，在HCV多蛋白背景下表达的NS2也对蛋白酶体介导的降解和CK2抑制剂处理敏感。我们认为NS2是一种短寿命蛋白，其蛋白酶体的降解是通过蛋白激酶CK2以磷酸化依赖的方式调节的。
Hepatitis C virus (HCV) nonstructural 2 (NS2) protein is a hydrophobic transmembrane protein, described to be involved in different functions, such as apoptosis inhibition and gene transcription modulation. We investigated here NS2 protein turnover and found that NS2 was rapidly degraded by the proteasome in different cell lines, as in primary human hepatocytes. Since posttranslational modifications can influence protein turnover, we looked for potential phosphoacceptor sites in NS2. Computational sequence analysis in combination with screening of NS2 point mutants revealed that serine residue 168 was critical for degradation. In the quest of a protein kinase for NS2, we identified by sequence analysis that the serine residue 168 was part of a consensus casein kinase 2 (CK2) recognition site (S/TXXE). This motif was highly conserved since it could be found in the NS2 primary consensus sequences from all HCV genotypes. To verify whether CK2 is involved in NS2 phosphorylation, we showed by an in vitro kinase assay that CK2 phosphorylated NS2, as far as this CK2 motif was conserved. Interestingly, NS2 became resistant to protein degradation when the CK2 motif was modified by a single point mutation. Furthermore, inhibition of CK2 activity by curcumin decreased NS2 phosphorylation in vitro and stabilized NS2 expression in HepG2 cells. Finally, we showed in Huh-7.5 replicon cells that NS2, expressed in the context of the HCV polyprotein, was also sensitive to both proteasome-mediated degradation and CK2 inhibitor treatment. We suggest that NS2 is a short-lived protein whose degradation by the proteasome is regulated in a phosphorylation-dependent manner through the protein kinase CK2.