PROTEIN KINASE C AND YEAST GROWTH CONTROL

蛋白激酶 C 和酵母生长控制

• 批准号: 2186012
• 负责人: DAVID E. LEVIN
• 金额: $ 24.83万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186012
• 关键词: Saccharomyces cerevisiae; biological signal transduction; cell growth regulation; electron microscopy; enzyme activity; gene expression; glycosyltransferase; isozymes; mutant; phosphorylation; protein kinase C; protein structure function; suppressor mutations

DESCRIPTION (Adapted from Applicant's Abstract): Members of the family of serine/threonine-specific protein kinase known collectively as protein kinase C (PKC) are thought to play a pivotal role in the regulation of metazoan cell proliferation through their activation by growth factors and other agonists. The long-range goal of this project is to use a combined molecular genetic and biochemical approach to understand the role of PKC in the growth control of the budding yeast, S. cerevisiae. The PKC1 gene of S. cerevisiae encoded a homolog of mammalian isozyme of PKC.Loss of PKC1 function results in a cell cycle-specific osmotic stability defect. Specifically, mutants in PKC1 undergo cell lysis shortly after bud emergence. Thin section electron microscopy of pkc1-arrested cells reveals small holes in the cell walls located at the bud tips - the site of new growth in budding yeast. This suggests a defect in the ability of pkc1 mutants to remodel their cell walls to accommodate new growth. Electron micrographs also reveal a thinning of the beta-glucan layer of the cell wall in the region surrounding the bud tip. The level of beta- glucan synthase and beta-glucanase activities in yeast cell extracts lacking PKC1 function will be examined to determine if either activity is regulated by PKC1. A class of dominant extragenic suppressors of a pkc1 deletion results from activation of novel protein kinase that may act downstream of Pkc1p. Deletion of the suppressor gene, designated BCK1, results in a cell lysis defect similar to that of pkc1 mutants. The sequence changes identified in suppressor alleles of BCK1 suggest that, under normal conditions, Bck1 may be activated in response to phosphorylation by Pkc1p. To test this hypothesis, protein kinase assays for Pkc1 and for Bck1p will be developed initially. Then, the ability of Pkc1p to phosphorylate Bck1 will be tested, and the effect of such phosphorylation on the protein kinase activity associated with Bck1p will be examined. Finally, if these initial experiments indicate that Bck1p is regulated by Pkc1p, dominant suppressor forms of Bck1p will be tested for Pkc1p-independent activity. A distinguishing feature of PKC isozymes is their requirement of several and 3) diacylgylcerol (DAG). PKC isozymes possess a catalytic domain, which is responsible for their protein kinase activity and a regulatory domain to which activating cofactors bind. Exogenous phorbolester tumor promoter can substitute for DAG in the activation of PKC. it is this feature of phorbol esters that is believed to be responsible for their tumorigenic properties. Mutant alleles of the PKC1 gene will be generated by oligonucleotide-directed mutagenesis for use in structure/function analysis of the regulatory domain of Pkc1p. The biological effects of these mutations (as measured in yeast cells bearing chromosomal deletion of PKC1) should provide insight into the specific amino acid residues involved in cofactor activation of Pkc1p. Enzymological studies of mutant Pkc1 forms will allow direct examination of the cofactor requirements of these enzymes. Existing pkc1 mutants will be exploited for the purpose of isolating additional pathway components. Expression of presumptive cofactor-independent forms of Pkc1p results in growth inhibition of yeast cells. Genes that are involved in the regulation of Pkc1p, or that encode proteins that modulate the effects of pkc1 activity, such as specific protein phosphatases, will be isolated through their ability to suppress this growth defect. Other known cell lysis mutants will also be examined for genetic interactions with PKC1 and BCK1.

描述（根据申请人的摘要改编）： 丝氨酸/苏氨酸特异性蛋白激酶统称为蛋白 激酶C（PKC）被认为在调节中起关键作用 通过生长因子的激活和 其他激动剂。该项目的远程目标是使用合并的 分子遗传和生化方法了解PKC的作用 在萌芽酵母菌的生长控制中，酿酒酵母。 PKC1基因 酿酒酵母编码了PKC的哺乳动物同工酶的同源物。 PKC1功能导致细胞周期特异性渗透稳定性缺陷。 具体而言，PKC1中的突变体在芽后不久会发生细胞裂解 出现。 PKC1预处细胞的薄截面电子显微镜 揭示位于芽尖的细胞壁上的小孔 - 位置 萌芽酵母的新增长。 这表明能力的缺陷 PKC1突变体重塑其细胞壁以适应新的生长。 电子显微照片还揭示了β-葡聚糖层的变薄 芽尖区域的细胞壁。 β- 酵母细胞提取物中的葡聚糖合酶和β-葡聚糖活性 缺乏PKC1功能将检查以确定这两个活动是否是 由PKC1调节。 PKC1缺失的一类主要的外部抑制因子是由 可能在PKC1P下游作用的新型蛋白激酶的激活。 抑制基因的缺失，指定为BCK1，导致细胞裂解 与PKC1突变体类似的缺陷。 确定的序列变化 在BCK1的抑制等位基因中，表明在正常条件下BCK1 可以通过PKC1P响应磷酸化而激活。 测试这个 假设，将开发用于PKC1和BCK1P的蛋白激酶测定 最初。然后，PKC1P磷酸化BCK1的能力将是 测试，以及这种磷酸化对蛋白激酶的影响 将检查与BCK1P相关的活动。 最后，如果这些 初始实验表明BCK1P受PKC1P的调节 BCK1P的抑制形式将测试与PKC1P无关的活性。 PKC同工酶的一个显着特征是它们对几个的要求 3）二酰基藻醇（DAG）。 PKC同工酶具有催化域， 这是其蛋白激酶活性和调节性的原因 激活辅助因子结合的域。 外源性育伐酯肿瘤 启动子可以在PKC的激活中代替DAG。 就是这样 据信负责其的凤凰酯的特征 肿瘤性特性。将生成PKC1基因的突变等位基因 通过寡核苷酸指导的诱变，用于结构/功能 PKC1P的调节域分析。 生物学作用 这些突变（如带有染色体缺失的酵母细胞中测量 PKC1）应深入了解特定的氨基酸残基 参与PKC1P的辅因子激活。 突变体的酶学研究 PKC1表格将允许直接检查辅助因素 这些酶。 现有的PKC1突变体将被利用 隔离其他途径组件。 推定的表达 pkc1p的辅助因子非依赖性形式导致酵母的生长抑制 细胞。 涉及PKC1P调节或编码的基因 调节PKC1活性作用的蛋白质，例如特定 蛋白质磷酸酶将通过抑制能力隔离 这种增长缺陷。 还将检查其他已知的细胞裂解突变体 用于与PKC1和BCK1的遗传相互作用。