Elion E A, Satterberg B, Kranz J E
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Mol Biol Cell. 1993 May;4(5):495-510. doi: 10.1091/mbc.4.5.495.
The mitogen-activated protein (MAP) kinase homologue FUS3 mediates both transcription and G1 arrest in a pheromone-induced signal transduction cascade in Saccharomyces cerevisiae. We report an in vitro kinase assay for FUS3 and its use in identifying candidate substrates. The assay requires catalytically active FUS3 and pheromone induction. STE7, a MAP kinase kinase homologue, is needed for maximal activity. At least seven proteins that specifically associate with FUS3 are phosphorylated in the assay. Many of these substrates are physiologically relevant and are affected by in vivo levels of numerous signal transduction components. One substrate is likely to be the transcription factor STE12. A second is likely to be FAR1, a protein required for G1 arrest. FAR1 was isolated as a multicopy suppressor of a nonarresting fus3 mutant and interacts with FUS3 in a two hybrid system. Consistent with this FAR1 is a good substrate in vitro and generates a FUS3-associated substrate of expected size. These data support a model in which FUS3 mediates transcription and G1 arrest by direct activation of STE12 and FAR1 and phosphorylates many other proteins involved in the response to pheromone.
有丝分裂原激活蛋白（MAP）激酶同源物FUS3在酿酒酵母的信息素诱导信号转导级联反应中介导转录和G1期阻滞。我们报道了一种针对FUS3的体外激酶分析方法及其在鉴定候选底物中的应用。该分析需要具有催化活性的FUS3和信息素诱导。MAP激酶激酶同源物STE7是实现最大活性所必需的。在该分析中，至少有七种与FUS3特异性结合的蛋白质被磷酸化。这些底物中的许多在生理上具有相关性，并受到多种信号转导成分体内水平的影响。一种底物可能是转录因子STE12。另一种可能是FAR1，一种G1期阻滞所需的蛋白质。FAR1是作为非阻滞性fus3突变体的多拷贝抑制子分离出来的，并在双杂交系统中与FUS3相互作用。与此一致的是，FAR1在体外是一种良好的底物，并产生预期大小的与FUS3相关的底物。这些数据支持了一个模型，即FUS3通过直接激活STE12和FAR1来介导转录和G1期阻滞，并磷酸化许多其他参与信息素反应的蛋白质。
