Parra-Palau Josep-Lluis, Scheper Gert C, Wilson Mary L, Proud Christopher G
Division of Molecular Physiology, Faculty of Life Sciences, University of Dundee, Dow St., Dundee DD1 5EH, United Kingdom.
J Biol Chem. 2003 Nov 7;278(45):44197-204. doi: 10.1074/jbc.M302398200. Epub 2003 Aug 28.
Eukaryotic initiation factor eIF4E binds to the 5'-cap structure of the mRNA and also to the molecular scaffold protein eIF4G. eIF4E is a phosphoprotein, and the kinases that act on it have been identified as the MAPK-interacting kinases Mnk1 and Mnk2. Mnk1/2 also bind to the scaffold protein eIF4G. The N-terminal region of Mnk1 has previously been shown to bind to importin alpha, a component of the nuclear transport machinery, although Mnk1 itself is cytoplasmic. Here we identify a CRM1-type nuclear export motif in the C-terminal part of Mnk1. Substitution of hydrophobic residues in this motif results in Mnk1 becoming nuclear. This has allowed us to study the features of Mnk1 that are involved in its transport to the nucleus. This process requires part, but not all, of a polybasic region near the N terminus of Mnk1. Residues required for nuclear transport are also required for its interaction with importin alpha. This polybasic region also serves a second function in that it is required for the binding of Mnk1 to eIF4G, although the residues involved in this interaction are not identical to those involved in the binding of Mnk1 to importin alpha. Interaction of Mnk1 with eIF4G promotes the phosphorylation of eIF4E. Mutations that reduce the binding of Mnk1 to eIF4G in vivo and in vitro also decrease the ability of Mnk1 to enhance eIF4E phosphorylation in vivo, underlining the importance of the eIF4G-Mnk1 interaction in this process.
真核生物起始因子eIF4E与mRNA的5'-帽结构结合，也与分子支架蛋白eIF4G结合。eIF4E是一种磷蛋白，作用于它的激酶已被鉴定为与丝裂原活化蛋白激酶相互作用的激酶Mnk1和Mnk2。Mnk1/2也与支架蛋白eIF4G结合。尽管Mnk1本身位于细胞质中，但先前已表明Mnk1的N端区域与核转运机制的一个组成部分输入蛋白α结合。在这里，我们在Mnk1的C端部分鉴定出一个CRM1型核输出基序。该基序中疏水残基的替换导致Mnk1进入细胞核。这使我们能够研究Mnk1转运至细胞核所涉及的特征。这个过程需要Mnk1 N端附近部分（而非全部）多碱性区域的参与。核转运所需的残基也是其与输入蛋白α相互作用所必需的。这个多碱性区域还具有第二个功能，即它是Mnk1与eIF4G结合所必需的，尽管参与这种相互作用的残基与参与Mnk1与输入蛋白α结合所涉及的残基不同。Mnk1与eIF4G的相互作用促进了eIF4E的磷酸化。在体内和体外降低Mnk1与eIF4G结合的突变也会降低Mnk1在体内增强eIF4E磷酸化的能力，这突出了eIF4G-Mnk1相互作用在此过程中的重要性。
