Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms
Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms
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DOI：
10.1074/jbc.274.34.24038
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发表时间：
1999-08-20
影响因子：
4.8
通讯作者：
Hemmings, BA
中科院分区：
文献类型：
--
作者：
Evans, DRH;Myles, T;Hemmings, BA
蛋白磷酸酶 2A (PP2A) 全酶在真核生物中结构保守。这反映了体内功能的保守性，因为人催化亚基 (PP2Ac) 在功能上取代了酿酒酵母的内源 PP2Ac，并与酵母调节 PR65/A 亚基 (Tpd3p) 结合形成二聚体。酵母被用作人类 PP2Ac 诱变和功能分析的新系统，揭示了不变的 C 端亮氨酸残基（调节甲基化的位点）对于蛋白质功能显然是可有可无的。然而，缺乏较大部分 C 末端的截短形式的人 PP2Ac 发挥了显着的干扰作用，一些含有取代突变的突变形式也是如此。 PP2Ac 结构的计算机模型显示，干扰氨基酸取代聚集在活性位点，并且一致地，PP2Ac-L199P 突变蛋白尽管结合了 Tpd3p，但催化受损。因此，干扰形式的PP2Ac滴定调节亚基和/或底物形成非生产性复合物，并将作为研究哺乳动物细胞中PP2A功能的有用工具。这里采用的转基因方法，包括对干扰突变体的简单筛选，可能普遍适用于分析蛋白磷酸酶和其他保守蛋白内的结构功能关系，并进一步证明了酵母在分析基因功能方面的效用。
The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes. This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac of Saccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (Tpd3p) forming a dimer. Yeast was employed as a novel system for mutagenesis and functional analysis of human PP2Ac, revealing that the invariant C-terminal leucine residue, a site of regulatory methylation, is apparently dispensable for protein function. However, truncated forms of human PP2Ac lacking larger portions of the C terminus exerted a dominant interfering effect, as did several mutant forms containing a substitution mutation. Computer modeling of PP2Ac structure revealed that interfering amino acid substitutions clustered to the active site, and consistently, the PP2Ac-L199P mutant protein was catalytically impaired despite binding Tpd3p. Thus, interfering forms of PP2Ac titrate regulatory subunits and/or substrates into non-productive complexes and will serve as useful tools for studying PP2A function in mammalian cells. The transgenic approach employed here, involving a simple screen for interfering mutants, may be applicable generally to the analysis of structure-function relationships within protein phosphatases and other conserved proteins and demonstrates further the utility of yeast for analyzing gene function.