G2/M TRANSITION REQUIRES MULTISITE PHOSPHORYLATION OF ONCOPROTEIN-18 BY 2 DISTINCT PROTEIN-KINASE SYSTEMS
G2/M TRANSITION REQUIRES MULTISITE PHOSPHORYLATION OF ONCOPROTEIN-18 BY 2 DISTINCT PROTEIN-KINASE SYSTEMS
复制标题
DOI：
10.1074/jbc.270.23.14175
复制
发表时间：
1995-06-09
影响因子：
4.8
通讯作者：
GULLBERG, M
中科院分区：
文献类型：
--
作者：
LARSSON, N;MELANDER, H;GULLBERG, M
癌蛋白18（Op 18）是一种保守的细胞溶质蛋白，其是细胞周期和细胞表面受体调节的磷酸化事件的靶标。四个残基Ser（16）、Ser（25）、Ser（38）和Ser（63）都受到细胞周期调节的磷酸化，Ser（25）和Ser（38）是细胞周期蛋白依赖性激酶（CDK）的靶标，虽然Ser（16）和Ser（63）被一种未鉴定的蛋白激酶磷酸化，但我们最近表明CDK靶位点缺陷突变体Op 18-S25 A、S38 A的诱导表达会在G2/M转变期间阻断人类细胞系，在本报告中，我们发现有丝分裂与两个Op 18 CDK靶位点Ser（25）和Ser（38）的完全磷酸化有关，而Ser（16）和Ser（为了评估Op 18多位点磷酸化的功能，我们表达并分析了不同激酶靶位点缺陷突变体的细胞周期表型。数据显示，S16 A、S63 A、S25 A、S38 A和S16 A、S25 A、S38 A、S63 A突变体的诱导表达均导致不可区分的表型，即立即的G2/M阻滞和随后的核内复制，G2相对于M期阻滞细胞的给定分数，以及M期阻滞细胞的特征性核形态。然而，通过Op 18磷酸异构体的分析提供了可能的解释，其揭示了CDK位点的突变干扰Ser（16）和Ser（63）的磷酸化。我们的结果的最简单的解释是，Ser（16）和Ser（63）的磷酸化在G2/M转换过程中是必不可少的，S25 A，S38 A突变体的表型是由观察到的Ser（16）/Ser（63）磷酸化的阻断介导的。
Oncoprotein 18 (Op18) is a conserved cytosolic protein that is a target for both cell cycle and cell surface receptor-regulated phosphorylation events, The four residues Ser(16), Ser(25), Ser(38), and Ser(63) are all subject to cell cycle-regulated phosphorylation, Ser(25) and Ser(38) are targets for cyclin dependent kinases (CDKs), while Ser(16) and Ser(63) are phosphorylated by an unidentified protein kinase, We have recently shown that induced expression of a CDK target site deficient mutant, Op18-S25A,S38A, blocks human cell lines during G2/M transition, In the present report we show that mitosis is associated with complete phosphorylation of the two Op18 CDK target sites Ser(25) and Ser(38) and that Ser(16) and Ser(63) are also phosphorylated to a high stoichiometry, To evaluate the function of multisite phosphorylation of Op18, we expressed and analyzed the cell cycle phenotype of different kinase target site-deficient mutants. The data showed that induced expression of the S16A,S63A, S25A,S38A, and S16A,S25A,S38A,S63A mutants all resulted in an indistinguishable phenotype, i.e. immediate G2/M block and subsequent endoreduplication, a given fraction of G2 versus M-phase blocked cells, and a characteristic nuclear morphology of M-blocked cells, This result was unexpected; however, a likely explanation was provided by analysis of Op18 phosphoisomers, which revealed that mutations of the CDK sites interfere with phosphorylation of Ser(16) and Ser(63). The simplest interpretation of our results is that phosphorylation of Ser(16) and Ser(63) is essential during G2/M transition and that the phenotype of the S25A,S38A mutant is mediated by the observed block of Ser(16)/Ser(63) phosphorylation.