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首页> 外文期刊>Molecular and Cellular Biology >Yeast phenotype classifies mammalian protein kinase C cDNA mutants.
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Yeast phenotype classifies mammalian protein kinase C cDNA mutants.

机译：酵母表型将哺乳动物蛋白激酶C cDNA突变体分类。

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The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of eukaryotic cellular signals. Molecular dissection of their mechanisms of action has been limited in part by the lack of random mutagenesis approaches and by the complexity of signaling pathways in mammalian cells which involve multiple PKC isoforms. Here we present a rapid screen which permits the quantification of mammalian PKC activity phenotypically in the yeast Saccharomyces cerevisiae. Bovine PKC alpha cDNA is functionally expressed in S. cerevisiae. This results in a phorbol ester response: a fourfold increase in the cell doubling time and a substantial decrease in yeast colony size on agar plates. We have expressed pools of bovine PKC alpha cDNAs mutagenized by Bal 31 deletion of internal, amino-terminal, or carboxyl-terminal sequences and have identified three classes of mutants on the basis of their distinct yeast phenotypes. Representatives of each class were analyzed. An internal deletion of amino acids (aa) 172 to 225 displayed ligand-dependent but reduced catalytic activity, an amino-terminal truncation of aa 1 to 153 displayed elevated and ligand-independent activity, and a carboxyl-terminal 26-aa truncation (aa 647 to 672) lacked activity under any conditions. Additional mutations confirmed the distinct functional characteristics of these classes. Our data show that deletion of the V1 and C1 regions results in elevated basal catalytic activity which is still Ca2+ responsive. Internal deletions in the V2 and C2 regions do not abolish phorbol ester or Ca2+ regulation of PKC activity, suggesting that most of the C2 domain is not essential for phorbol ester stimulation and most of the regulatory domain is dispensable for Ca2+ regulation of PKC activity. These distinct activities od the PKC mutants correlate with a specific and proportional yeast phenotype and are quantified on agar plates by yeast colony size. This provides a phenotypic screen which is suitable to identity rare, randomly altered but active mammalian PKC mutants. It quantifies their catalytic and biological activities in response to PKC activators or inhibitors for a systematic mapping of PKC structure and function or PKC-drug interaction.
机译：佛波酯受体蛋白激酶C（PKC）基因家族编码真核细胞信号的重要介体。缺乏随机诱变方法以及哺乳动物细胞中涉及多种PKC同工型的信号通路的复杂性，部分限制了其作用机理的分子解剖。在这里，我们提出了一种快速筛选技术，该技术可以对酿酒酵母中的表型进行哺乳动物PKC活性定量分析。牛PKCαcDNA在酿酒酵母中功能性表达。这导致佛波醇酯反应：琼脂平板上细胞倍增时间增加四倍，酵母菌落大小大幅减少。我们已经表达了由内部，氨基末端或羧基末端序列的Bal 31缺失诱变的牛PKCαcDNA库，并根据其独特的酵母表型鉴定了三类突变体。分析了每个班级的代表。氨基酸（aa）172至225的内部缺失显示配体依赖性但催化活性降低，aa 1至153的氨基末端截短显示升高且不依赖配体的活性，以及​​羧基末端26-aa截短（aa 647至672）在任何情况下都没有活动。其他突变证实了这些类别的独特功能特征。我们的数据表明，删除V1和C1区域会导致基础催化活性升高，但仍对Ca2 +具有响应性。 V2和C2区的内部缺失不会消除佛波酯或PKC活性的Ca2 +调节，这表明大部分C2域对佛波酯刺激不是必不可少的，并且大多数调节域对于PKC活性的Ca2 +调节都是可有的。 PKC突变体的这些独特活性与特定且成比例的酵母表型相关，并在琼脂平板上通过酵母菌落大小进行定量。这提供了适合于鉴定稀有，随机改变但有活性的哺乳动物PKC突变体的表型筛选。它可以量化其对PKC激活剂或抑制剂的催化和生物学活性，从而系统地绘制PKC的结构和功能或PKC-药物相互作用。

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