de Nadal Eulàlia, Casadomé Laura, Posas Francesc
Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona E-08003, Spain.
Mol Cell Biol. 2003 Jan;23(1):229-37. doi: 10.1128/MCB.23.1.229-237.2003.
Exposure of Saccharomyces cerevisiae to increases in extracellular osmolarity activates the stress-activated Hog1 mitogen-activated protein kinase (MAPK), which is essential for cell survival upon osmotic stress. Yeast cells respond to osmotic stress by inducing the expression of a very large number of genes, and the Hog1 MAPK plays a critical role in gene transcription upon stress. To understand how Hog1 controls gene expression, we designed a genetic screen to isolate new transcription factors under the control of the MAPK and identified the MEF2-like transcription factor, Smp1, as a target for Hog1. Overexpression of SMP1 induced Hog1-dependent expression of osmoresponsive genes such as STL1, whereas smp1Delta cells were defective in their expression. Consistently, smp1Delta cells displayed reduced viability upon osmotic shock. In vivo coprecipitation and phosphorylation studies showed that Smp1 and Hog1 interact and that Smp1 is phosphorylated upon osmotic stress in a Hog1-dependent manner. Hog1 phosphorylated Smp1 in vitro at the C-terminal region. Phosphorylation of Smp1 by the MAPK is essential for its function, since a mutant allele unable to be phosphorylated by the MAPK displays impaired stress responses. Thus, our data indicate that Smp1 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK. Moreover, Smp1 concentrates in the nucleus during the stationary phase, and the lack of SMP1 results in cells that lose viability in the stationary phase. Localization of Smp1 depends on HOG1, and consistently, hog1Delta cells also lose viability during this growth phase. These data suggest that Smp1 could be mediating a role for the Hog1 MAPK during the stationary phase.
将酿酒酵母暴露于细胞外渗透压升高的环境中会激活应激激活的Hog1丝裂原活化蛋白激酶（MAPK），这对于酵母细胞在渗透应激下的存活至关重要。酵母细胞通过诱导大量基因的表达来应对渗透应激，而Hog1 MAPK在应激时的基因转录中起关键作用。为了了解Hog1如何控制基因表达，我们设计了一个遗传筛选实验，以分离受MAPK调控的新转录因子，并鉴定出MEF2样转录因子Smp1是Hog1的一个靶标。SMP1的过表达诱导了诸如STL1等渗透压响应基因的Hog1依赖性表达，而smp1Δ细胞在这些基因的表达上存在缺陷。同样，smp1Δ细胞在渗透冲击后显示出活力降低。体内共沉淀和磷酸化研究表明，Smp1和Hog1相互作用，并且Smp1在渗透应激下以Hog1依赖性方式被磷酸化。Hog1在体外C末端区域磷酸化Smp1。MAPK对Smp1的磷酸化对其功能至关重要，因为一个不能被MAPK磷酸化的突变等位基因表现出应激反应受损。因此，我们的数据表明Smp1在Hog1的下游起作用，控制由MAPK诱导的一部分反应。此外，Smp1在稳定期集中在细胞核中，缺乏SMP1会导致细胞在稳定期失去活力。Smp1的定位依赖于HOG1，同样，hog1Δ细胞在这个生长阶段也会失去活力。这些数据表明，Smp1可能在稳定期介导Hog1 MAPK的作用。
