Yamano H, Ishii K, Yanagida M
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
EMBO J. 1994 Nov 15;13(22):5310-8. doi: 10.1002/j.1460-2075.1994.tb06865.x.
We show that the fission yeast dis2 protein phosphatase, which is highly similar to mammalian type 1 phosphatase, is a phosphoprotein containing phosphoserine (phospho-S) and threonine (phospho-T). It has several phosphorylation sites, two of which locate in the C-terminus. Phospho-T was abolished in the alanine substitution mutant at the C-terminal T316, which is conserved as a residue in the cdc2 consensus, TPPR, in a number of type 1-like phosphatases. In G2-arrested cdc2-L7 cells, the degree of T316 phosphorylation was reduced, whereas it was enhanced in metaphase-arrested nuc2-663 mutant cells. Phospho-T was produced in dis2 by fission yeast cdc2 kinase, but not in the substitution mutant A316, indicating that the T316 residue was the site for cdc2 kinase in vitro. Phosphatase activity of wild type dis2 was reduced by incubation with cdc2 kinase, but that of mutant dis2-A316 was not. Phosphorylation of T316 hence has a potential significance in cell cycle control in conjunction with cdc2 kinase activation and inactivation. Overexpression phenotypes of wild type dis2+, sds21+ and mutant dis2-A316, sds21-TPPR genes were consistent with negative regulation of dis2 by phosphorylation. This type of regulation would explain why cells harboring the dis2-11 mutation enter mitosis but fail to exit from it.
我们发现，裂殖酵母的dis2蛋白磷酸酶与哺乳动物1型磷酸酶高度相似，是一种含有磷酸丝氨酸（p-S）和磷酸苏氨酸（p-T）的磷蛋白。它有多个磷酸化位点，其中两个位于C末端。在C末端T316的丙氨酸替代突变体中，p-T消失，在许多1型样磷酸酶中，该位点作为cdc2共有序列TPPR中的一个残基保守存在。在G2期停滞的cdc2-L7细胞中，T316的磷酸化程度降低，而在中期停滞的nuc2-663突变体细胞中则增强。裂殖酵母cdc2激酶可使dis2产生p-T，但在替代突变体A316中则不能，这表明T316残基是体外cdc2激酶的作用位点。与cdc2激酶孵育后，野生型dis2的磷酸酶活性降低，但突变体dis2-A316的磷酸酶活性未降低。因此，T316的磷酸化在细胞周期调控中与cdc2激酶的激活和失活具有潜在的重要意义。野生型dis2+、sds21+以及突变体dis2-A316、sds21-TPPR基因的过表达表型与磷酸化对dis2的负调控一致。这种调控类型可以解释为什么携带dis2-11突变的细胞进入有丝分裂但无法从中退出。
