Ostrander D B, Gorman J A
Department of Leads Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
J Bacteriol. 1999 Apr;181(8):2527-34. doi: 10.1128/JB.181.8.2527-2534.1999.
The function of the extracellular domain (ECD) of Sln1p, a plasma membrane two-transmembrane domain (TMD) sensor of the high-osmolarity glycerol (HOG) response pathway, has been studied in the yeast Saccharomyces cerevisiae. Truncations of SLN1 that retain an intact kinase domain are capable of complementing the lethality of an sln1Delta strain. By observing levels of Hog1p phosphorylation as well as the phosphorylation state of Sln1p, the kinase activities of various SLN1 constructions were determined. In derivatives that do not contain the first TMD, Sln1p activity was no longer dependent on medium osmolarity but appeared to be constitutively active even under conditions of high osmolarity. Removal of the first TMD (DeltaTMD1 construct) gave a protein that was strongly phosphorylated whereas Hog1p was largely dephosphorylated, as expected if the active form of Sln1p is phosphorylated. When both TMDs as well as the ECD were deleted, so that the kinase domain is cytosolic, Sln1p was not phosphorylated whereas Hog1p became constitutively hyperphosphorylated. Surprisingly, this hyperactivity of the HOG mitogen-activated protein kinase signaling pathway was not sufficient to result in cell lethality. When the ECD of the DeltaTMD1 construct was replaced with a leucine zipper motif, Sln1p was hyperactive, so that Hog1p became mostly unphosphorylated. In contrast, when the Sln1p/leucine zipper construct was crippled by a mutation of one of the internal leucines, the Sln1 kinase was inactive. These experiments are consistent with the hypothesis that the ECD of Sln1p functions as a dimerization and activation domain but that osmotic regulation of activity requires the presence of the first TMD.
Sln1p是高渗甘油（HOG）应答途径的质膜双跨膜结构域（TMD）传感器，其胞外结构域（ECD）的功能已在酿酒酵母中得到研究。保留完整激酶结构域的SLN1截短体能够弥补sln1Δ菌株的致死性。通过观察Hog1p的磷酸化水平以及Sln1p的磷酸化状态，确定了各种SLN1构建体的激酶活性。在不含第一个TMD的衍生物中，Sln1p活性不再依赖于培养基渗透压，而是即使在高渗条件下似乎也组成性激活。去除第一个TMD（ΔTMD1构建体）产生了一种强烈磷酸化的蛋白质，而Hog1p在很大程度上脱磷酸化，正如预期的那样，如果Sln1p的活性形式被磷酸化。当两个TMD以及ECD都被删除，使得激酶结构域位于胞质中时，Sln1p未被磷酸化，而Hog1p变成组成性过度磷酸化。令人惊讶的是，HOG丝裂原活化蛋白激酶信号通路的这种过度激活不足以导致细胞致死。当ΔTMD1构建体的ECD被亮氨酸拉链基序取代时，Sln1p过度活跃，使得Hog1p大部分未被磷酸化。相反，当Sln1p/亮氨酸拉链构建体因内部亮氨酸之一的突变而受损时，Sln1激酶无活性。这些实验与以下假设一致：Sln1p的ECD作为二聚化和激活结构域发挥作用，但活性的渗透调节需要第一个TMD的存在。
