Ser2194 is a highly conserved major phosphorylation site of the hepatitis C virus nonstructural protein NS5A
Ser2194 is a highly conserved major phosphorylation site of the hepatitis C virus nonstructural protein NS5A
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DOI：
10.1006/viro.2000.0662
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发表时间：
2000-12-20
期刊：
影响因子：
3.7
通讯作者：
Aebersold, R
中科院分区：
文献类型：
--
作者：
Katze, MG;Kwieciszewski, B;Aebersold, R
非结构NS 5A蛋白的磷酸化在丙型肝炎病毒（HCV）基因型中高度保守。然而，NS 5A磷酸化的精确位点尚未确定，磷酸化的功能意义仍然未知。在这里，我们表明，通过二维磷酸肽映射，蛋白激酶或激酶存在于酵母，昆虫和哺乳动物细胞磷酸化高度纯化的HCV基因型Ib NS 5A从昆虫细胞上相同的丝氨酸残基。我们发现了一种主要的磷酸肽（对应于HCV Ib多聚蛋白的氨基酸2193-2212），通过负离子电喷雾电离-质谱法测定的磷酸肽的洗脱时间对应于大部分P-32-的洗脱时间。通过体外激酶反应掺入磷酸肽中的标记。随后通过自动正离子电喷雾串联质谱法对峰级分进行分析，结果显示Ser（2194）是磷酸肽Gp-SPPSLASSSASQLSAPSLK上的主要磷酸化残基。用Ala取代Ser（2194）导致主要体内磷酸化肽的伴随消失。Ser（2194）及其周围氨基酸在所有HCV基因型中高度保守，表明NS 5A在Ser（2194）的磷酸化可能是调节NS 5A生物学功能的重要机制。(C)北京大学出版社.
Phosphorylation of the nonstructural NS5A protein is highly conserved among hepatitis C virus (HCV) genotypes. However, the precise site or sites of phosphorylation of NS5A have not been determined, and the functional significance of phosphorylation remains unknown. Here, we showed by two-dimensional phosphopeptide mapping that a protein kinase or kinases present in yeast, insect, and mammalian cells phosphorylated a highly purified HCV genotype Ib NS5A from insect cells on identical serine residues. We identified a major phosphopeptide (corresponding to amino acids 2193-2212 of the HCV Ib polyprotein) by using negative-ion electrospray ionization-microcapillary high performance liquid chromatography-mass spectrometry, The elution time of the phosphopeptide determined by negative-ion electrospray ionization-mass spectrometry corresponded with the elution time of the majority of P-32-label that was incorporated into the phosphopeptide by an in vitro kinase reaction. Subsequent analysis of the peak fraction by automated positive-ion electrospray ionization-tandem mass spectrometry revealed that Ser(2194) was the major phosphorylated residue on the phosphopeptide Gp-SPPSLASSSASQLSAPSLK. Substitution for Ser(2194) with Ala resulted in the concomitant disappearance of major in vivo phosphorylated peptides. Ser(2194) and surrounding amino acids are highly conserved in all HCV genotypes, suggesting NS5A phosphorylation at Ser(2194) may be an important mechanism for modulating NS5A biological functions. (C) 2000 Academic Press.