Tatebayashi Kazuo, Takekawa Mutsuhiro, Saito Haruo
Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku,Tokyo 108-8639, Japan.
EMBO J. 2003 Jul 15;22(14):3624-34. doi: 10.1093/emboj/cdg353.
Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules composed of three sequentially activated kinases (MAPKKK, MAPKK and MAPK). Because individual cells contain multiple MAPK cascades, mechanisms are required to ensure the fidelity of signal transmission. In yeast, external high osmolarity activates the HOG (high osmolarity glycerol) MAPK pathway, which consists of two upstream branches (SHO1 and SLN1) and common downstream elements including the Pbs2 MAPKK and the Hog1 MAPK. The Ssk2/Ssk22 MAPKKKs in the SLN1 branch, when activated, exclusively phosphorylate the Pbs2 MAPKK. We found that this was due to an Ssk2/Ssk22-specific docking site in the Pbs2 N-terminal region. The Pbs2 docking site constitutively bound the Ssk2/Ssk22 kinase domain. Docking site mutations drastically reduced the Pbs2-Ssk2/Ssk22 interaction and hampered Hog1 activation by the SLN1 branch. Fusion of the Pbs2 docking site to a different MAPKK, Ste7, allowed phosphorylation of Ste7 by Ssk2/Ssk22. Thus, the docking site contributes to both the efficiency and specificity of signaling. During these analyses, we also found a nuclear export signal and a possible nuclear localization signal in Pbs2.
丝裂原活化蛋白激酶（MAPK）级联是由三种依次激活的激酶（MAPKKK、MAPKK和MAPK）组成的保守信号模块。由于单个细胞含有多个MAPK级联，因此需要一些机制来确保信号传递的保真度。在酵母中，外部高渗透压激活HOG（高渗透压甘油）MAPK途径，该途径由两个上游分支（SHO1和SLN1）和包括Pbs2 MAPKK和Hog1 MAPK在内的共同下游元件组成。SLN1分支中的Ssk2/Ssk22 MAPKKKs被激活时，会专门磷酸化Pbs2 MAPKK。我们发现这是由于Pbs2 N端区域存在一个Ssk2/Ssk22特异性对接位点。Pbs2对接位点持续结合Ssk2/Ssk22激酶结构域。对接位点突变极大地降低了Pbs2与Ssk2/Ssk22的相互作用，并阻碍了SLN1分支对Hog1的激活。将Pbs2对接位点与另一个MAPKK Ste7融合，可使Ssk2/Ssk22对Ste7进行磷酸化。因此，对接位点对信号传导的效率和特异性都有贡献。在这些分析过程中，我们还在Pbs2中发现了一个核输出信号和一个可能的核定位信号。
