Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein
Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein
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DOI：
10.1128/jvi.76.16.8124-8137.2002
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发表时间：
2002-08-01
影响因子：
5.4
通讯作者：
Cotten, M
中科院分区：
文献类型：
--
作者：
Daub, H;Blencke, S;Cotten, M
乙肝病毒核心蛋白的磷酸化是前基因组RNA被包裹到病毒衣壳中的先决条件，但介导这一复制周期的关键步骤的宿主细胞激酶尚未确定。我们在HH-7细胞总裂解物中检测到两个95 kDa和115 kDa的激酶，它们与乙肝病毒核心蛋白特异性地相互作用，并使其富含精氨酸的C末端结构域磷酸化。纯化了95 kDa的蛋白激酶，并经质谱仪鉴定为SR蛋白特异性蛋白激酶I(SRPK1)。根据这一发现，通过免疫印迹分析，可以将115 kDa的激酶鉴定为相关的蛋白激酶SRPK2。在体外，两个SRPKs在体内发现被磷酸化的相同丝氨酸残基上磷酸化HBVc蛋白。此外，在总细胞裂解物中检测到的主要细胞HBVK活性显示出与SRPK1和SRPK2相同的生化性质，通过测量与一组层析介质的结合来检验。我们还清楚地证明，无论是细胞周期蛋白依赖的CDC2和CDK2，还是蛋白激酶C，都不能解释乙肝病毒核心蛋白的活性。我们认为，SRPK1和SRPK2很可能是在病毒感染过程中介导HBVc蛋白磷酸化的细胞蛋白激酶，因此是治疗干预的重要宿主细胞靶点。
Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase I (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.