Identification of a physiological phosphorylation site of the herpes simplex virus 1-encoded protein kinase Us3 which regulates its optimal catalytic activity in vitro and influences its function in infected cells
Identification of a physiological phosphorylation site of the herpes simplex virus 1-encoded protein kinase Us3 which regulates its optimal catalytic activity in vitro and influences its function in infected cells
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DOI：
10.1128/jvi.00044-08
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发表时间：
2008-07-01
影响因子：
5.4
通讯作者：
Kawaguchi, Yasushi
中科院分区：
文献类型：
--
作者：
Kato, Akihisa;Tanaka, Michiko;Kawaguchi, Yasushi
Us3是由单纯疱疹病毒I （HSV-1）编码的丝氨酸/苏氨酸蛋白激酶。在这里，我们报道了丝氨酸147位（Ser-147）的生理Us3磷酸化位点的鉴定，该位点在体外调节其蛋白激酶活性。此外，该位点的突变影响Us3功能，包括酶的正确定位和诱导hsv -1感染细胞的通常形态变化。这些结论是基于以下观察：(i)在体外激酶实验中，含有Ser-147的Us3结构域被Us3和蛋白激酶a特异性磷酸化，而Ser-147被丙氨酸取代的突变结构域则没有被磷酸化；（ii）在体外实验中，Ser-147 （S147A）在Us3中的丙氨酸替代导致杆状病毒系统中表达的纯化分子的激酶活性显著受损；（iii）用一种抗体特异性检测了被编码VenusA206K-Us3的重组HSV-1感染的Vero细胞中，单个荧光蛋白（FP） VenusA206K （VenusA206K-Us3）标记的Us3中Ser-147的磷酸化，该抗体可识别-3和-2位置的精氨酸磷酸化的丝氨酸或苏氨酸残基；（iv） S147A突变影响了部分但不是全部的Us3功能，包括该蛋白正确定位自身的能力和在感染细胞中诱导野生型细胞病变效应的能力。我们的研究结果表明，Us3在感染细胞中的一些调控活性是由Ser-147位点的磷酸化控制的。
Us3 is a serine/threonine protein kinase encoded by herpes simplex virus I (HSV-1). Here, we report the identification of a physiological Us3 phosphorylation site on serine at position 147 (Ser-147) which regulates its protein kinase activity in vitro. Moreover, mutation of this site influences Us3 function, including correct localization of the enzyme and induction of the usual morphological changes in HSV-1-infected cells. These conclusions are based on the following observations: (i) in in vitro kinase assays, a domain of Us3 containing Ser-147 was specifically phosphorylated by Us3 and protein kinase A, while a mutant domain in which Ser-147 was replaced with alanine was not; (ii) in vitro, alanine replacement of Ser-147 (S147A) in Us3 resulted in significant impairment of the kinase activity of the purified molecule expressed in a baculovirus system; (iii) phosphorylation of Ser-147 in Us3 tagged with the monomeric fluorescent protein (FP) VenusA206K (VenusA206K-Us3) from Vero cells infected with a recombinant HSV-1 encoding VenusA206K-Us3 was specifically detected using an antibody that recognizes phosphorylated serine or threonine residues with arginine at the -3 and -2 positions; and (iv) the S147A mutation influenced some but not all Us3 functions, including the ability of the protein to localize itself properly and to induce wild-type cytopathic effects in infected cells. Our results suggest that some of the regulatory activities of Us3 in infected cells are controlled by phosphorylation at Ser-147.