Ferrigno P, Posas F, Koepp D, Saito H, Silver P A
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, and the Dana-Farber Cancer Institute, Boston, MA 02115, USA.
EMBO J. 1998 Oct 1;17(19):5606-14. doi: 10.1093/emboj/17.19.5606.
MAP kinase signaling modules serve to transduce extracellular signals to the nucleus of eukaryotic cells, but little is known about how signals cross the nuclear envelope. Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 MAP kinase cascade, which is composed of three tiers of protein kinases, namely the SSK2, SSK22 and STE11 MAPKKKs, the PBS2 MAPKK, and the HOG1 MAPK. Using green fluorescent protein (GFP) fusions of these kinases, we found that HOG1, PBS2 and STE11 localize to the cytoplasm of unstressed cells. Following osmotic stress, HOG1, but neither PBS2 nor STE11, translocates into the nucleus. HOG1 translocation occurs very rapidly, is transient, and correlates with the phosphorylation and activation of the MAP kinase by its MAPKK. HOG1 phosphorylation is necessary and sufficient for nuclear translocation, because a catalytically inactive kinase when phosphorylated is translocated to the nucleus as efficiently as the wild-type. Nuclear import of the MAPK under stress conditions requires the activity of the small GTP binding protein Ran-GSP1, but not the NLS-binding importin alpha/beta heterodimer. Rather, HOG1 import requires the activity of a gene, NMD5, that encodes a novel importin beta homolog. Similarly, export of dephosphorylated HOG1 from the nucleus requires the activity of the NES receptor XPO1/CRM1. Our findings define the requirements for the regulated nuclear transport of a stress-activated MAP kinase.
丝裂原活化蛋白激酶（MAP激酶）信号转导模块负责将细胞外信号传导至真核细胞的细胞核，但对于信号如何穿过核膜却知之甚少。将酵母细胞暴露于细胞外渗透压升高的环境中会激活HOG1 MAP激酶级联反应，该级联反应由三层蛋白激酶组成，即SSK2、SSK22和STE11 MAPKKK、PBS2 MAPKK以及HOG1 MAPK。利用这些激酶的绿色荧光蛋白（GFP）融合体，我们发现HOG1、PBS2和STE11定位于未受应激细胞的细胞质中。在渗透应激后，HOG1会转移至细胞核，而PBS2和STE11则不会。HOG1的转移非常迅速，是短暂的，并且与MAP激酶被其MAPKK磷酸化和激活相关。HOG1磷酸化对于核转位是必要且充分的，因为磷酸化后的无催化活性激酶与野生型一样有效地转移至细胞核。应激条件下MAPK的核输入需要小GTP结合蛋白Ran-GSP1的活性，但不需要与核定位信号（NLS）结合的输入蛋白α/β异二聚体。相反，HOG1的输入需要一个基因NMD5的活性，该基因编码一种新型的输入蛋白β同源物。同样，去磷酸化的HOG1从细胞核输出需要NES受体XPO1/CRM1的活性。我们的研究结果确定了应激激活的MAP激酶的调节性核转运的要求。
