Institute for Systems Biology, Seattle, Washington 98103, USA.
Mol Cell Proteomics. 2010 Sep;9(9):2076-88. doi: 10.1074/mcp.M000116-MCP201. Epub 2010 Apr 15.
Phosphorylation of proteins is a key posttranslational modification in cellular signaling, regulating many aspects of cellular responses. We used a quantitative, integrated, phosphoproteomics approach to characterize the cellular responses of the yeast Saccharomyces cerevisiae to the fatty acid oleic acid, a molecule with broad human health implications and a potent inducer of peroxisomes. A combination of cryolysis and urea solubilization was used to minimize the opportunity for reorientation of the phosphoproteome, and hydrophilic interaction liquid chromatography and IMAC chemistries were used to fractionate and enrich for phosphopeptides. Using these approaches, numerous phosphorylated peptides specific to oleate-induced and glucose-repressed conditions were identified and mapped to known signaling pathways. These include several transcription factors, two of which, Pip2p and Cst6p, must be phosphorylated for the normal transcriptional response of fatty acid-responsive loci encoding peroxisomal proteins. The phosphoproteome data were integrated with results from genome-wide assays studying the effects of signaling molecule deletions and known protein-protein interactions to generate a putative fatty acid-responsive signaling network. In this network, the most highly connected nodes are those with the largest effects on cellular responses to oleic acid. These properties are consistent with a scale-free topology, demonstrating that scale-free properties are conserved in condition-specific networks.
蛋白质磷酸化是细胞信号转导中的一种关键的翻译后修饰,调节细胞反应的许多方面。我们使用一种定量的、综合的磷酸蛋白质组学方法来描述酵母酿酒酵母对脂肪酸油酸的细胞反应,油酸是一种对人类健康有广泛影响的分子,也是过氧化物酶体的有效诱导剂。采用低温裂解和尿素溶解的组合来最小化磷酸蛋白质组重定向的机会,并使用亲水相互作用液相色谱和 IMAC 化学来分离和富集磷酸肽。使用这些方法,鉴定并映射到已知信号通路的许多特定于油酸诱导和葡萄糖抑制条件的磷酸化肽。这些包括几个转录因子,其中两个,Pip2p 和 Cst6p,必须磷酸化才能正常转录脂肪酸响应基因编码的过氧化物酶体蛋白。磷酸蛋白质组数据与研究信号分子缺失和已知蛋白质-蛋白质相互作用对细胞对油酸反应的影响的全基因组分析结果相结合,生成一个假定的脂肪酸响应信号网络。在这个网络中,连接度最高的节点是对油酸细胞反应影响最大的节点。这些特性与无标度拓扑一致,表明无标度特性在特定条件的网络中是保守的。