Larsson N, Melander H, Marklund U, Osterman O, Gullberg M
Department of Cell and Molecular Biology, University of Umeå, Sweden.
J Biol Chem. 1995 Jun 9;270(23):14175-83. doi: 10.1074/jbc.270.23.14175.
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 Ser16, Ser25, Ser38, and Ser63 are all subject to cell cycle-regulated phosphorylation. Ser25 and Ser38 are targets for cyclin dependent kinases (CDKs), while Ser16 and Ser63 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 Ser25 and Ser38 and that Ser16 and Ser63 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 Ser16 and Ser63. The simplest interpretation of our results is that phosphorylation of Ser16 and Ser63 is essential during G2/M transition and that the phenotype of the S25A,S38A mutant is mediated by the observed block of Ser16/Ser63 phosphorylation.
癌蛋白18（Op18）是一种保守的胞质蛋白，是细胞周期和细胞表面受体调节的磷酸化事件的靶点。丝氨酸16、丝氨酸25、丝氨酸38和丝氨酸63这四个残基均受到细胞周期调节的磷酸化作用。丝氨酸25和丝氨酸38是细胞周期蛋白依赖性激酶（CDK）的作用靶点，而丝氨酸16和丝氨酸63则由一种未知的蛋白激酶磷酸化。我们最近发现，诱导表达一种缺乏CDK作用靶点的突变体Op18-S25A,S38A，会在G2/M期转换过程中阻断人类细胞系。在本报告中，我们表明有丝分裂与Op18的两个CDK作用靶点丝氨酸25和丝氨酸38的完全磷酸化有关，并且丝氨酸16和丝氨酸63也以高化学计量比被磷酸化。为了评估Op18多位点磷酸化的功能，我们表达并分析了不同激酶作用靶点缺陷突变体的细胞周期表型。数据表明，诱导表达S16A,S63A、S25A,S38A和S16A,S25A,S38A,S63A突变体均导致难以区分的表型，即立即发生G2/M期阻断以及随后的核内复制、一定比例的G2期与M期阻断细胞，以及M期阻断细胞的特征性核形态。这一结果出乎意料；然而，对Op18磷酸异构体的分析提供了一个可能的解释，即CDK位点的突变会干扰丝氨酸16和丝氨酸63的磷酸化。我们结果的最简单解释是，丝氨酸16和丝氨酸63的磷酸化在G2/M期转换过程中至关重要，并且S25A,S38A突变体的表型是由观察到的丝氨酸16/丝氨酸63磷酸化的阻断所介导的。
