Kranz J E, Satterberg B, Elion E A
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Genes Dev. 1994 Feb 1;8(3):313-27. doi: 10.1101/gad.8.3.313.
Activation of the Saccharomyces cerevisiae MAP kinase Fus3 is thought to occur via a linear pathway involving the sequential action of three proteins: Ste5, a protein of unknown function, Ste11, a MAPKK kinase homolog, and Ste7, a MAPK kinase homolog which phosphorylates and activates Fus3. In this report, we present evidence for a novel mechanism of Fus3 activation that involves a direct association with Ste5, a protein not predicted to interact with Fus3. First, overexpression of Ste5 suppresses fus3 point mutations in an allele-specific manner and increases Fus3 kinase activity in vitro. Second, Ste5 associates with Fus3 in vivo as demonstrated by the two-hybrid system and by two methods of copurification. Third, Ste5 and Fus3 associate prior to pheromone stimulation even when Fus3 is inactive, and in strains lacking Ste7 and Ste11. Fourth Ste5 is phosphorylated by Fus3 in purified complexes and copurifies with an additional protein kinase(s). These observations suggest the possibility that Ste5 promotes signal transduction by tethering Fus3 to its activating protein kinase(s).
酿酒酵母丝裂原活化蛋白激酶Fus3的激活被认为是通过一条线性途径发生的，该途径涉及三种蛋白质的顺序作用：Ste5，一种功能未知的蛋白质；Ste11，一种丝裂原活化蛋白激酶激酶激酶同源物；以及Ste7，一种丝裂原活化蛋白激酶激酶同源物，它磷酸化并激活Fus3。在本报告中，我们提供了Fus3激活新机制的证据，该机制涉及与Ste5直接结合，Ste5是一种未被预测与Fus3相互作用的蛋白质。首先，Ste5的过表达以等位基因特异性方式抑制fus3点突变，并在体外增加Fus3激酶活性。其次，通过双杂交系统和两种共纯化方法证明，Ste5在体内与Fus3结合。第三，即使Fus3无活性，在缺乏Ste7和Ste11的菌株中，Ste5和Fus3在信息素刺激之前就结合。第四，在纯化的复合物中，Ste5被Fus3磷酸化，并与另一种蛋白激酶共纯化。这些观察结果表明，Ste5可能通过将Fus3拴系到其激活蛋白激酶上来促进信号转导。
