The physical association and phosphorylation of Cdc25C protein phosphatase by Prk
The physical association and phosphorylation of Cdc25C protein phosphatase by Prk
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DOI：
10.1038/sj.onc.1202983
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发表时间：
1999-10-28
期刊：
影响因子：
8
通讯作者：
Dai, W
中科院分区：
文献类型：
--
作者：
Bin, OY;Li, WQ;Dai, W
PrK编码一种蛋白丝氨酸/苏氨酸激酶，参与调节细胞周期中的M期功能。我们利用杆状病毒载体表达系统表达了His6-PrK和His6-CDc25C蛋白。纯化的重组His6-PrK对His6-CDc25C具有较强的体外磷酸化能力，而不是一个突变的His6-PrK(K52R)。免疫共沉淀和亲和柱层析实验证明GST-PrK与天然的CDC25C相互作用。当与His6-PrK和His6-CDc25C重组杆状病毒混合感染时，SF-9细胞产生His6-CDc25C抗原，与单独感染His6-CDc25C杆状病毒的细胞相比，在变性聚丙烯酰胺凝胶上有一条额外的慢速迁移带。此外，与His6-PrK(K52R)和His6-CDc25C杆状病毒共感染的SF-9细胞中的His6-CDc25C免疫共沉淀物含有显著增强的磷酸化His6-CDc25C的激酶活性，而与His6-PrK(K52R)和His6-CDc25C杆状病毒不同。此外，磷酸肽图谱显示，His6-PrK在体外有两个位点使His6-CDc25C磷酸化，并且在异步化细胞中，主要的磷酸化位点与体内磷酸化的位点共同迁移。进一步的研究表明，His6-PrK使丝氨酸(216)上的CDc25C磷酸化，丝氨酸(216)上的残基也被Chk1和Chk2磷酸化。综上所述，这些观察有力地表明，PrK在有丝分裂中的作用至少部分是通过直接调控CDC25C来调节的。
prk encodes a protein serine/threonine kinase involved in regulating M phase functions during the cell cycle. We have expressed His6-Prk and His6-Cdc25C proteins using the baculoviral vector expression system. Purified recombinant His6-Prk, but not a kinase-defective mutant His6-Prk(K52R), is capable of strongly phosphorylating His6-Cdc25C in vitro. Co-immunoprecipitation and affinity column chromatography experiments demonstrate that GST-Prk and native Cdc25C interact. When co-infected with His6-Prk and His6-Cdc25C recombinant baculoviruses, sf-9 cells produce His6-Cdc25C antigen with an additional slower mobility band on denaturing polyacrylamide gels compared with cells infected with His6-Cdc25C baculovirus alone. In addition, His6-Cdc25C immunoprecipitated from sf-9 cells co-infected with His6-Prk and His6-Cdc25C baculoviruses, but not with His6-Prk(K52R) and His6-Cdc25C baculoviruses, contains a greatly enhanced kinase activity that phosphorylates His6-Cdc25C in vitro. Moreover, phosphopeptide mapping shows that His6-Prk phosphorylates His6-Cdc25C at two sites in vitro and that the major phosphorylation site co-migrates with the one that is phosphorylated in vivo in asynchonized cells. Further studies reveal that His6-Prk phosphorylates Cdc25C on serine(216), a residue also phosphorylated by Chk1 and Chk2. Together, these observations strongly suggest that Prk's role in mitosis is at least partly mediated through direct regulation of Cdc25C.